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https://openalex.org/W2615318521
http://journals.ums.ac.id/index.php/dayasaing/article/download/3788/2450
Indonesian
null
PENGARUH DISIPLIN KERJA, MOTIVASI KERJA, KEPUASAN KERJA DAN KOMPETENSI TERHADAP KOMITMEN ORGANISASI
Jurnal Manajemen Dayasaing/Jurnal manajemen dayasaing
2,017
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4,502
Abstract The purpose of this study to determine and analyze the effect of work discipline, work motivation, job satisfaction, and work competence of the organization’s commitment to employees in RSO Prof. Dr. R Soeharso Surakarta. This research was explanatory research with survey research design. Data used primary data. The population of all employees who work in RSO Prof. Dr. R. Soeharso totaling 626 people, were taken a sample of 244 people with purposive sampling technique. The research instrument used a questionnaire with a scale linkert. The data analysis includes validation test, reliability and multiple regression analysis, t-test, F and R2. The results showed discipline work positive and significant effect on organizational commitment, motivation positive and significant effect on organizational commitment, job satisfaction positive and significant effect on organizational commitment, competence work positive and significant effect on organizational commitment, and the discipline of work, work motivation , job satisfaction and work competence significant effect together against the organization’s commitment to employee hospital Orthopedics Prof. Dr. R. Soeharso Surakarta Keywords: Work discipline, work motivation, job satisfaction, and competence, organization commitment. Kata Kunci: Disiplin kerja, motivasi kerja, kepuasan kerja, kompetensi kerja, komitmen organisasi PENGARUH DISIPLIN KERJA, MOTIVASI KERJA, KEPUASAN KERJA DAN KOMPETENSI TERHADAP KOMITMEN ORGANISASI Nurlaely M. dan Asri Laksmi Riani Pascasarjana Universitas Sebelas Maret Surakarta, Jl. Ir. Sutami 36 A Surakarta Email: nurlelimanurung@yahoo.com Abstrak Tujuan penelitian ini untuk mengetahui dan menganalisis pengaruh disiplin kerja, motivasi kerja, kepuasan kerja, dan kompetensi kerja terhadap komitmen organisasi pada karyawan di RSO Prof. Dr. R Soeharso Surakarta. Penelitian ini berjenis penelitian eksplanatori dengan desain penelitian survei. Jenis data yang digunakan data primer. Populasi yang digunakan seluruh karyawan yang bekerja di RSO Prof Dr R. Soeharso yang berjumlah 626 orang, diambil sampel sebanyak 244 orang dengan teknik purposive sampling. Instrumen penelitian menggunakan kuesioner dengan skala linkert. Analisa data meliputi uji validasi, reliabilitas dan analisa regresi berganda, uji t, uji F dan R2. Hasil penelitian menunjukkan disiplin kerja berpengaruh positif dan signifikan terhadap komitmen organisasi, motivasi kerja berpengaruh positif dan signifikan terhadap komitmen organisasi, Kepuasan kerja berpengaruh positif dan signifikan terhadap komitmen organisasi, Kompetensi kerja berpengaruh positif dan signifikan terhadap komitmen organisasi, dan Disiplin kerja, motivasi kerja, kepuasan kerja dan kompetensi kerja berpengaruh signifikan secara bersama-sama terhadap komitmen organisasi pada karyawan RS. Ortopedi Prof. Dr. R. Soeharso Surakarta. ata Kunci: Disiplin kerja, motivasi kerja, kepuasan kerja, kompetensi kerja, komitmen ganisasi 10 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 18, No. 1, Juni 2016 Pendahuluan Penelitian ini merupakan pengembangan dari hasil Studi pada PT. Dada Indonesia yang dilakukan Mangkunegara dan Octorend (2015) dengan Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja dan Komitmen Organisasi pada karyawan di Perusahaan tahun 2015 yang dilakukan oleh Sriekaningsih dan Setyadi (2015) mengenai pengaruh kompetensi, motivasi dan budaya organisasi kearah komitmen organisasi dan kinerja pada dosen state university di Kalimantan Timur tahun 2015. Era globalisasi berdampak pada segala aspek dan membawa berbagai perubahan paradigma dan persaingan bisnis terlebih lagi dengan dicanangkannya MEA. Semakin banyaknya kompetitor dan tingginya persaingan dalam segala aspek, maka SDM dalam organisasi dituntut memiliki pengetahuan, kemampuan, motivasi dan kompetensi yang tinggi, bahkan SDM perlu peningkatan dalam bidang spesialisasinya. Motivasi kerja yang ada pada diri seseorang menjadi frame of reference dalam mewujudkan suatu perilaku yang diarahkan pada tujuan pribadi. Hal ini perlu ditunjang oleh disiplin kerja yang bagus dan komitmen terhadap segala peraturan yang ada dan mentaati perintah serta kebijakan organisasi dan pimpinan sehingga setiap karyawan yang ada mampu berperan aktif dalam mewujudkan visi dan misi dari organisasi. Tujuan penelitian ini sebagai berikut: (1) Menganalisis pengaruh disiplin kerja terhadap komitmen organisasi; (2) Menganalisis pengaruh motivasi terhadap komitmen organisasi; (3) Menganalisis pengaruh kepuasan kerja terhadap terhadap komitmen organisasi; (4) Menganalisis pengaruh kompetensi terhadap komitmen organisasi; (5) Menganalisis pengaruh disiplin kerja, motivasi kerja, kepuasan kerja, dan kompetensi kerja terhadap komitmen organisasi pada karyawan di RSO Prof. Dr. R Soeharso Surakarta. Tuntutan masyarakat yang semakin kompleks, peningkatan kemampuan dan pemberdayaan sumber daya manusia merupakan modal penting bagi kelancaran organisasi, disamping adanya kelengkapan sarana dan prasarana. Menurut Handoko (2014) manusia harus bertindak sebagai pengelola dan pelaksananya sebab manusia merupakan kunci keberhasilan dalam mengerjakan suatu pekerjaan, untuk itu dibutuhkan Karyawan yang mampu dan terampil, dalam melaksanakan tugas. Metode Penelitian Penelitian dilakukan di Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta. Penelitian ini merupakan penelitian eksplanatori dengan desain penelitian survei. Jenis data yang digunakan data primer. Populasi yang digunakan seluruh karyawan yang bekerja di RSO Prof Dr R. Soeharso yang berjumlah 626 orang, diambil sampel sebanyak 244 orang dengan teknik purposive sampling. Variabel bebas dalam penelitian ini adalah disiplin kerja, motivasi kerja, kepuasan kerja dan kompetensi kerja, sedangkan variabel terikatnya adalah komitmen organiasi. Instrumen penelitian menggunakan kuesioner dengan skala linkert. Analisa data meliputi uji validasi, reliabilitas dan analisa regresi berganda, uji t, uji F dan R2 . 3. Kinerja Menurut Minner (dalam Pramudita 2008) kinerja adalah tingkat keberhasilan seseorang Karyawan dalam melaksanakan pekerjaan, jadi kinerja merupakan hasil yang dicapai oleh seseorang menurut ukuran yang berlaku untuk suatu pekerjaan. 1. Motivasi Motivasi merupakan masalah yang kompleks dalam perubahan sebab kebutuhan dan keinginan setiap anggota rumah sakit berbeda-beda. Rumah sakit perlu memahami motivasi para Karyawannya, sebab faktor ini akan menentukan jalannya rumah sakit dalam mencapai tujuannya, agar perilaku Karyawan sesuai dengan tujuan visi dan misi organisasi maka harus ada perpaduan yang serasi antara motivasi dengan pemenuhan kebutuhan mereka serta keinginan rumah sakit. Untuk memenuhi kebutuhan Karyawan, rumah sakit menuangkannya dalam kebijakan. Kebijakan rumah sakit yang sesuai dengan kebutuhan, upaya, keinginan serta harapan Karyawan akan berdampak positif terhadap kinerja Karyawan (Halim, 2013). Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta sebagai institusi layanan sosial dan merupakan rumah sakit pusat rujukan nasional yang berperan aktif dalam pelaksanaan pemberian jasa layanan masyarakat. Sebagai pemberi jasa layanan sosial masyarakat harus mampu mengelola sumber daya manusia atau karyawan agar menjadi pelayan masyarakat yang baik khususnya mereka yang sedang membutuhkan perhatian yang lebih karena kondisinya yang sedang kurang sehat secara fisik. Manajemen sumber daya manusia merupakan bagian dari organisasi. 11 Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja Dan Kompetensi Terhadap Komitmen Organisasi (10-19) Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja Dan Kompetensi Terhadap Komitmen Organisasi (10-19) Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja Dan Kompetensi Terhadap Komitmen Organisasi (10-19) 2. Kepuasan Kerja rumah sakit. Karyawan yang memiliki komitmen yang kuat mempunyai keterikatan yang besar dengan institusi ia bekerja, sebagai bukti tidak berkeinginan untuk meninggalkan tempat kerja karena mempunyai loyalitas yang tinggi. Hasil penelitian Schein yang dikutip oleh Celine (2007) menunjukkan para pekerja akan merasa lebih puas dan memegang teguh komitmennya sesuai dengan nilai-nilai rumah sakit, dalam interaksi sehari- hari antara atasan dengan bawahan kadang muncul berbagai asumsi dan harapan lain. Adanya kesesuaian asumsi dan harapan Karyawan akan menimbulkan kepuasan kerja dan menjadi salah satu faktor penentu keberhasilan rumah sakit. Menurut Spencer (1993) salah satu indikator kompetensi akan terlihat dari motivasi karakteristik individu; semakin tinggi motivasi seseorang maka semakin berkompeten hal ini akan terlihat dari hasil kerja yang maksimal sesuai dengan kemampuan dan cara kerja yang dilakukan. Kepuasan kerja adalah perasaan seseorang terhadap pekerjaannya (As’ad, 2007). Kepuasan kerja bagi seorang Karyawan baik itu sebagai pimpinan maupun hanya pelaksana laksana bensin pada kendaraan bermotor, sebab kendaraan tanpa bensin tidak akan mampu berjalan dengan lancar. Menurut Davis (2008) kepuasan kerja adalah suatu perasaan yang menyokong diri Karyawan yang berhubungan dengan pekerjaan maupun dengan dirinya. Konsepsi kepuasan kerja Karyawan sebagai hasil interaksi manusia dengan lingkungannya dan terkait dengan masing-masing individu serta situasi lingkungan kerja. Menurut Bloom (dalam As’ad, 2007) kepuasan kerja adalah sikap umum yang merupakan hasil dari beberapa sikap khusus terhadap faktor-faktor pekerjaaan, penyesuaian diri dan hubungan sosial individual di luar kerja. Adanya motivasi, kedisiplinan yang tinggi serta kepuasan kerja diharapkan seorang Karyawan mampu menghasilkan kinerja yang optimal. 4. Komitmen Komitmen sebagai derajat keterikatan relative dari individu terhahap kepentingan organisasi dan derajat keterlibatannya dalam organisasi, menurut Luthans (2008) memiliki 3 faktor yaitu : a) Keinginan kuat untuk tetap menjadi anggota suatu organisasi; b) Kesediaan untuk berusaha sebaik mungkin demi kebaikan organisasi; c) Kepercayaan dan penerimaan yang kuat terhadap nilai- nilai dan tujuan organisasi tersebut. Untuk mencapai komitmen yang tinggi dibutuhkan pengelolaan Karyawan dengan benar. Menurut Trianggraery (2013) komitmen terhadap kerja sangat berperan dalam mencapai keberhasilan 1. Analisis Koefisien Determinasi (R2)i 1. Analisis Koefisien Determinasi (R2)i Nilai koefisien derterminasi (R2) model persamaan ini sebagaimana terlihat pada tabel 1 berikut. 12 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 18, No. 1, Juni 2016 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 18, No. 1, Juni 2016 Tabel 1. Hasil Analisis Koefisien Determiasi (R2) Model Summary 2. Uji F Perhitungan nilai R2 diperoleh angka sebesar 0,378, artinya bahwa 37,80% variasi variabel komitmen organisasi dapat dijelaskan oleh variabel disiplin kerja, motivasi kerja, kepuasan kerja dan kompetensi kerja, sedangkan sisanya yaitu 62,2% merupakan variabel yang tidak diteliti dalam penelitian ini. Uji F dimaksudkan untuk menguji apakah model regresi dengan variabel dependen dan variabel independen mempunyai pengaruh signifikan secara simultan. Berdasarkan uji hipotesis dengan uji F diketahui seperti tampak pada tabel berikut : Tabel 2. Hasil Uji F-test ANOVAa Model Sum of Squares Df Mean Square F Sig. 1 Regression 3190,918 4 797,729 36,262 .000b Residual 5257,765 239 21,999 Total 8448,672 243 a. Dependent Variable: Komitmen organisasi (Y) b. Predictors: (Constant), Kompetensi Kerja (X4), Kepuasan Kerja (X3), Disiplin Kerja (X1), Motivasi Kerja (X2) Sumber: Analisis Data primer, 2016. Tabel 2. Hasil Uji F-test ANOVAa berpengaruh signifikan terhadap variabel terikat yaitu komitmen organisasi. berpengaruh signifikan terhadap variabel terikat yaitu komitmen organisasi. Berdasarkan hasil analisis didapatkan Fhitung > Ftabel ; 36,262 > 2,760 dan nilai sign. 0,000 < 0,05 maka Ho ditolak dan Ha diterima, hal ini berarti variabel disiplin kerja, motivasi kerja, kepuasan kerja, dan kompetensi kerja berpengaruh signifikan terhadap variabel terikat yaitu komitmen organisasi. 3. Uji t Berdasarkan hasil analisis dapat diketahui hasil uji t seperti tampak pada tabel berikut: 3. Uji t i variabel disiplin kerja, motivasi san kerja, dan kompetensi kerja Berdasarkan hasil analisis dapat diketahui hasil uji t seperti tampak pada tabel berikut: Berdasarkan hasil analisis dapat diketahui hasil uji t seperti tampak pada tabel berikut: Tabel 3. Rekapitulasi Hasil Uji t Variabel Koefisien Regresi thitung ttabel Sig. (a=0,05) Disiplin Kerja (X1) Motivasi Kerja (X2) Kepuasan Kerja (X3) Kompetensi Kerja (X4) 0,501 1,072 0,777 0,725 3,125 7,161 6,606 5,002 2,042 2,042 2,042 2,042 0,002 0,000 0,000 0,000 Sumber: Analisis Data Primer Tabel 3. Rekapitulasi Hasil Uji t Tabel 3. Rekapitulasi Hasil Uji t Sumber: Analisis Data Primer pengaruh tiap-tiap variabel independen yaitu disiplin kerja, motivasi kerja, kepuasan kerja dan kompetensi kerja yang baik akan dapat meningkatkan komitmen organisasi pada karyawan di Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta. Berdasarkan Tabel 1. menunjukkan hasil uji signifikan secara parsial bahwa variabel disiplin kerja, motivasi kerja, kepuasan kerja dan kompetensi kerja berpengaruh positif dan signifikan terhadap variabel komitmen organisasi. Uji ini dilakukan untuk mengetahui signifikansi 13 Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja Dan Kompetensi Terhadap Komitmen Organisasi (10-19) Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja Dan Kompetensi Terhadap Komitmen Organisasi (10-19) hasil analisis dapat diuraikan pengaruh tiap- tiap variabel independen terhadap variabel dependen sebagai berikut: Pengaruh kompetensi kerja (X4) terhadap komitmen organisasi. Hasil uji-t diperoleh nilai thitung sebesar 5,002 > ttabel (2,042) dengan nilai probabilitas (r = 0,000) < 0,05 berarti H4 diterima kebenarannya, artinya bahwa ada pengaruh yang signifikan antara kompetensi kerja terhadap komitmen organisasi pada karyawan di Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta, adapun besarnya pengaruhnya antara kompetensi kerja terhadap komitmen organisasi pada karyawan sebesar 0,725, artinya jika kompetensi kerja meningkat satu persen maka komitmen organisasi meningkat sebesar 0,725 dengan asumsi variabel X1, X2, dan X4 = 0. Pengaruh disiplin kerja (X1) terhadap komitmen organisasi. Hasil uji-t diperoleh nilai thitung sebesar 3,125 > ttabel (2,042) dengan nilai probabilitas (r = 0,002) < 0,05 berarti H2 diterima kebenarannya, artinya bahwa ada pengaruh yang signifikan antara disiplin kerja terhadap komitmen organisasi pada karyawan di Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta, adapun besarnya pengaruhnya antara disiplin kerja terhadap komitmen organisasi pada karyawan sebesar 0,501, artinya jika disiplin kerja meningkat satu satuan maka komitmen organisasi meningkat sebesar 0,501 dengan asumsi variabel X2, X3, dan X3 = 0. 3. Uji t Berdasarkan hasil analisis tersebut terbukti bahwa hipotesis yang menyatakan ”ada pengaruh yang signifikan variabel independen (disiplin kerja, motivasi kerja, kepuasan kerja dan kompetensi kerja) terhadap variabel dependen (komitmen organisasi) pada karyawan di RS. Ortopedi Prof. Dr. R. Soeharso Surakarta secara parsial”, terbukti kebenarannya. 3 Pengaruh motivasi kerja (X2) terhadap komitmen organisasi. Hasil uji-t diperoleh nilai thitung sebesar 7,161 > ttabel (2,042) dengan nilai probabilitas (r = 0,000) < 0,05 berarti H3 diterima kebenarannya, artinya bahwa ada pengaruh yang signifikan antara motivasi kerja terhadap komitmen organisasi pada karyawan di Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta, adapun besarnya pengaruhnya antara motivasi kerja terhadap komitmen organisasi pada karyawan sebesar 1,072, artinya jika motivasi kerja meningkat satu persen maka komitmen organisasi meningkat sebesar 0,301 dengan asumsi variabel X1, X3, dan X4 = 0. 4. Pengaruh disiplin kerja terhadap komitmen organisasi Berdasarkan pengujian hipotesis dengan uji t diketahui nilai sign. 0,002 < 0,05, maka hipotesis pertama (H1) terbukti kebenarannya, artinya ada pengaruh positif dan signifikan secara parsial antara disiplin kerja terhadap variabel terikat yaitu komitmen organisasi. y g Kedisiplinan kerja yang ditunjukan karyawan juga mempengaruhi kinerjanya (Amran, 2009). Karena dengan kedisiplinan yang baik dengan cara mengikuti aturan-aturan perusahaan karyawan dapat mengerjakan tugasnya dengan tepat waktu dan tidak mengahambat bidang kerja lain dalam perusahaan. Menurut Mangkunegara (2015) disiplin pada dasarnya adalah kemampuan untuk mengendalikan diri dan mendukung sesuatu yang telah dibuat. Manajemen untuk menegakkan standar organisasi yang dinyatakan dalam pelaksanaan pedoman manajemen untuk memperkuat organisasi. Disiplin kerja sebagai salah satu alat yang digunakan oleh manajer untuk 1 3 4 Pengaruh kepuasan kerja (X3) terhadap komitmen organisasi. Hasil uji-t diperoleh nilai thitung sebesar 6,606 > ttabel (2,042) dengan nilai probabilitas (r = 0,000) < 0,05 berarti H4 diterima kebenarannya, artinya bahwa ada pengaruh yang signifikan antara kepuasan kerja terhadap komitmen organisasi pada karyawan di Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta, adapun besarnya pengaruhnya antara kepuasan kerja terhadap komitmen organisasi pada karyawan sebesar 0,777, artinya jika kepuasan kerja meningkat satu persen maka komitmen organisasi meningkat sebesar 0,777 dengan asumsi variabel X1, X2, dan X4 = 0. 14 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 18, No. 1, Juni 2016 motivasi dengan komitmen organisasi dapat diuaraikan sebagai brrikut semakin tinggi motivasi seseorang untuk bertahan dan terlibat dalam suatu organisasi maka akan semakin meningkat komitmen seseorang, Motivasi kerja yang timbul dapat dipengaruhi oleh beberapa faktor, antara lain, atasan, fisik fasilitas, kebijakan, aturan, penghargaan dan uang non-tunai layanan, jenis bekerja dan tantangan. berkomunikasi dengan karyawan sehingga mereka bersedia mengubah perilaku dalam upaya mematuhi semua peraturan perusahaan dan norma-norma sosial yang berlaku sosial. tanpa pengecualian. Hasil penelitian ini didukung oleh hasil penelitian sebelumnya yang dilakukan Amran (2009), Zesbendri dan Aryanti (2009) menyatakan bahwa disiplin kerja berpengaruh secara parsial terhadap komitmen organisasi. Di samping itu hasil penelitian ini diperkuat oleh penelitian yang dilakukan oleh Prabasari (2013) yang menyimpulkan bahwa tersapat pengaruh positif dan signifikan antara disiplin kerja terhadap komitmen organisasi. Hasil penelitian ini sejalan dengan penelitian yang dilakukan oleh Nurcahyo (2011) menyatakan bahwa motivasi berpengaruh signifikan secara parsial terhadap komitmen organisasi, dan juga Prabasari (2013) yang menyatakan bahwa ada pengaruh positif dan signifikan variabel motivasi terhadap komitmen organisasi. 4. Pengaruh disiplin kerja terhadap komitmen organisasi Hasil penelitian ini sejalan dengan penelitian yang dilakukan oleh Sriekaningsih dan Setyadi (2008) mengenai pengaruh kompetensi, motivasi dan budaya organisasi kearah komitmen organisasi dan kinerja pada Dosen State University di Kalimantan Timur tahun 2015, hasil penelitiannya menyimpulkan bahwa ternyata motivasi, disiplin kerja dan kepuasan kerja mempunyai pengaruh terhadap prestasi kerja pegawai, sehingga dapat dijelaskan pula bahwa disiplin kerja dan kepuasan kerja dapat meramalkan perubahan pada kinerja pegawai tersebut. Secara teori motivasi mempengaruhi aktivitas dan tujuan organisasi, dorongan- dorongan yang ada pada diri seseorang yang mengarah pada tercapainya tujuan, dorongan yang paling kuat menghasilkan adanya perilaku, baik yang berupa aktivitas terarah ke tujuan atau aktivitas tujuan, adanya motivasi maka pegawai akan memberikan rangsangan yang positif terhadap pekerjaannya, sehingga disiplin kerja pegawai akan mempe-ngaruhi prestasi atau kinerjanya, demikian halnya adanya kepuasan kerja secara teori kepuasan kerja mencakup pemahaman pegawai terhadap misi, komitmen, komunikasi, sense of belonging (rasa memiliki) dan penghargaan yang proporsional akan mendorong terciptanya efektivitas organisasi. Hal ini dapat ditempuh dengan memberikan dorongan yang positif 5. Pengaruh Motivasi Kerja terhadap Komitmen Organisasi Berdasarkan pengujian hipotesis dengan uji t diketahui nilai sign. 0,000 < 0,05, maka hipotesis kedua (H2) terbukti kebenarannya, artinya ada pengaruh positif dan signifikan secara parsial antara motivasi kerja terhadap variabel terikat yaitu komitmen organisasi. Hal ini berarti semakin baik dan meningkat dari motivasi kerja yang dimiliki karyawan maka semakin meningkat pula komitmen organisasi tersebut.Hal yang perlu dilakukan dalam upaya meningkatkan motivasi kerja terutama berkaitan dengan motivasi yang berhubungan dengan kegiatan baik itu pemasaran maupun hasil kerja garment terhadap kinerja seperti memberikan gaji yang lebih apabila ada karyawan yang melebihi target hasil kerja yang ditetapkan perusahaan, mengikutsertakan karyawan pada kegiatan-kegiatan sosial dan lain sebagainya, dengan disiplin kerja yang dimiliki karyawan akan menyebabkan karyawan menggunakan semaksimal mungkin terhadap hasrat untuk bekerja secara benar sehingga diperoleh komitmen organisasi yang maksimal pula. Menurut Mangkunegara (2015) motivasi merupakan proses yang mendorong seseorang dalam menentukan intensitas, arah, dan ketekunan individu dalam upaya untuk mencapai target baik target individu maupun organisasi dimana ia berada. Hubungan 15 Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja Dan Kompetensi Terhadap Komitmen Organisasi (10-19) Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja Dan Kompetensi Terhadap Komitmen Organisasi (10-19) serta memberikan interaksi antara sikap, kebutuhan, persepsi dan keputusan yang ada pada diri pegawai dalam melaksanakan pekerjaannya, sebab motivasi mendorong jiwa dan sikap mental manusia sehingga memberikan energi kearah pencapaian suatu tujuan yang telah ditentukan. terhadap komitmen organisasi pada Industri Roko di Jawa Timur. Jadi kepuasan kerja mempunyai hubungan secara positif dan searah berpengaruh terhadap kinerja, dengan demikian bahwa kepuasan kerja yang kuat akan menghasilkan kinerja yang positif. 7. Pengaruh Kompetensi Kerja terhadap Komitmen Organisasi 6. Pengaruh Kepuasan Kerja terhadap Komitmen Organisasi Berdasarkan pengujian hipotesis dengan uji t diketahui nilai sign. 0,005 < 0,05, maka hipotesis keempat (H4) terbukti kebenarannya, artinya ada pengaruh positif dan signifikan secara parsial antara kepuasan kerja terhadap variabel terikat yaitu komitmen organisasi. Hal ini berarti semakin baik kepuasan kerjanya maka semakin meningkat pula komitmen organisasi tersebut. Hasil penelitian ini mendukung teori yang dikemukakan oleh Streuner dan Bjoruest (1998) kompetensi merupakan kemampuan individu melakukan tugas-tugas yang telah ditetapkan pimpinan kepadanya. Kompetensi dipahami sebagai pengetahuan, keterampilan, dan identitas profesional sesuai komitmen karyawan yang bersangkutan, Menurut Michael Zwell (2005) ada lima kategori kompetensi, yaitu: task achievement, relationship, personal attribute, managerial dan leadership. Berdasarkan pengujian hipotesis dengan uji t diketahui nilai sign. 0,005 < 0,05, maka hipotesis ketiga (H3) terbukti kebenarannya, artinya ada pengaruh positif dan signifikan secara parsial antara kepuasan kerja terhadap variabel terikat yaitu komitmen organisasi. Hal ini berarti semakin baik kepuasan kerjanya maka semakin meningkat pula komitmen organisasi tersebut. Hasil penelitian ini mendukung teori yang dikemukakan oleh Robbins (2008) bahwa kepuasan kerja dapat menyatukan berbagai karakteristik individu kedalam suatu wadah perilaku yang didasarkan nilai-nilai kebersamaan dan diyakini kebenaranya. Upaya untuk meningkatkan komitmen organisasi diantaranya karyawan telah menggunakan metode kerja yang efektif, karyawan mempunyai kemampuan untuk menyelesaikan pekerjaan, karyawan cukup memahami terhadap pekerjaan yang dibebankan kepada pimpinan atau perusahaan, dan lain-lainnya. Hal ini dapat ditempuh dengan memberikan penjelasan dari perusahaan/pimpinan untuk bekerja lebih efektif, memahamkan karyawan untuk bekerja lebih baik dari hari kemarin, dan memberikan kebijakan kepada karyawan untuk mampu bekerja sesuai dengan arahan dan metode yang benar yang sudah ditetapkan oleh perusahaan. Hubungan kompetensi kerja terhadap komitmen organisasibahwa semua kemampuan dan keterampilan melaksanakan tugas yang diberikan atasan, hubungan yang baik dengan atasan maupun sesama akan sangat berpengaruh terhadap peningkatan komitmen organisasi. 7. Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja dan Kompetensi Kerja terhadap Komitmen Organisasi 7. Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja dan Kompetensi Kerja terhadap Komitmen Organisasi Hasil penelitian ini sejalan dengan hasil penelitian yang dilakukan Amran (2009) bahwa terdapat pengaruh positif antara kepuasan kerja dengan kinerja pegawai Dinas Kebudayaan dan Pariwisata di Jawa Timur. Dan penelitian yang dilakukan oleh Usman (2011) terdapat pengaruh sangat kuat dan signifikan antara kepuasan kerja Berdasarkan pengujian hipotesis dengan uji F diketahui nilai sign. 0,000 < 0,05, maka hipotesis kelima (H5) terbukti kebenarannya, artinya ada pengaruh signifikan secara bersama-sama antara disiplin kerja, motivasi kerja, kepuasan kerja dan kompetensi kerja terhadap variabel terikat yaitu komitmen 16 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 18, No. 1, Juni 2016 organisasi. Hal ini dikarenakan karyawan berkerja dengan sesuai dengan tugas dan tanggung jawab, berkerja sesuai dengan aturan yang berlaku, menggunakan waktu dengan baik, menyelesaikan pekerjaan tepat waktu dan mematuhi perintah atasan serta taat dan tertib dalam melaksanakan tugas dan tanggung jawab yang diberikan kepadanya. Hasil penelitian ini mendukung terori yang dikemukakan oleh Hasibuan (2009). Dengan disiplin yang baik akan mencerminkan besarnya tanggung jawab seseorang terhadap tugas-tugas yang diberikan karena disiplin merupakan operatif manajemen sumber daya manusia yang terpenting karena semakin baik disiplin karyawan, semakin tinggi prestasi kerja yang dapat dicapainya, dan tanpa disiplin karyawan yang baik sulit bagi organisasi mencapai hasil yang optimal. terdapat pengaruh signifikan motivasi kerja, disiplin kerja dan kepuasan kerja terhadap kinerja pegawai kesehatan di RS Secanti Gisting. Selain itu hasil penelitian ini juga diperkuat oleh penelitian yang dilakukan oleh Novitasari (2013) yang menyimpulkan bahwa terdapat pengaruh signifikan secara simultan antara motivasi, kepuasan kerja dan disiplin kerja terhadap kinerja pegawai di Dinas Perindustrian dan Perdagangan Provinsi Jawa Tengah. Penutup p Berdasarkan pada penyajian hasil penelitian serta pembahasan hasil penelitian, maka dapat dikemukakan beberapa kesimpulan sebagai berikut. Disiplin kerja berpengaruh positif dan signifikan terhadap komitmen organisasi pada karyawan Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta. Motivasi kerja berpengaruh positif dan signifikan terhadap komitmen organisasi pada karyawan Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta. Kepuasan kerja berpengaruh positif dan signifikan terhadap komitmen organisasi di Rimah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta. Kompetensi kerja berpengaruh positif dan signifikan terhadap komitmen organisasi pada karyawan Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta. Disiplin kerja, motivasi kerja, dan kepuasan kerja berpengaruh signifikan secara bersama-sama terhadap komitmen organisasi pada karyawan Rumah Sakit Ortopedi Prof. Dr. R. Soeharso Surakarta. Demikian juga motivasi, keberhasilan suatu organisasi sangat dipengaruhi oleh faktor angota-anggotanya dalam melakukan fungsinya masing-masing. Faktor yang sangat berpengaruh terhadap kualitas kerja dari anggota organisasi adalah komitmen organisasi yang dimiliki. Motivasi adalah suatu proses yang mendorong, mengarahkan dan memelihara perilaku manusia ke arah pencapaian suatu tujuan. Batasan tersebut memperlihatkan kepada kita bahwa setiap gerak individu sadar atau tidak pasti memiliki motivasi (Djatmiko, 2005). Di samping kedisiplinan dan motivasi, faktor budaya juga berpengaruh signifikan terhadap timbulnya komitmen organisasi. Hal ini sesuai dengan teori yang dikemukakan oleh Robbins (2008) bahwa kepuasan kerja dapat menyatukan berbagai karakteristik individu kedalam suatu wadah perilaku yang didasarkan nilai-nilai kebersamaan dan diyakini kebenaranya. Saran dalam penelitian ini sebagai berikut. Bagi Karyawan berkaitan dengan motivasi kerja diharapkan dapat meningkatkan motivasi kerja terutama berkaitan dengan kemampuan yang berhubungan dengan kegiatan penjualan maupun penyelesaian kerja konfeksi seperti mengikutsertakan karyawan dalam pelatihan- pelatihan/diklat dan studi banding bagi karyawan baru agar mampu melaksanakan tugasnya dengan semaksimal mungkin. Pimpinan dapat memberikan motivasi tinggi terhadap kinerja diri karyawan dengan cara memberikan penghargaan bagi yang Dari hasil penelitian tersebut disimpulkan bahwa secara simultan terdapat pengaruh kedisiplinan, motivasi kerja dan kepuasan kerja terhadap komitmen organisasi. Hasil penelitian ini sejalan dengan penelitian yang dilakukan oleh Widiati (2012), yang menyimpulkan bahwa secara simultan 17 Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja Dan Kompetensi Terhadap Komitmen Organisasi (10-19) Pengaruh Disiplin Kerja, Motivasi Kerja, Kepuasan Kerja Dan Kompetensi Terhadap Komitmen Organisasi (10-19) mempunyai kinerja baik, serta memberikan pembinaan bagi yang berkinerja rendah, untuk itu pihak rumah sakit perlu membina hubungan kerja yang baik dan selaras antara pimpinan dan karyawan sehingga akan timbul kebersamaan dan kerjasama yang baik sehingga dapat meningkatkan kinerja perawat. Penutup Pimpinan perlu menggali faktor- faktor yang dapat mendukung terbentuknya komitmen organisasi seperti memperhatikan kepuasan perawat/karyawan, kebijakan yang transparan dalam pembagian remunerasi, jaminan kesehatan, pengembangan karier, sehingga mampu menghasilkan karyawan yang berkinerja yang baik serta mempunyai komitmen yang tinggi terhadap rumah sakit. Pimpinan memberikan dukungan yang tinggi bagi karyawan di tempat kerja sehingga menyebabkan mereka memiliki komitmen yang tinggi pada pekerjaannya sekaligus memungkinkan rumah sakit dapat menciptakan kepuasan kerja karyawan sehingga akan meningkatkan kinerja rumah sakit yang pada akhirnya akan menguntungkan rumah sakit itu sendiri. Bagi Peneliti selanjutnya diharapkan dapat meneliti faktor-faktor yang mempengaruhi komitmen organisasi selain faktor disiplin kerja, motivasi kerja, kepuasan kerja dan kompetensi kerja, misalnya faktor lingkungan kerja, kepemimpinan, dan karakteristik individu. Penelitian ini bisa diharapkan menggeneralisasi secara luas beberapa pengaruh yang dapat memberikan kontribusi terhadap faktor yang berpengaruh terhadap komitmen organisasi. Daftar Pustaka Amran. 2009. Pengaruh Disiplin Kerja Terhadap Kinerja Pegawai Departemen Sosial Kabupaten Gorontalo. Jurnal Ichasan Gorontalo. Vol. 4, No. 2, Hal 2397-2413. Brahmasari, I. A. dan Suprayetno, A. 2008. Pengaruh Motivasi Kerja, Kepemimpinan dan Budaya Organisasi Terhadap Kepuasan Kerja Karyawan serta Dampaknya pada Kinerja Perusahaan (Studi kasus pada PT. Pei Hai International Wiratama Indonesia). Jurnal Manajemen dan Kewirausahaan. Vol. 10, No. 2. September 2008: 124-135. Gibson, I. D.. 2005. Organisasi, alih bahasa Nunuk Adiarni, edisi Kedelapan, Jakarta: Binarupa Aksara oko, T. H. 2010. Manajemen Personalia dan Sumber Daya Manusia. Yogyakarta: BPFE. Hardjosoedarmo. S. 2006. Total Quality Manage-ment. Yogyakarta: Penerbit Andi. Hasibuan, M.S.P. 2006. Manajemen Sumber Daya Manusia. Edisi Ketujuh. Jakarta: Bumi Aksara. Ranupandoyo, H. dan Husnan, S. 2007. Manajemen Personalia, BPFE, Yogyakarta: BPFE Hidayat. 2007. Pengaruh Motivasi Kerja terhadap Kinerja Karyawan dengan Variabel Moderator Etos Kerja Spritual. Jurnal Ekonomi dan Manajemen. Volume 8, Nomor 1. Februari 2007. Luthans. 2007. Perilaku Organisasi. Yogyakarta: Andy Offset. ns. 2007. Perilaku Organisasi. Yogyakarta: Andy Offset. Miner, J.B. 2006. Organizational Behavior: Performance and Productivity, First Edition. Inc. New York: random House, Inc Moekiyat. 2007. Manajemen Kepegawaian. Yogyakarta: BPFE UGM. iyat. 2007. Manajemen Kepegawaian. Yogyakarta: BPFE UGM. Novitasari, 2013. Pengaruh Motivasi, Budaya Organisasi dan Disiplin Kerja terhadap Kinerja Pegawai di Dinas Perindustrian dan Perdagangan Provinsi Jawa Tengah. Jurnal Ekonomi dan Bisnis. Semarang: Unsep. 18 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya Vol. 18, No. 1, Juni 2016 Nurcahyo, A. 2011. Analisis Variabel-Variabel Yang Menpengaruhi Kinerja Karyawan Pada PT. Quadra Mitra Perkasa Balikpapan. Jurnal Eksis. Vol.7 No.2, Hal. 1972 – 1982. Olsen, R.W. 2006. Seni Berpikir Kreatif: Sebuah Pedoman Praktis. Jakarta:Erlangga Prabasari. 2013. Pengaruh Motivasi, Disiplin Kerja dan Komunikasi terhadap Kinerja Karyawan pada PT. PLN (Persero) Distribusi Bali”. Jurnal Ekobis. Fakultas Ekonomi Universitas Udayana (UNUD). Volume Reksohadiprodjo, H.T. 2007. Perilaku Organisasi Konsep Dasar dan Aplikasinya, Jakarta: PT Raja Grafindo Persada. Robbin, S., 2008, Perilaku Organisasi, Konsep, Kontroversi, Aplikasi, Alih bahasa Handyana Pujaatmaka. Edisi Kedelapan. Jakarta: Prihallindo Sekaran, U. 2007. Research Methods for Businnes. New York: John Wiley & Sons. Inc. Setiadi. 2008. Metode Kuantitatif untuk Pengendalian Manajemen. Yogyakarta: Bina Ilmu. Stoner, J.A.F. 2006. Human Resources Management. New York: West Publishing Compa Sudarmayanti. 2011. Manajemen Sumber Daya Manusia Reformasi Birokrasi dan Manajemen Pegawai Negeri Sipil. Bandung: Refka Aditama. Sugiyono. 2008. Metodologi Penelitian. Bandung: Alfabeta. Sugiyono. 2008. Metodologi Penelitian. Bandung: Alfabeta. Timpe, A. D. 2005. Kinerja, Seri Ilmu dan Seni Manajemen Bisnis. Alih Bahasa Sofyan Cikmat. Jakarta: PT. Daftar Pustaka Elex Media Komputindo, Wibowo. 2002. “Hubungan Karakteristik Pekerjaan terhadap Kinerja (Studi pada Kantor Unit Caang BRI Pattimura Semarang)”. Tesis Magister Manajemen Undip. (Tidak dipublikasikan). Semarang: UNDIP. Winardi, 2010, Motivasi dan Pemotivasian dalam Manajemen, Cetakan Pertama, Jakarta: PT. Raja Grafindo Persada. 19
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Storage Method for Parts from End of Life Vehicles' Dismantling Process According to Sustainable Development Requirements: Polish Case Study
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World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 M. K. is with the Faculty of Engineering Management Poznan University of Technology, 60-965 Poznan, Poland (phone: +48 61 665 34 14; fax: +48 61 665-33-75; e-mail: monika.kosacka@put.poznan.pl). I. K. is with the Faculty of Engineering Management Poznan University of Technology, 60-965 Poznan, Poland (e-mail: izabela.kudelska@put.poznan.pl). International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 Poland is a country with a high potential in ELVs recycling according to the age structure and vehicles’ quantity, what was studied in [6]. In Poland, similar to other countries in the EU, ELVs are a major stream of waste1 [4] which should be managed in the recycling network [7]. In the structure of polish recycling network, the dismantling/disassembling facility (DF) is the only one, that is authorized to manage ELVs. The processes realized in the DF include obtaining vehicles; depollution of ELVs, disassembling and storage of ELVs, spare parts and waste. In the result of all activities realized in DF there are obtained two streams: used parts ready for redistribution as spare parts (reuse scenario) and waste, prepared for recycling or disposal. Keywords—Dismantling, end of life vehicle, sustainability, storage. DFs are running a specific business, where sell of the used parts is the basic source of revenues. What is more, the reuse scenario is, in the authors’ opinion, the best from all ELV’s waste management possibilities, from the sustainability perspective. Although there are specific guidelines for some parts storage process, however there is lack of method which would support decision makers in the dismantling facility in the warehouse management area, what could improve their performance. 1 Every year, ELVs generate between 7 and 8 million tons of waste [8]. Storage Method for Parts from End of Life Vehicles' Dismantling Process According to Sustainable Development Requirements: Polish Case Study M. Kosacka, I. Kudelska discarded by its last owner, what makes that ELV is waste according to the law [4]. As many researches claim, waste from ELV is the issue of a world-wide concern, because of its rapidly growing quantity and special composition of substances, including different toxic substances such as lubricants, acid solutions, and coolants [5]. In the previous work [6], authors proved that ELV should be perceived as a valuable source of materials and used parts. Hence, ELVs’ management is significant for resource conservation, environmental protection and economy growth. Abstract—Vehicle is one of the most influential and complex product worldwide, which affects people’s life, state of the environment and condition of the economy (all aspects of sustainable development concept) during each stage of lifecycle. With the increase of vehicles’ number, there is growing potential for management of End of Life Vehicle (ELV), which is hazardous waste. From one point of view, the ELV should be managed to ensure risk elimination, but from another point, it should be treated as a source of valuable materials and spare parts. In order to obtain materials and spare parts, there are established recycling networks, which are an example of sustainable policy realization at the national level. The basic object in the polish recycling network is dismantling facility. The output material streams in dismantling stations include waste, which very often generate costs and spare parts, that have the biggest potential for revenues creation. Both outputs are stored into warehouses, according to the law. In accordance to the revenue creation and sustainability potential, it has been placed a strong emphasis on storage process. We present the concept of storage method, which takes into account the specific of the dismantling facility in order to support decision-making process with regard to the principles of sustainable development. The method was developed on the basis of case study of one of the greatest dismantling facility in Poland. M. K. is with the Faculty of Engineering Management Poznan University of Technology, 60-965 Poznan, Poland (phone: +48 61 665 34 14; fax: +48 61 665-33-75; e-mail: monika.kosacka@put.poznan.pl). I. K. is with the Faculty of Engineering Management Poznan University of Technology, 60-965 Poznan, Poland (e-mail: izabela.kudelska@put.poznan.pl). A. Dismantling Facility in the polish Recycling Network Polish recycling network is structured according to the guidelines defined in the European Directive 2000/53/EC [7]. It consists of many enterprises involved in ELVs management, what is presented in Fig. 1. As it is presented in Fig. 1, the main entity in the polish recycling network is DF, which obtains ELVs from different sources directly including last owners, insurance companies, public authorities, vehicle return stations as well indirect with the use of return stations. On the 10th May, 2016 Polish recycling network consisted of 1008 DF [10] and 136 return stations [11] In comparison, at International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 Fig. 1 The participants and the flows in the polish recycling network Source: [9] II. ELVS DISMANTLING FACILITY – POLISH EXAMPLE the beginning of recovery network creation period (at the end of 2005) those numbers were: 360DF and 50 return stations, what demonstrates that there is a demand for services offered by DF. It is related to the vehicle fleet characteristic in Poland what creates the potential for ELVs recycling (mentioned in the Introduction section). A. Dismantling Facility in the polish Recycling Network I. INTRODUCTION A UTOMOTIVE industry is one of the most influential sectors of economy worldwide. Automobiles are integral part of the current generation’s life; however, there is a growing concern over the impact of vehicles through end of life phase, which is referred to management of ELVs [1]. With the increase of vehicles worldwide, it is observed the growth of ELVs’ number, what results treatment ELVs as an international emerging problem [2]. Meanwhile, there is a rapidly increasing awareness in industry that today’s companies have to seriously consider the impact on People, Environment and Economy, particularly in automotive sector. At the same time, when issues on “sustainable development” have been valid globally, the recycling, reuse, recovery or disposal of ELVs have attracted greater attention [3]. A Taking into consideration all presented aspects, we present a concept of storage method for parts obtained from ELVs disassembling which would consider sustainability issues. Paper consists of 5 sections. Section II provides information about the DF from the perspective of business specification in Poland. In the Section III, there are presented considerations of reusable parts storage in dismantling facility on the basis of case study. Section IV describes the concept of the proposed method for parts storage. Section V summarizes contributions and directions of future work. ELV is a specified vehicle, which is discarded or is to be scholar.waset.org/1307-6892/10004977 2387 International Scholarly and Scientific Research & Innovation 10(7) 2016 World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 Fig. 1 The participants and the flows in the polish recycling network Source: [9] International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 Storage process is according to the polish norm, a set of activities related to the stocks’ placement on the storage space of the storage building (e.g. in storage equipment) in a systematic way according to the inventory’s characteristic and the existing conditions [14]. Reusable parts are all parts, which according to the polish law may be used in the same function as they were designed for. Those parts are used spare parts. We focused on the reusable parts storage process. It is a result of the value of the reusable parts from the perspective of the sustainability. First of all, the best strategy for the sustainable development policy introducing is reuse. Reuse is equal to saving natural resources, protecting the environment and saving money, what in a case of the automotive industry is significant (high material – and energy consuming). It brings positives from all sustainability dimensions. Fig. 3 Core activities realized in DF In order to process ELVs, DF has to obtain and take supplied vehicles, what results in ELV’s reception and development all required documents for supplier. Those activities require additional actions including marketing and ELV’s transport from the supplier. International Science Index, Economics and Management Enginee Secondly, used spare parts are the basic source of revenues for DF. In accordance to the research conducted in one of the biggest DF in Poland processing around 1200 ELVs per year [6], authors identified that only metals sale is profitable, although DF are addicted to the price level of the materials which are changing very often and it is poor low. Comparison of the income from spare parts sale and materials sale prove that around 70% of all incomes generate the first group. Taking that into consideration, there should be an emphasis on the storage process realization due to the economic potential. Upon ELV’s reception, the ELVs are inspected, drained from fluids (i.e. engine oil, fuel, etc.) and air conditioning, batteries, wheels/tires and catalytic convertors are removed. International Science Index, Economics and M Prepared ELV’s are placed into Sale Yard, where they are temporary stored. Stored ELVs are available for walk-in customers who are able to dismantle only required parts on their own or with Employee’s help (ELVs are treated as outdoor warehouse). On the other hand, whole ELVs are disassembled into components after dismantling order, which does not have to be related to the customer’s order for part. B. Specific of the Dismantling Facility Business In author’s opinion, DF is dealing with the same functions as traditionally manufacturing company, including procurement, processing and distribution, what is presented in Fig. 2. Fig. 2 Comparison of dismantling facility vs traditional manufacturing company International Science Index, Economics and Manag Fig. 2 Comparison of dismantling facility vs traditional manufacturing company Taking into consideration the specificity of the DF business, there are different material flows as opposed to traditional manufacturing company, where instead of low level of complexity of input stream, there are complex products – vehicles, hazardous for the Environment. Moreover, categories of suppliers and customers of DF are also a difference. The DF is the only officially approved entity authorized by polish law to ELVs processing. In the light of considerations on ELVs as dangerous waste, the dismantling stations are under constant monitoring and are required to achieve the scholar.waset.org/1307-6892/10004977 International Scholarly and Scientific Research & Innovation 10(7) 2016 World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 goals related to the recycling and recovery rates, in accordance to UE guidelines. of ELVs implementation. of ELVs implementation. In previous work [12], authors have analyzed the DF from the perspective of the social, economic and environmental perspective, what confirms importance of DF for sustainable development. Although a high significance of DF, there are a lot of problems related to the DF business in Poland what was described [13]. The core activities realized in DF are related to the ELV’s management including the following operations presented in Fig. 3. Fig. 3 Core activities realized in DF We focus on the storage process of reusable parts, due to the importance for the DF considering sustainability issues, what is discussed in the next section. III. REUSABLE PARTS STORAGE IN DISMANTLING FACILITY – CASE STUDY International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 , Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 International Scholarly and Scientific Research & Innovation 10(7) 2016 International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 Provisions on objects moving in the type of mechanized work Internal transport 0 Warehouse equipment 0 Labeling locations - agement Engineering Vol:10, No:7, 2016 waset.org/Publicati International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.or Considering mechanization of warehousing related to the introduction of machines and mechanical devices by which handmade warehouse operations will be performed mechanically, that issue is subject in one and in the second type of the company. Moreover, in automotive manufacturers, there are an automated job type storage, what is impossible to use in the DF. It is a result of variety of types, shapes and sizes of parts as well as the variety of working methods. Type of storage is primarily defined by the equipment used in the warehouse. Hardware stores are the technical and organizational measures: means of transport, equipment, tools, installations. Both, manufacturing company and DF use specified means of transport in storage process. In the DF there are used: platform trucks and forklifts trucks. These devices are the most often used, although they are not only used in storage process. However, in manufacturing companies, there can be found carriages designed for use in narrow aisles between the shelves. In contrast to DF, these devices are used only for storage and work for one type of SKU. International Science Index, Economics and Management In any enterprise, goods should be stored according to their type of store. One of the criteria that should be taken into account are properties of natural products, e.g.: sensitivity to atmospheric conditions, susceptibility to corrosion susceptibility to temperature. In addition, products’ group which occur in a manufacturing company and DF, may have aggressive properties e.g.: flammable, explosive, toxic, etc. It is a threat for human life and health as well as for the Environment. It can also be a cause of damage of a storage facility. The described feature occurs in both cases – DF and manufacturing company. Another very important feature that distinguishes mentioned types of enterprise are devices used for storage, which are used in both cases. However, due to the diversity of stored parts in DF, storage takes place primarily on the universal racks. Unfortunately, the weight and appearance of the assortment does not always allow to use this type of equipment (e.g. some goods with very unusual shape). Thus, there are used special station racks. International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 u o s ve co p ed w e d o u c u g company with the use of following criteria presented in Table II. TABLE II THE COMPARISON FEATURES PRESENT IN STORAGE Criterion The presence of the features Comments Properties + The shape, dimensions, weight - Packaging unit - Warehouse space 0 Zoning in the warehouse 0 Type of storage 0 Adjustments in the rules for mass objects moved by hand. Provisions on objects moving in the type of mechanized work Internal transport 0 Warehouse equipment 0 Labeling locations - In any enterprise, goods should be stored according to their type of store. One of the criteria that should be taken into account are properties of natural products, e.g.: sensitivity to atmospheric conditions, susceptibility to corrosion susceptibility to temperature. In addition, products’ group which occur in a manufacturing company and DF, may have aggressive properties e.g.: flammable, explosive, toxic, etc. It is a threat for human life and health as well as for the Environment. It can also be a cause of damage of a storage facility. The described feature occurs in both cases – DF and manufacturing company. Another issue is number of the assortment stored in a warehouse. In the DF, there is no single assortment. Products stored in DF are diverse in terms of shape, dimensions and International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004 Manual work, in both companies, is based on the physical strength of a man which performs such tasks as lifting, carrying, packing or filing. All manual operations are in both types of companies. However, in manufacturing company, there are standards applying maximum weight of goods. In DF, in accordance to law, there are requirements of manual handling of goods which are harmful and dangerous. TABLE II THE COMPARISON FEATURES PRESENT IN STORAGE Criterion The presence of the features Comments Properties + The shape, dimensions, weight - Packaging unit - Warehouse space 0 Zoning in the warehouse 0 Type of storage 0 Adjustments in the rules for mass objects moved by hand. International Scholarly and Scientific Research & Innovation 10(7) 2016 International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 What is more, the business model of selling used parts is well developed in Poland as well as other post-communist countries in the Europe. It is mainly the result of low salaries and the opportunity to acquire parts which are no longer produced. Those aspects may be considered from the social context of sustainability. During disassembling there are emitted: During disassembling there are emitted: g  air pollution; p  noise;  solid waste;  sewage; As it was proved, there are many benefits for People, Economy and Environment due to the reusable parts. In order to take the advantage of that it is necessary, in authors opinion to storage parts appropriate.  segregated materials;  reusable parts. From the list of presented outputs of disassembly process there are two main flows: waste and reusable parts, both are stored, what is strictly regulated by law and then sold to customers (individual as well as business) via different distribution channels, mostly direct sale. B. Storage in Dismantling Facility and Traditional Manufacturing Company - Comparison B. Storage in Dismantling Facility and Traditional Manufacturing Company - Comparison In the every production company, there is storage. However, in dismantling stations, there are differences resulting from stored and at the same time very different parts. DF is relevant entity in sustainability policy realization at the national level, because of the reuse and recycling scenario scholar.waset.org/1307-6892/10004977 International Scholarly and Scientific Research & Innovation 10(7) 2016 2389 World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 would allow to carry out parts easier from the warehouse. Regarding to the storage space in manufacturing company, there can be isolated storage area, surface handling, auxiliary surface, the surface area of communication and design. However in the DF, each part is different (weight, volume), which results in not enough storage area. Moreover, each part requires proper storage. The fulfilment of these conditions is difficult in business reality. Therefore, for the comparison analysis there was created a scale in order to verify similarity of the storage process in traditional production enterprise and DF, presented in Table I. International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 TABLE I THE COMPARISON SCALE MATRIX Scale Explanation - There are only differences 0 There are similarities but also differences + There are only similarities TABLE I THE COMPARISON SCALE MATRIX Scale Explanation - There are only differences 0 There are similarities but also differences + There are only similarities The same problem as above, applies to zones of acceptance, completion and release. There is no possibility of separation specific storage area in DF warehouse. Moreover, sometimes there are no storage areas. The sign "-" in Table I indicates no similarities of storage process in traditional production facility and DF. The "0" means that there are some similarities as well as differences. With the sign “+” there were pointed out only similarities. Authors have compared DF with the traditional manufacturing company with the use of following criteria presented in Table II. Type of storage is primarily connected with devices with which operations are performed in the warehouse. In manufacturing companies, as well in DF, there is a manual system of warehousing and mechanized one, too. International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 The variety assortment causes the use of pallets, load units, boxes and containers. In addition, in the manufacturing companies, entering and exiting of the products from the warehouse is appropriately tagged. The location of the goods in the warehouse is also known. Each load unit is assigned to a location in the system. In the analyzed DF, there are a lot of different kinds of parts, the Another issue is number of the assortment stored in a warehouse. In the DF, there is no single assortment. Products stored in DF are diverse in terms of shape, dimensions and weight. In addition, each group of assortment requires proper storage, which is not always easy in real life and made according to the law requirements. Every product in the traditional production company has a specific shape or before storage, it is packed into the packaging unit. In the case of the DF, parts obtained in the result of disassembly process are stored in the same state as they were disassembled. There is no packaging unit, which scholar.waset.org/1307-6892/10004977 International Scholarly and Scientific Research & Innovation 10(7) 2016 2390 World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 observations made in the polish representing of DF; interviews with Employees, including company’s owner; brainstorming. observations made in the polish representing of DF; storage area is limited and there is lack of addresses location. Information flow is discharged by warehouse’s Employees, who knows where the part is regarding to his experience and knowledge. • interviews with Employees, including company’s owner; • brainstorming. • interviews with Employees, including company’s owner; • brainstorming. The basic method used in the research methodology was a case study, of one of the greatest DF in Poland, with about 1200 processed ELVs per year. C. The Specifics of Storage in Dismantling Facility – Case Study The proposed method for vehicle parts storage is intended for use by a disassembly worker, who dismantles ELV into parts and waste. The method is prepared according to two assumptions: As it was pointed out in Section II, one of the core activity in the DF is storage of parts and waste. International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publicati The SMEP consists of 6 steps presented in Fig. 4. Fig. 4 SMEP procedure In the analyzed DF, there is used universal equipment, including shelfing racks, console racks as well as special racks (e.g. for glass, doors, etc.). All operations are made by hand, including transport with the exception of movement of engine or transmission, where the forklift is used. From the perspective of the mode of storage, authors perceived that there is no procedure for storage process. There are no containers for parts which are stored at the shelf. Many of elements are stored direct on the floor, sometimes without required protection and equipment (e.g. palette). Storage area are divided into zones designated for kind of part (e.g. for engines, starters, seats, etc.), although there is lack of formal instructions, what results excess transport and defects of parts (muda). It may cause Employee’s overload and in the consequence less efficiency, what may be considered in the sustainability context. Taking into consideration all presented above information, authors assumed that there is a need for a method supporting decision makers in the DF in the storage process, what could improve DF performance concerning sustainability issues. In order to find a storage method dedicated for specific business of DF, authors have made a literature review with the use of research bases including: SCOPUS, Science Direct and Google Scholar. In the result, authors did not find any example of the work considering reusable parts storage method description. Fig. 4 SMEP procedure Fig. 4 SMEP procedure Each of step in the presented procedure is described in the particular subsection. B. Step 1: Susceptibility to Reuse Scenario Verification It leads to development of the storage method for parts (SMEP) from ELVs’ dismantling process considering sustainability requirements, described in details in Section IV. The first stage of the method was to verify susceptibility to reuse scenario. The basic question of that stage is: “Should part be stored?” IV. SMEP FROM ELVS’ DISMANTLING PROCESS In order to answer the previous question, authors propose simple tool – Reuse Matrix to check if part is able to be stored. The Reuse Matrix is presented in Table III. International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 In accordance to polish recycling law, all reusable parts as well as waste are stored in appropriate conditions, including the following criteria [15]: A1. Employee disassembles a vehicle according to the Disassembly list (hereafter: DL). It contains a list of parts which should be stored. DL should be developed according to the demand analysis for parts or Employee’s knowledge about the potential demand, excluding all parts which regarding to the law, cannot be reuse because of security threat in case of their reuse.  C1: Storage location (where?);  C2: Equipment (with what?);  C3: Mode of storage (how?).  C3: Mode of storage (how?). International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 The basic requirement is related to the storage location, what is the result of the requirements regarding land use. In the DF there should be separated storage areas for reusable parts, waste and ELVs. The most important issue is to storage reusable parts in the covered object, what is very often not respected due to the increasing quantity of stored parts and limited storage area. A2. Stored parts are clearly marked what makes the element’s identification easy. C. Step 2: Classification Parts into Storage Groups In the result, in Table V, there were distinguished 7 different groups of parts with the required equipment. In the second stage of the procedure, there are identified storage groups of parts intended for storage (result of the first stage of the SMEP). w n o C a s a s s a o a r i f o d e International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 where: f(s)= 1  part is susceptible to reuse; f(s)= 0  part is not susceptible to reuse. Simplicity of the reuse matrix is a result of requirements obtained from the interview with Employees and DF owner. Consequently, only correct and not damaged parts from DL are qualified to storage. Rest of them are intended for material segregation (waste) and they should be placed in the appropriate container. D. Step 3: Required space calculation International Science Index, Economics and Management Engineering Vol:10, N In the third stage of the method, there is estimated required space (m3) for each of the following groups of parts (A-G). In order to make the calculation, there should be an access to the actual plan of the company, including warehouse area. The basic question of that stage is:” how to storage?”. With a view to answer the question, authors used results of observations (possible storage equipment and storage parts in analyzed company) and brainstorming. The estimation of the space required for the chosen group of parts should be calculated according to (2): Authors have made a brainstorming session with two representatives of the University and three members of DF including the owner. j j j i c S *  (2) (2) The first stage of the brainstorming was to determine list of factors for parts classification. In the result there were obtained the following factors (F1-F5), including (Table IV): where: Sj- required space for the j-th group of parts, j={A,B,C,D,E,F,G}; cj –average space required for one part from j-th group; ij – actual number of parts in j-th group (it is acceptable to add 10%). TABLE IV FACTORS DETERMINING STORAGE GROUPS Factor Values Id Description 1 2 3 F1 Weight up to 5 kg 5-30 kg over 30 kg 2 F2 Size a,b,c <0,2 m a,b,c ϵ<0,2-0,6) m a,b,c>0,6 m F3 Shape Regular Irregular, but not long timber Long timber F4 Content of harmful substances no - yes F5 Required storage equipment no - yes F6 Sensitiveness to damages no yes TABLE IV FACTORS DETERMINING STORAGE GROUPS There should be known the required space in the rack for one part and the information about the number of actual inventory to calculate the total required space. Taking into account the information about the available equipment (particularly rack capacity of stored parts) and the required space for the each group of parts there will be available information about the required area for the j-th group of parts (aj) in [m2]. A. Method Description The presented storage method (SMEP) was prepared with the use of the following research methods: There were used only two criteria for determination susceptibility to reuse scenario for part. Authors assumed that Employees will only disassembly those parts which are • systematic literature review according to storage of reusable vehicle’s parts; • systematic literature review according to storage of reusable vehicle’s parts; scholar.waset.org/1307-6892/10004977 2391 International Scholarly and Scientific Research & Innovation 10(7) 2016 World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 required according to DL. required according to DL. respect to Employees opinion, who deal each day with parts transport. Factors F4 and F5 were extracted in conjunction with law requirements, particularly “Required storage equipment” which may be related to the security of part during storage, separate storage space or container/palette usage). TABLE III REUSE MATRIX Condition Id Value Description Efficiency C1 0 incorrect 1 correct Technical Condition C2 0 damaged 1 undamaged Each part stored in DF was analyzed with the use of Factors (F1-F5) presented in Table IV. As a result of analysis made by participants of brainstorming, there were prepared following categories of parts (Table V): To summarize, susceptibility to reuse scenario is the function of two conditions presented in (1): TABLE V STORAGE PARTS GROUPS Parts Equipment engine, transmission  palette with protection, console racks tire, wheel trim, wheel, rim  separated are with fire extinguisher, racks or stands drive shaft, pipe, damper, spring, bumper swingarm, gas installation, exhaust, wiper cantilever racking, hooks, hangers  reflector, navigation, radio,  containers, shelving racks  seats  foiled, console racks  painting elements, glass, including doors, mask, special racks, spacers between parts  alternator, starter, wheel  shelving racks TABLE V STORAGE PARTS GROUPS 2 * 1 ) ( C C s f  , f(s) ϵ {0,1} (1) (1) V. CONCLUSION This paper refers to the research conducted under Statutory activity, financed by MNiSW/Poznan University of Technology, project: “Storage method for auto parts dismantled from End Of Life Vehicles management according to sustainable development” (Project ID: 503225/11/140/DSMK/4136). This paper refers to the research conducted under Statutory activity, financed by MNiSW/Poznan University of Technology, project: “Storage method for auto parts dismantled from End Of Life Vehicles management according to sustainable development” (Project ID: 503225/11/140/DSMK/4136). Warehouse management and phenomena that occur in each company are treated as one of the most important cells in an field of logistics. Well-organized warehouse processes cause the flow of materials. Warehouse as the supply chain fulfils a series of functions that need to carry out basic tasks. These tasks are the storage of goods and handling activities. G. Step 6: Cost Review The last stage in the method includes the cost analysis of the storage, repeated each day automatically. The presented storage method takes into account the specific of the dismantling facility business as well as sustainability issues. International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Pu Authors suggest that inventories should be examined in the context of profitability of storage. In practice sometimes parts are stored although their costs are higher than potential income. In the consequence authors recommend cost review of inventories. In the result DF should get the information when the total cost of the part in warehouse is higher than potential income from selling it. In that case there should be made a decision of material segregation, in order to restore a value of part. Unfortunately, this method is only a concept and it require to carry out further studies. These studies should be aimed at the realization of this method even in the form of a program that will help the Employee to decide about storage of parts. The increase in the number of vehicles in Poland is still growing and therefore dismantlers also gaining in importance. Therefore, the problems of storage process in DF also acquire greater value. The last step is at the same time a direction of further research for authors. E. Step 4: Storage Groups Allocation in Warehouse The further two steps (4,5) are directed to obtain the answer for the question ”where to storage?”. The basis is a combined ABC analysis by Gelders and van Looy named FSN analysis [17], where are three types of products: fast moving (F), slow moving (S) and no moving (N) [18]. The FSN analysis is made for storage groups (A-G). There are assigned places in the warehouse according to the rule “transport distance According to the presented results, there were arbitrary defined five important factors. Factor “weight” was established in accordance to the law requirements included in [16]. Factors: “size” and “shape” were distinguished with 2 According to [16] scholar.waset.org/1307-6892/10004977 International Scholarly and Scientific Research & Innovation 10(7) 2016 2392 World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 process in traditional manufacturing company and DF, proved that they are different. Moreover analyzed DF has limited financial resources and do not always have the possibility to invest in the warehouse equipment. minimization”. Transport distance - tj is equal to the distance between dismantling workstation and warehouse zone for j-th storage group of parts. In practice it is equal to assign groups of parts from the “F” category as close as it is possible to the warehouse entrance/exit, in accordance to the warehouse layout. Reusable disassembled parts can bring financial benefits for the DF. In order to achieve that, the storage process, as in a typical manufacturing company, should be improved from the perspective of the material flow, to not be a bottleneck. Therefore, the authors have created the concept of storage method for DF called SMEP. This method is aimed at decision support in the process of storing parts. It is not easy to make good decision, what is related to the very large number of removable parts, and large variety of them in order to ensure properly storage for each part. International Science Index, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977  In the middle - parts from the group: F; SMEP method consists of six steps. It aims to bring decision-maker answers to three questions: where, what and how to store reusable parts. Furthermore, the concept also involves an analysis of the cost of the stored parts, what is appealing for the DF.  In the middle parts from the group: F;  at the bottom: parts from the group: S;  at the bottom: parts from the group: S; 0049  at the bottom: parts from the group:  at the top: parts from the group: N.  at the top: parts from the group: N. F. Step 5: Parts Allocation F. Step 5: Parts Allocation In the next step, the same analysis (FSN) is used but the purpose is to assign place at rack for each part from the analyzed storage group. Considering demand on particular parts, there are following guidelines: dex, Economics and Management Engineering Vol:10, No:7, 2016 waset.org/Publication/10004977 2 International Scholarly and Scientific Research & Innovation 10(7) 2016 World Academy of Science, Engineering and Technology International Journal of Economics and Management Engineering Vol:10, No:7, 2016 2394 International Scholarly and Scientific Research & Innovation 10(7) 2016 scholar.waset.org/1307-6892/10004977 REFERENCES [1] P. Golińska, M. Kosacka, “Environmental friendly practices in the automotive industry”, In Environmental Issues in Automotive Industry, Springer Berlin Heidelberg, 2014, pp. 3-22. [1] P. Golińska, M. Kosacka, “Environmental friendly practices in the automotive industry”, In Environmental Issues in Automotive Industry, Springer Berlin Heidelberg, 2014, pp. 3-22. [1] P. Golińska, M. Kosacka, “Environmental friendly practices in the automotive industry”, In Environmental Issues in Automotive Industry, Springer Berlin Heidelberg, 2014, pp. 3-22. Warehouse processes also occur in the dismantling facility. Poland is a type of a country, where the potential of sustainable policy realization is related to the recycling network, particularly dismantling facilities which are managing ELVs with the reuse and recycling scenarios. It results in increased demand for services in the field of dismantling and recovery of materials from ELVs, including storage process. [2] V. Simic, B. Dimitrijevic, “Perspectives for application of RFID on ELV CLSC”, Proc of the 1st Olympus int conf on supply chains, Katerini, Greece; 2010, http://www.teicm.gr/logistics/images/logisticsdocs/ icsc2010/fullabstracts/8_4_ICSC2010_009_Simic_Dimitrijevic.pdf (accessed January, 20, 2015). [3] Y. Pan, H. Li, “Sustainability evaluation of end-of-life vehicle recycling based on emergy analysis: A case study of an end-of-life vehicle recycling enterprise in China”, Journal of Cleaner Production, 2016. The modern vehicle is made of many different materials with different properties. Disassembled components can also be a source of financial gain. From an environmental point of view, properly conducted storage is more than just a material benefit. [4] European Commission Directorate-General Environment (ECDGE), “End-of-life vehicles: influence of production costs on recycling rates”. Sci. Environ. Policy News Alert 282, 2012, 1. y [5] V. Simic, “A multi-stage interval-stochastic programming model for planning end-of-life vehicles allocation”, Journal of Cleaner Production 115, 2016, pp. 366-381. [6] M. Kosacka, I. Kudelska, P. Golińska-Dawson, “How properly value ELVs? – concept of the tool of ELVs assessment for dismantling station. Case study”, Proceedings of the 25th International Conference on [6] M. Kosacka, I. Kudelska, P. Golińska-Dawson, “How properly value ELVs? – concept of the tool of ELVs assessment for dismantling station. Case study”, Proceedings of the 25th International Conference on Disassembled parts, due to the different size (the volume), weight, shape, require appropriate storage conditions, what with limited space, is difficult. Comparison of the storage International Scholarly and Scientific Research & Innovation 10(7) 2016 scholar.waset.org/1307-6892/10004977 2393 Flexible Automation and Intelligent Manufacturing (FAIM 2015), volume I, 2015, pp 208 -215. pp [7] European Parliament and the Council, 2000. REFERENCES Directive 2000/53/EC of pp [7] European Parliament and the Council, 2000. Directive 2000/53/EC of the European Parliament and of the Council of 18 September 2000 on end-of-life Vehicles. the European Parliament and of the Council of 18 September 2000 on end-of-life Vehicles. [8] http://ec.europa.eu/environment/waste/elv/ (accessed: May, 27, 2016). [9] A. Merkisz – Guranowska, “End-of-life vehicles recycling network design”, Journal of KONES, 18, 2011, pp. 261-268. [10] http://fors.pl/wp-content/uploads/2014/01/stacje-2.pdf (accessed: May, 10, 2016). [11] http://fors.pl/wp-content/uploads/2016/03/punkty.pdf (accessed: May, 10, 2016). [12] M. Kosacka, P. Golińska, “Assessment of sustainability in dismantling station - case study”, Research in Logistics & Production, Vol. 4, No. 2 2014, pp. 135-145. pp [13] P. Golińska, “Implementation of ELV Directive in Poland, as an Example of Emerging Market Country”, Environmental Issues in Automotive Industry. Springer Berlin Heidelberg, 2014, pp. 247-259. [14] PN-N-01800:1984 Stock management – basic terminology [15] Polish Law on Recycling of ELVs (Dz. U. z 2005. No 25, item 202 with further amendments. [16] Ordinance of the Minister of Labour and Social Policy of 14 March, 2000 on occupational health and safety during manual handling, Dz.U. 2000 no 26 item. 313 [17] L. F. Gelders, P. M. Van Looy, “An inventory policy for slow and fast movers in a petrochemical plant: a case study”, Journal of the Operational Research Society 29 (9), 1978,pp. 867–874 . [18] J. Stoll, R. Kopf, J. Schneider, G. Lanza, “Criticality analysis of spare parts management: a multi-criteria classification regarding a cross-plant central warehouse strategy”, Production Engineering, 9(2), 2015, pp. 225-235. 2394 ational Scholarly and Scientific Research & Innovation 10(7) 2016 scholar.waset.org/1307-6892/10004977 2394 International Scholarly and Scientific Research & Innovation 10(7) 2016 scholar. scholar.waset.org/1307-6892/10004977
https://openalex.org/W2064956722
https://europepmc.org/articles/pmc3149022?pdf=render
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Using Detergent to Enhance Detection Sensitivity of African Trypanosomes in Human CSF and Blood by Loop-Mediated Isothermal Amplification (LAMP)
PLoS neglected tropical diseases
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cc-by
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Abstract doi:10.1371/journal.pntd.000 Editor: Jayne Raper, New York University School of Medicine, United States of America Received February 5, 2011; Accepted June 8, 2011; Published August 2, 2011 ebruary 5, 2011; Accepted June 8, 2011; Published August 2, 201 Received February 5, 2011; Accepted June 8, 2011; Published August 2, 2011 b et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright:  2011 Grab 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. Funding: This research was supported in part by grants from the National Institutes of Health (5R01AI082695 and 1R21AI079282) to D in study design, data collection and analysis, decision to publish, or preparation of the manuscript. was supported in part by grants from the National Institutes of Health (5R01AI082695 and 1R21AI079282) to DJG. The funders had no role ection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dgrab1@jhmi edu * E-mail: dgrab1@jhmi.edu Dennis J. Grab1*, Olga V. Nikolskaia1, Noboru Inoue2, Oriel M. M. Thekisoe3, Liam J. Morrison4, Wendy Gibson5, J. Stephen Dumler1 1 Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan, 3 Department of Zoology and Entomology, University of the Free State, Qwaqwa Campus, Phuthaditjhaba, South Africa, 4 Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom, 5 School of Biological Sciences, University of Bristol, Bristol, United Kingdom www.plosntds.org www.plosntds.org Abstract Background: The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per mL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay. Methodology/Principal Findings: For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 mL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards. Conclusions/Significance: This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world. Citation: Grab DJ, Nikolskaia OV, Inoue N, Thekisoe OMM, Morrison LJ, et al. (2011) Using Detergent to Enhance Detection Sensitivity of African Trypanosomes in Human CSF and Blood by Loop-Mediated Isothermal Amplification (LAMP). PLoS Negl Trop Dis 5(8): e1249. doi:10.1371/journal.pntd.0001249 ation: Grab DJ, Nikolskaia OV, Inoue N, Thekisoe OMM, Morrison LJ, et al. (2011) Using Detergent to Enhance Detection Sensitivity of Af man CSF and Blood by Loop-Mediated Isothermal Amplification (LAMP). PLoS Negl Trop Dis 5(8): e1249. LAMP Two LAMP primer sets were tested. Data on analytical sensitivity and specificity of a LAMP primer set for trypanosome DNA based on the multicopy (approximately 500 copies) repetitive insertion mobile element (RIME) of subgenus Trypanozoon (GenBank Accession No. K01801) is well-documented [16,26,29]. Using between 2–4 mL sample, this pan-T. brucei assay is reported to detect DNA from ,0.001 trypanosome [16]. LAMP primers based on the serum resistance associated (SRA) gene (GenBank AJ560644) (see Table S1 for gene sequence) were designed using PrimerExplorer version 4 software (http://primerexplorer.jp/e/) to create the basic F3, B3, FIP, BIP [30] and loop LF, LB [23] primers (Fig. 1A). As this assay recognized more than the SRA gene, this primer set is designated PSEUDO-SRA. All RIME and PSEUDO-SRA LAMP primers were synthesized and HPLC purified. For comparison, we also used the SRA gene (GenBank accession number Z37159)- specific LAMP assay [17]. Genomic DNA was prepared by using Qiagen DNeasy Blood & Tissue Kits. gambiense infections, which constitute over 90% of all HAT cases, typically show very low parasitemias, and concentration tech- niques such as centrifugation or mini-anion exchange columns are usually necessary [6,7,8]. Stage determination still relies on lumbar puncture to examine CSF for trypanosomes or white cell count/ protein concentration suggestive of chronic meningoencephalitis. Threshold values for these parameters are controversial, with the conventional value for stage 2 (.5 cells/mL) now increased to .10 or even 20 cells/mL [9]. In summary, diagnosis and staging of HAT is currently time consuming, intensive and difficult. gambiense infections, which constitute over 90% of all HAT cases, typically show very low parasitemias, and concentration tech- niques such as centrifugation or mini-anion exchange columns are usually necessary [6,7,8]. Stage determination still relies on lumbar puncture to examine CSF for trypanosomes or white cell count/ protein concentration suggestive of chronic meningoencephalitis. Threshold values for these parameters are controversial, with the conventional value for stage 2 (.5 cells/mL) now increased to .10 or even 20 cells/mL [9]. In summary, diagnosis and staging of HAT is currently time consuming, intensive and difficult. The LAMP reaction was performed as previously described [11,14,15]. LAMP Briefly, the reaction contained 12.5 mL of 2x LAMP buffer (40 mM Tris-HCl [pH 8.8], 20 mM KCl, 16 mM MgSO4, 20 mM [NH4]2SO4, 0.2% Tween 20, 1.6 M Betaine, 2.8 mM of each deoxyribonucleotide triphosphate), 1.0 mL primer mix (5 pmol each of F3 and B3, 40 pmol each of FIP and BIP) or 1.3 mL when LF and LB (20 pmol each) were included, 1 mL (8 units) Bst DNA polymerase (New England Biolabs, Tokyo, Japan), 1 mL of template DNA. Final volumes were adjusted to 25 mL with distilled water. All reactions were conducted in 2 to 4 replicates and were monitored in real-time in a LoopampH real-time turbidimeter LA320C (Teramecs, Tokyo, Japan). The optimal reaction temperatures were 62uC (RIME LAMP) and 63uC (PSEUDO-SRA LAMP). The reaction was terminated by increasing the temperature to 80uC for 5 min. In addition to turbidity, the amplified products were analyzed on 2% agarose gels using the E-Gel EX system with ethidium bromide or SYBR green incorporated into the gels (Invitrogen), and/or after addition of hydroxynaphthol blue (HNB) [26] to enable visual detection. The HNB color change from violet to sky blue has been consistently interpreted by independent observers as the easiest to see [26]. DNA-based diagnostic methods such as PCR and LAMP now offer greater sensitivity than existing diagnostic methods, detecting DNA from the equivalent of 0.01 parasites or less. Based on PCR protocols for HAT [10], we described LAMP targeting the conserved paraflagellar rod A (PRFA) gene in all T. brucei subspecies and T. evansi [11]. LAMP is an isothermal DNA amplification method with excellent analytical sensitivity and specificity when employed for the detection of a variety of microorganisms (reviewed in [12]), including human and animal infective African trypanosomes [11,13,14,15,16,17,18,19,20,21]. LAMP relies on autocycling strand displacement coupled to DNA synthesis by Bst DNA polymerase, a reaction similar to rolling- circle amplification [22] but with the added advantage that a heat- denaturing step is not necessary to initiate rounds of amplification. Specificity is dictated by four primers (F3, B3, FIP and BIP), and the addition of two loop primers (LoopF and LoopB) significantly reduces the reaction time [23]. Author Summary Author Summary Human African trypanosomiasis or sleeping sickness is a fatal disease (if untreated) spread by bloodsucking tsetse flies. Trypanosome parasites first enter the blood and lymph and eventually invade the brain. In rural clinical settings, diagnosis still relies on the detection of these microbes in blood and cerebrospinal fluid (CSF) by microscopy. LAMP, or loop-mediated isothermal amplifi- cation of DNA, is a technique that can specifically detect very small amounts of DNA from an organism. It is similar to PCR, the polymerase chain reaction, another DNA amplification technique widely used for diagnosis of infectious diseases. LAMP’s advantages are that the reaction works at one temperature, whereas PCR needs a thermocycler, and LAMP is not affected by blood components that inhibit PCR. We show that by simply adding detergent during sample preparation, the an- alytical sensitivity of LAMP targeting many gene copies is greatly improved, presumably because DNA is released from the pathogen cells and dispersed through the sample. To demonstrate proof of principle, we used pathogenic trypanosomes in different human body fluids (CSF or blood), but this simple modification should be applicable for diagnosis of other microbial infections where cells are sensitive to detergent lysis. www.plosntds.org LAMP for Detection of Live African Trypanosomes parasite/mL (103 parasites/mL), the practical detection limit based on a 1 mL sample volume often used in LAMP or PCR assays. While sensitivity can be increased 5–10 fold by adding more sample volume, significant improvement in the assay system for the detection of live parasites in biologically relevant samples would clearly be of benefit for diagnosis. Here, we introduce a very simple modification to the LAMP assay recognizing multi-copy gene targets that can increase the analytical sensitivity for the detection of live parasites 100-fold or more. Introduction A key issue in the treatment of HAT is to distinguish stage 1 from stage 2 disease, as the drugs used for the treatment of stage 2 need to cross the blood-brain barrier [1,2]. The most widely used drug is melarsoprol (developed in 1949), which is effective for T. b. gambiense and T. b. rhodesiense HAT, but unfortunately, melarsoprol leads to severe and fatal encephalitis in about 5–10% of recipients despite treatment for this condition [3,4,5]. Therefore, where HAT is endemic, accurate staging is critical, because failure to treat CNS involvement leads to death, yet inappropriate CNS treatment exposes an early-stage patient unnecessarily to highly toxic and life-threatening drugs. Tsetse fly-transmitted African trypanosomes are major pathogens of humans and livestock. Two subspecies of Trypanosoma brucei (T. b. rhodesiense and T. b. gambiense) cause human African trypanosomiasis (HAT, commonly called sleeping sickness). After replicating at the tsetse fly bite site, trypanosomes enter the hemolymphatic system (early stage or stage 1) (5, 9). Without treatment, the parasites go on to invade the central nervous system (CNS; late stage or stage 2), a process that takes months to years with T. b. gambiense (West and Central African HAT) or weeks to months with T. b. rhodesiense (East African HAT). The parasites cause a meningoencephalitis leading to progressive neurologic involvement with concomitant psychiatric disorders, fragmentation of the circadian sleep-wake cycle and ultimately to death if untreated (4, 5, 9). The diagnosis of HAT in the rural clinical setting, where most patients are found, still relies largely on the detection of parasitemia by blood smear and/or CSF microscopy [6,7]. While T. b. rhodesiense detection in blood is frequently successful, T. b. August 2011 | Volume 5 | Issue 8 | e1249 1 August 2011 | Volume 5 | Issue 8 | e1249 LAMP for Detection of Live African Trypanosomes Analytical sensitivity using human CSF spiked with trypanosomes LAMP is cost-effective (,1 US dollar/test), simple (the isothermal reaction requires a simple heating device), and rapid (within 60 minutes) [12,24]. Furthermore, Bst DNA polymerase can be stored for weeks at ambient temperatures, a critical property where maintaining a cold chain is difficult [13]. Positive reactions are indicated by turbidity [25], color changes after addition of hydroxynaphthol blue (HNB) [26], or changes in fluorescence using indicator dyes [26,27,28]. Human CSF was obtained as discarded samples from The Johns Hopkins Hospital Microbiology laboratory with approval of the Johns Hopkins Medicine IRB. CSF were adjusted to contain either 1/20 volume deionized water (untreated CSF) or 1/20 volume 10% (w/v) Triton X-100 (final concentration 0.5% Triton). A 10% (w/v) Triton X-100 stock solution was made by adding 1 g Triton X-100 to a final volume of 10 mL DNase/RNase free water (Qiagen). We used bloodstream form T. b. rhodesiense IL1852, a CSF isolate from a patient in Kenya [31,32]. Originally thought to be T. b. gambiense it Despite its advantages, the usefulness of LAMP for HAT diagnosis is handicapped in the clinical setting by its inability to directly detect live trypanosomes in blood or CSF below 1 August 2011 | Volume 5 | Issue 8 | e1249 2 LAMP for Detection of Live African Trypanosomes Figure 1. PSEUDO-SRA LAMP for the detection of T. b. rhodesiense genomic DNA. The PSEUDO-SRA LAMP primer set (Pa with 1:10 serially diluted T. b. rhodesiense IL1852 DNA (1700 fg to 0.017 fg) (Panel B). Replicates: Samples with DNA, n = 2; without DN for each individual sample is presented as real-time turbidity values versus LAMP reaction time. As shown in Panel C, 5 mL reac PSEUDO-SRA LAMP or SRA LAMP amplification of differing concentrations of T. b. rhodesiense IL1852 template were electropho agarose gel containing ethidium bromide. From left to right: Lane 1, 1 kb DNA ladder (Fermentas); Lane 2, 17 pg IL 1852 DNA afte [18]). Lane 3, 17 pg IL 1852 DNA after PSEUDO-SRA LAMP. Lanes 4–10, show a dilution series of template IL1852 DNA as follows Lane 5, 170 fg; Lane 6,17 fg; Lane 7, 1.7 fg; Lane 8, 0.17 fg; Lane 9, 0.017 fg; Lane 10, no DNA template. doi:10.1371/journal.pntd.0001249.g001 Figure 1. PSEUDO-SRA LAMP for the detection of T. b. rhodesiense genomic DNA. The PSEUDO-SRA LAMP primer set (Panel A) was tested with 1:10 serially diluted T. b. www.plosntds.org Analytical sensitivity using human CSF spiked with trypanosomes rhodesiense IL1852 DNA (1700 fg to 0.017 fg) (Panel B). Replicates: Samples with DNA, n = 2; without DNA, n = 4. The data for each individual sample is presented as real-time turbidity values versus LAMP reaction time. As shown in Panel C, 5 mL reaction product after PSEUDO-SRA LAMP or SRA LAMP amplification of differing concentrations of T. b. rhodesiense IL1852 template were electrophoresed through 2% agarose gel containing ethidium bromide. From left to right: Lane 1, 1 kb DNA ladder (Fermentas); Lane 2, 17 pg IL 1852 DNA after SRA LAMP (Njiru [18]). Lane 3, 17 pg IL 1852 DNA after PSEUDO-SRA LAMP. Lanes 4–10, show a dilution series of template IL1852 DNA as follows: Lane 4, 1700 fg; Lane 5, 170 fg; Lane 6,17 fg; Lane 7, 1.7 fg; Lane 8, 0.17 fg; Lane 9, 0.017 fg; Lane 10, no DNA template. doi:10.1371/journal.pntd.0001249.g001 ww August 2011 | Volume 5 | Issue 8 | e1249 3 www.plosntds.org LAMP for Detection of Live African Trypanosomes Figure 2. Detergent increases analytical sensitivity of RIME LAMP for the direct detection of T. b. rhodesiense in human CSF. Fifty mL water (DNAse/RNAse free) or 10% Triton X-100 was added to human CSF. T. b. rhodesiense IL1852 was spiked into 950 mL human CSF without and with 0.5% Triton X-100. The samples were serially diluted in duplicate in normal or detergent treated CSF and incubated at ambient temperature for 60 min. One mL aliquots were assayed for 1 hr at 62uC using RIME LAMP primers. Normal CSF (1 mL) with or without Triton X-100 was used as a control. Each panel shows hydroxynaphthol blue reaction tubes (top), agarose gel (center) and real-time turbidity data (bottom) from the same samples. doi:10 1371/journal pntd 0001249 g002 Figure 2. Detergent increases analytical sensitivity of RIME LAMP for the direct detection of T. b. rhodesiense in human CSF. Fifty mL water (DNAse/RNAse free) or 10% Triton X-100 was added to human CSF. T. b. rhodesiense IL1852 was spiked into 950 mL human CSF without and with 0.5% Triton X-100. The samples were serially diluted in duplicate in normal or detergent treated CSF and incubated at ambient temperature for 60 min. One mL aliquots were assayed for 1 hr at 62uC using RIME LAMP primers. Normal CSF (1 mL) with or without Triton X-100 was used as a control. Analytical sensitivity using human CSF spiked with trypanosomes Each panel shows hydroxynaphthol blue reaction tubes (top), agarose gel (center) and real-time turbidity data (bottom) from the same samples. doi:10 1371/journal pntd 0001249 g002 doi:10.1371/journal.pntd.0001249.g002 doi:10.1371/journal.pntd.0001249.g002 Microbiology laboratory with approval of the Johns Hopkins Medicine IRB was spiked with T. b. rhodesiense (104 to 1022 parasites/mL). Protein Saver 903 cards were pretreated with 50 mL 0.5% Triton X-100, which was sufficient to fill the designated circle on the cards, and dried overnight prior to whole blood spotting. For assay standardization, three 2 mm punches made from the 1 cm2 dried blood spots (DBS) and DNA was extracted using standard methods [37]. The LAMP assays were done using 1 mL DBS DNA template. Alternatively, untreated and 0.5% Triton X-100 treated trypanosome-spiked blood were spotted on untreated Protein Saver 903 cards and dried overnight with subsequent DBS DNA extraction as above. has been reclassified as T. b. rhodesiense [33] based on the presence of the SRA gene [34] and the absence of the TgsGP gene [35,36] (Fig. S1). Human CSF was spiked with bloodstream form T. b. rhodesiense IL1852 and the samples serially diluted 1:10 in CSF with or without 0.5% detergent to cover a range of parasite concentrations from 104 to 1021 parasites/mL. After 60 min incubation at ambient temperature to allow for lysis, the LAMP assays were done using 1 mL CSF. Analytical sensitivity using human blood spiked with trypanosomes In the field, biological samples are often shipped to another geographical site for later analyses. They are often preserved by spotting on paper cards designed for short-term protein, RNA and DNA storage (2 weeks at ambient temperature) such as Whatman Protein Saver 903, or long-term (years) DNA storage/archiving on Whatman FTA cards. To simulate these conditions, human blood obtained as discarded samples from The Johns Hopkins Hospital www.plosntds.org Results and Discussion LAMP with genomic parasite DNA www.plosntds.org LAMP with genomic parasite DNA Based on experiments repeated at least 3 times, LAMP assays successfully amplified T. b. rhodesiense DNA within 55–60 min at 62uC (RIME LAMP) or 63uC (PSEUDO-SRA LAMP). As August 2011 | Volume 5 | Issue 8 | e1249 August 2011 | Volume 5 | Issue 8 | e1249 4 Figure 3. Analytical sensitivity of real-time PSEUDO-SRA LAMP for the direct detection of T. b. rhodesiense in human CSF. T. b. rhodesiense IL1852 was spiked into human CSF without and with 0.5% Triton X-100 (+Tx). The samples were serially diluted in normal or detergent treated CSF. After 60 min incubation at ambient temperature, 1 mL aliquots were assayed using the PSEUDO-SRA LAMP primers. Normal CSF with or without Triton X-100 was used as a control. The data for each individual sample is presented as real-time turbidity values versus LAMP reaction time. The number of parasites/mL CSF originally present in the sample used for the assays in the panels shown are: [A], 104/mL; [B], 103/mL; [C], 102/mL; [D], 101/mL; [E], 100/mL; [F], CSF alone. doi:10.1371/journal.pntd.0001249.g003 LAMP for Detection of Live African Trypanosomes LAMP for Detection of Live African Trypanosomes Figure 3. Analytical sensitivity of real-time PSEUDO-SRA LAMP for the direct detection of T. b. rhodesiense in human CSF. T. b. rhodesiense IL1852 was spiked into human CSF without and with 0.5% Triton X-100 (+Tx). The samples were serially diluted in normal or detergent treated CSF. After 60 min incubation at ambient temperature, 1 mL aliquots were assayed using the PSEUDO-SRA LAMP primers. Normal CSF with or without Triton X-100 was used as a control. The data for each individual sample is presented as real-time turbidity values versus LAMP reaction time. The number of parasites/mL CSF originally present in the sample used for the assays in the panels shown are: [A], 104/mL; [B], 103/mL; [C], 102/mL; [D], 101/mL; [E], 100/mL; [F], CSF alone. doi:10.1371/journal.pntd.0001249.g003 reported previously [16], we found that RIME LAMP detected 0.1 fg genomic DNA (0.001 parasite) from T. b. rhodesiense IL1852 (not shown). PSEUDO-SRA LAMP was as sensitive and reliably detected 0.1 fg (0.001 parasite) or less T. b. rhodesiense IL1852 genomic DNA (Fig. 1B and 1C). Nonetheless, when using the SRA gene specific LAMP assay [17] the detection limit for T. b. rhodesiense IL1852 genomic DNA was 0.1–1.0 pg (1–10 parasites), comparing favorably to reported values [17]. genomic components besides the SRA gene. www.plosntds.org www.plosntds.org LAMP with genomic parasite DNA As SRA is a truncated VSG, it is likely that the PSEUDO-SRA LAMP is amplifying other VSG sequences, albeit not efficiently (see below). Although the PSEUDO-SRA LAMP primer sequences were verified as being unique by BLAST analysis of the T. b. brucei TREU 927 genome sequence and the VSG database (TriTrypDB: http://tritrypdb. org/tritrypdb/), VSG repertoires are diverse between strains, and we were unable to assess the primers against sequences of the full IL1852 VSG repertoire as its genome has not been sequenced. The standard curves with PSEUDO-SRA LAMP seem to display biphasic kinetics with an early initial phase (15–20 min) followed by a late second phase (35 and 55 min) with a break point around 1.7 fg DNA (Fig. 1B) suggesting that it targets other The PSEUDO-SRA LAMP was specific and recognized DNA equally well from other T. b. rhodesiense strains (LouTat 1A, GYBO, IL1501), but did not recognize DNA from T. b. gambiense isolates August 2011 | Volume 5 | Issue 8 | e1249 5 August 2011 | Volume 5 | Issue 8 | e1249 LAMP for Detection of Live African Trypanosomes (IL 3258, DAL 972, DAL 072, DAL 069, IPR SG-1020, FONT l993, JUA, MOS, MA 002) (not shown). It also recognized T. b. brucei strain 927 genomic DNA at very high concentrations (i.e. .1 ng DNA/mL equivalent to .104 parasites/mL), but it was specific for T. b. rhodesiense at the concentrations tested (10 pg to 0.1 fg DNA/mL equivalent to 102 to 1023 parasites/mL). Negative controls included the eukaryotic protozoan parasites Babesia microti, Plasmodium falciparum, Plasmodium ovale, and Toxoplasma gondii, as well as DNA from clinical samples or spiked blood samples, such as Borrelia burgdorferi, Borrelia crocidurae, Enterococcus spp., Ehrlichia chaffeensis, Escherichia coli, Pseudomonas aeruginosa, Rickettsia parkeri, Staphylococcus spp., and DNA from mouse and human blood. Furthermore, using PSEUDO-SRA LAMP under carefully con- trolled conditions, no false positives were found when DNA from 192 normal human CSF samples was tested. Although it is possible to detect very low parasite numbers using Psuedo-SRA LAMP, the assay’s sensitivity is a potential drawback because of risk for amplicon contamination. Therefore, until more validation is done, we do not propose PSEUDO-SRA LAMP for diagnosis of T. b. rhodesiense. However, the range of sensitivity made it an ideal choice to study the effects of detergent on increasing the ability of LAMP to detect live parasites in biological samples. LAMP in human CSF spiked with parasites To mimic a clinical situation, we first tested RIME LAMP and PSEUDO-SRA LAMP on human CSF spiked with live T. b. rhodesiense IL1852 and analyzed the reaction products on agarose gels, HNB reaction, and/or real-time LAMP based on turbidimetric readings. As predicted, the LAMP assays had a detection limit of 103 parasites per mL based on 1 mL assay samples for RIME (Fig. 2) and PSEUDO-SRA LAMP (Fig. 3). While sensitivity could be increased up to 10 fold by increasing CSF sample volume to 10 mL (not shown), the presence of detergent (i.e. 0.5% Triton X-100) alone added to the CSF samples improved detection to 10 and 1 parasite/ mL, representing a 100 to 1000-fold increase in RIME LAMP and PSEUDO-SRA LAMP assay analytical sensitivity, respectively (Figs 2 and 3; Table 1). Release of parasite DNA by 0.5% Triton required between 30 and 60 min incubation. LAMP assay using dried blood spots (DBS) of human blood spiked with parasites The transport and storage of DBS or CSF on filter paper cards is a common practice in the field. DBS on Whatman Protein Saver 903 cards are used for parasite pathogen detection (DNA, RNA and/or protein) and genotyping [38,39,40,41,42,43]. Depending on the paper matrix, DNA, RNA and/or protein to be tested are first extracted from defined diameter punches (e.g. 2 mm) and 1–5 uL are assayed. Assay sensitivity for trypanosomes is limited by the stoichiometric presence of the parasite in the assayed sample. Analytical sensitivity is further reduced since sample volumes in filter paper punches represent ,1% of the total captured on the paper itself [44]. We used parasite-spiked human blood spotted on dry Protein Saver 903 cards pretreated with detergent. Remarkably, the presence of detergent greatly enhanced LAMP detection limits for parasite DNA by about 100 fold for RIME and PSEUDO-SRA LAMP (Figs 4 and 5; Table 1). Enhanced detection sensitivity was also found when T. b. rhodesiense IL1852 DNA was extracted from DBS from Protein Saver 903 cards containing normal or detergent-treated parasite-spiked human blood (Figs S2 and S3). In general, replicates were more reproducible in assays where the detergent was present in the paper. The presence of detergent had no effect on analytical sensitivity by HNB [26], confirming its use for easy, inexpensive, accurate, and reliable field detection of LAMP-amplified DNA. As with any DNA amplification method, standard precautions for avoiding template contamination [45] also apply for LAMP-based assays. Added Advantages It has been shown that sensitivity, including detection of type 1 T. b. gambiense [20], can be greatly enhanced after heat denaturing the samples before LAMP assay [16,17,20]. However, this procedure is less convenient than simply incubating samples at ambient temperature with detergent or allowing samples to dry on detergent-pretreated filter cards. Aerosol effects by heating the samples could also increase the risk of cross-contamination prior to addition of the reaction mixture. Furthermore, the extra steps required for techniques such as quantitative buffy coat, microhe- Table 1. Summary of LAMP assays conducted using trypanosome spiked human CSF and blood. Table 1. Summary of LAMP assays conducted using trypanosome spiked human CSF and blood. Table 1. Summary of LAMP assays conducted using trypanosome spiked human CSF and blood. LAMP assay conditions Trypanosomes/mL Source of DNA How sample assayed Triton added to Primer set 104 103 102 101 100 1021 1022 None Sample Card CSF Direct No N/A RIME + + 2 2 2 2 nd 2 Yes N/A RIME + + + + 2 2 nd 2 No N/A PSEUDO-SRA + + – – – – nd 2 Yes N/A PSEUDO-SRA + + + + + +/– nd 2 Blood DBS No No RIME + + 2 2 2 2 nd 2 No Yes RIME + + + + 2 2 nd 2 No No PSEUDO-SRA nd + +/2 2 2 2 2 2 No Yes PSEUDO-SRA nd + + + + + + 2 Blood DBS No No RIME + + 2 2 2 2 nd 2 Yes No RIME + + + + 2 2 nd 2 No No PSEUDO-SRA + + + + 2 2 nd 2 Yes No PSEUDO-SRA + + + + + +/2 nd 2 + = All replicates positive; +/– = positive/negative mix; 2 = All replicates negative; nd = not done; N/A = not applicable; DBS = dried blood spot on 903 card. doi:10.1371/journal.pntd.0001249.t001 + = All replicates positive; +/– = positive/negative mix; 2 = All replicates negative; nd = not done; N/A = not applicable; DBS = dried blood spot on 903 card. doi:10.1371/journal.pntd.0001249.t001 August 2011 | Volume 5 | Issue 8 | e1249 6 www.plosntds.org LAMP for Detection of Live African Trypanosomes Figure 4. Analytical sensitivity of RIME LAMP for detection of T. b. rhodesiense DNA in human blood. T. b. Added Advantages rhodesiense IL1852 was spiked into whole human blood, serially diluted and spotted in duplicate on Protein Saver 903 cards or 903 cards pretreated with 0.5% Triton X-100 and allowed to dry overnight. DNA from the DBS was extracted as described in the methods [37] and 1 mL aliquots assayed using RIME LAMP primers. Normal blood (blood) or nuclease free water with or without Triton X-100 were used as controls. Each panel shows hydroxynaphthol blue reaction tubes (top), agarose gel (center) and real-time turbidity data (bottom) from the same samples. DBS DNA from uninfected blood was used as a negative control. doi:10.1371/journal.pntd.0001249.g004 Figure 4. Analytical sensitivity of RIME LAMP for detection of T. b. rhodesiense DNA in human blood. T. b. rhodesiense IL1852 was spiked into whole human blood, serially diluted and spotted in duplicate on Protein Saver 903 cards or 903 cards pretreated with 0.5% Triton X-100 and allowed to dry overnight. DNA from the DBS was extracted as described in the methods [37] and 1 mL aliquots assayed using RIME LAMP primers. Normal blood (blood) or nuclease free water with or without Triton X-100 were used as controls. Each panel shows hydroxynaphthol blue reaction tubes (top), agarose gel (center) and real-time turbidity data (bottom) from the same samples. DBS DNA from uninfected blood was used as a negative control. doi:10.1371/journal.pntd.0001249.g004 doi:10.1371/journal.pntd.0001249.g004 trypanosomes in blood and CSF approximately approaching or reaching the detection limits of LAMP for genomic DNA. matocrit centrifugation (mHCT), mini-anion-exchange centrifu- gation technique (mAECT) used to concentrate the parasites from blood or CSF [6,7] also increase contamination risk. The addition of samples directly in the reaction helps reduce contamination. www.plosntds.org www.plosntds.org Conclusion Recent findings by Deborggraeve et al. [46] suggest that while PCR performed better than, or similar to current parasite detection techniques for T. b. gambiense sleeping sickness diagnosis and staging, it cannot be used for post-treatment follow-up because of persistence of living or dead parasites or their DNA after successful treatment. The use of LAMP on serially diluted sample in the absence and presence of detergent could be useful for differentiating between these scenarios; large difference might indicate a recent infection with small differences indicating persistent or relapse infection. While we have not yet optimized conditions with regards to detergent concentration or class (nonionic, ionic or zwitterionic), our preliminary evidence supports the concept that a detergent such as Triton X-100 can be used in a variety of ways to enhance the analytical sensitivity of multi-copy gene LAMP-based assays for the detection of intact African In addition to LAMP, the implications of these findings are far reaching and should also be applicable for improved lateral-flow dipstick methods recently introduced [47], PCR, or other nucleic acid amplification-based [Recombinase Polymerase Amplification (TwistDX), Strand Displacement Amplification (Probetec ET, Becton-Dickinson), Nucleic Acid Sequenced Base Amplification (Primer Biosoft International)] technologies where microbial pathogen, including protozoan parasite (e.g. Plasmodium) DNA/ RNA could be easily released by detergents. Unlocking the potential power of LAMP for accurate HAT diagnosis presents an excellent option for the administration of effective anti-trypano- some treatment. In summary, the procedure paves the way for the adaptation of LAMP and similar technologies as simple cost-effective diagnostics for intact African trypanosomes in humans, animals and tsetse flies, and also for other protozoan August 2011 | Volume 5 | Issue 8 | e1249 August 2011 | Volume 5 | Issue 8 | e1249 7 LAMP for Detection of Live African Trypanosomes Figure 5. Analytical sensitivity of PSEUDO-SRA LAMP for detection of T. b. rhodesiense DNA in human blood. T. b. rhodesiense IL1852 was spiked into whole human blood, serially diluted and spotted in duplicate on Protein Saver 903 cards or 903 cards pretreated with 0.5% Triton X- 100 (+ Tx) and allowed to dry overnight. Control blood samples without trypanosomes were spotted in quadruplicate. DNA from individual DBS (n = 2) was extracted as described in the methods and 1 mL aliquots assayed using PSEUDO-SRA LAMP primers. DBS DNA from uninfected blood was used as a negative control (n = 4 +/2 Triton). Conclusion The number of parasites/mL blood originally present in the sample used for the assays in the panels shown are: [A], 102/mL; [B], 101/mL; [C], 100/mL; [D], 1021/mL; [E], 1022/mL; [F], blood alone. doi:10.1371/journal.pntd.0001249.g005 Figure 5. Analytical sensitivity of PSEUDO-SRA LAMP for detection of T. b. rhodesiense DNA in human blood. T. b. rhodesiense IL1852 was spiked into whole human blood, serially diluted and spotted in duplicate on Protein Saver 903 cards or 903 cards pretreated with 0.5% Triton X- 100 (+ Tx) and allowed to dry overnight. Control blood samples without trypanosomes were spotted in quadruplicate. DNA from individual DBS (n = 2) was extracted as described in the methods and 1 mL aliquots assayed using PSEUDO-SRA LAMP primers. DBS DNA from uninfected blood was used as a negative control (n = 4 +/2 Triton). The number of parasites/mL blood originally present in the sample used for the assays in the panels shown are: [A], 102/mL; [B], 101/mL; [C], 100/mL; [D], 1021/mL; [E], 1022/mL; [F], blood alone. doi:10.1371/journal.pntd.0001249.g005 parasites and microorganisms that cause diseases that plague the developing world. Fifty mL water (DNAse/RNAse free) or 10% Triton X-100 was added to 950 mL human blood. T. b. rhodesiense IL1852 was spiked into human blood without and with 0.5% (w/v) Triton X-100. The samples were serially diluted in normal or detergent treated blood and spotted on Protein Saver 903 cards. DNA from the DBS was extracted [37] and 1 mL aliquots assayed using RIME LAMP primers. Each panel shows hydroxynaphthol blue reaction tubes (top), agarose gel (center) and real-time turbidity data (bottom) from the same samples. DBS DNA from uninfected blood was used as a negative control. (TIF) References 9. Bisser S, Lejon V, Preux PM, Bouteille B, Stanghellini A, et al. (2002) Blood- cerebrospinal fluid barrier and intrathecal immunoglobulins compared to field diagnosis of central nervous system involvement in sleeping sickness. J Neurol Sci 193: 127–135. 27. Tomita N, Mori Y, Kanda H, Notomi T (2008) Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3: 877–882. 10. Solano P, Michel JF, Lefrancois T, de La Rocque S, Sidibe I, et al. (1999) Polymerase chain reaction as a diagnosis tool for detecting trypanosomes in naturally infected cattle in Burkina Faso. Vet Parasitol 86: 95–103. 28. Qiao YM, Guo YC, Zhang XE, Zhou YF, Zhang ZP, et al. (2007) Loop- mediated isothermal amplification for rapid detection of Bacillus anthracis spores. Biotechnol Lett 29: 1939–1946. 11. Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, et al. (2003) Loop- mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol 41: 5517–5524. 29. Matovu E, Kuepfer I, Boobo A, Kibona S, Burri C (2010) Comparative detection of trypanosomal DNA by loop-mediated isothermal amplification and PCR from flinders technology associates cards spotted with patient blood. J Clin Microbiol 48: 2087–2090. 12. Mori Y, Notomi T (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 15: 62–69. 30. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, et al. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28: E63. 13. Thekisoe OM, Bazie RS, Coronel-Servian AM, Sugimoto C, Kawazu S, et al. (2009) Stability of Loop-Mediated Isothermal Amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates. J Vet Med Sci 71: 471–475. 31. Mhando PJ, Yanagi T, Fukuma T, Nakazawa S, Kanaba H (1987) In vitro cultivation of Trypanosoma brucei subspecies with cells derived from brain and mouse of new born mouse. Trop Med 29. 14. Thekisoe OM, Kuboki N, Nambota A, Fujisaki K, Sugimoto C, et al. (2007) Species-specific loop-mediated isothermal amplification (LAMP) for diagnosis of trypanosomosis. Acta Trop 102: 182–189. 32. Eshita Y, Urakawa T, Hirumi H, Fish WR, Majiwa PA (1992) Metacyclic form- specific variable surface glycoprotein-encoding genes of Trypanosoma (Nannomonas) congolense. Gene 113: 139–148. 15. Thekisoe OM, Inoue N, Kuboki N, Tuntasuvan D, Bunnoy W, et al. References 20. Njiru ZK, Traub R, Ouma JO, Enyaru JC, Matovu E (2011) Detection of Group 1 Trypanosoma brucei gambiense by Loop-mediated isothermal amplification (LAMP). J Clin Microbiol 49: 1530–1536. 1. Docampo R, Moreno SN (2003) Current chemotherapy of human African trypanosomiasis. Parasitol Res 90(Supp 1): S10–13. 2. Enanga B, Burchmore RJ, Stewart ML, Barrett MP (2002) Sleeping sickness and the brain. Cell Mol Life Sci 59: 845–858. 21. Njiru ZK, Ouma JO, Bateta R, Njeru SE, Ndungu K, et al. (2011) Loop- mediated isothermal amplification test for Trypanosoma vivax based on satellite repeat DNA. Vet Parasitol. 3. Grab DJ, Kennedy PE (2008) Traversal of human and animal trypanosomes across the blood-brain barrier. J Neurovirology 14: 344–351. 22. Hafner GJ, Yang IC, Wolter LC, Stafford MR, Giffard PM (2001) Isothermal amplification and multimerization of DNA by Bst DNA polymerase. Biotechniques 30: 852-856, 858, 860 passim. 4. Kristensson K, Nygard M, Bertini G, Bentivoglio M (2010) African trypanosome infections of the nervous system: parasite entry and effects on sleep and synaptic functions. Prog Neurobiol 91: 152–171. q p 23. Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16: 223–229. 5. Kennedy PG (2004) Human African trypanosomiasis of the CNS: current issues and challenges. J Clin Invest 113: 496–504. 24. Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, et al. (2006) Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplifica- tion. Clin Chem 52: 303–306. g J 6. Chappuis F, Loutan L, Simarro P, Lejon V, Bu¨scher P (2005) Options for field diagnosis of human african trypanosomiasis. Clin Microbiol Rev 18: 133–146. agnosis of human african trypanosomiasis. Clin Microbiol Rev 18: 13 7. Bu¨scher P, Lejon V, eds (2004) Diagnosis of human African trypanosomiasis: CAB International. pp 203–218. 25. Mori Y, Nagamine K, Tomita N, Notomi T (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289: 150–154. 8. Koffi M, Solano P, Denizot M, Courtin D, Garcia A, et al. (2006) Aparasitemic serological suspects in Trypanosoma brucei gambiense human African trypanosomi- asis: a potential human reservoir of parasites? Acta Trop 98: 183–188. 26. Wastling SL, Picozzi K, Kakembo AS, Welburn SC (2010) LAMP for Human African Trypanosomiasis: A Comparative Study of Detection Formats. PLoS Negl Trop Dis 4: e865. Table S1 SRA gene 59-39 sequence targeted by PSEUDO- SRA (AJ560644). (DOC) Table S1 SRA gene 59-39 sequence targeted by PSEUDO- SRA (AJ560644). (DOC) Conceived and designed the experiments: DJG NI LJM JSD. Performed the experiments: OVN LJM NI. Analyzed the data: DJG WG NI LJM JSD OMMT. Contributed reagents/materials/analysis tools: DJG NI. Wrote the paper: DJG WG LJM JSD NI OMMT. Acknowledgments From Johns Hopkins University, we thank Dr. Rob Jensen for the gift of kinetoplast DNA, Dr. Rahul Bakshi for reading and manuscript editing, From Johns Hopkins University, we thank Dr. Rob Jensen for the gift of kinetoplast DNA, Dr. Rahul Bakshi for reading and manuscript editing, LAMP for Detection of Live African Trypanosomes spotted on paper cards as in Fig. 4. The DNA from the DBS was extracted and 1 mL aliquots assayed using PSEUDO-SRA LAMP primers. The data for each individual sample is presented as real- time turbidity values versus LAMP reaction time. DBS DNA from uninfected blood was used as a negative control. The number of parasites/mL blood in the panels shown are: [A], 103/mL; [B], 102/mL; [C], 101/mL; [D], 100/mL; [E], 1021/mL; [F], blood alone. (TIF) and Emily Clemens for excellent technical assistance. We also thank Dr. John Mansfield, University of Wisconsin (Madison) for his generous gift of highly virulent, pleomorphic T. b. rhodesiense LouTat 1A [48], as well as gratefully acknowledge Dr. Sylvie Bisser at the Institut de Neurologie Tropicale, Universite´ de Limoges (France), Dr. Ian Burbulis at the Molecular Science Institute (Oakland CA) and Dr. Rebecca Garabed, Ohio State University (OH) for reading and editing before manuscript submission. spotted on paper cards as in Fig. 4. The DNA from the DBS was extracted and 1 mL aliquots assayed using PSEUDO-SRA LAMP primers. The data for each individual sample is presented as real- time turbidity values versus LAMP reaction time. DBS DNA from uninfected blood was used as a negative control. The number of parasites/mL blood in the panels shown are: [A], 103/mL; [B], 102/mL; [C], 101/mL; [D], 100/mL; [E], 1021/mL; [F], blood alone. Supporting Information Figure S1 T. b. rhodesiense IL1852 contains the SRA gene. Genomic DNA isolated from IL1852 trypanosomes was checked by PCR using oligonucleotide primers directed against the SRA gene diagnostic for T. b. rhodesiense, and the TgsGP gene diagnostic for T. b. gambiense. Positive controls included in each reaction were ELIANE, a T. b. gambiense group 1 from Coˆte d’Ivoire [49], and Z222, a confirmed T. b. rhodesiense from Zambia. (TIF) Figure S3 Analytical sensitivity of PSEUDO-SRA for dried blood spot detection of T. b. rhodesiense DNA from detergent treated human blood spotted on 903 cards. T. b. rhodesiense IL1852 was spiked and serially diluted into human blood without and with 0.5% Triton X-100 (+Tx) and Figure S2 Analytical sensitivity of RIME LAMP for dried blood spot detection of T. b. rhodesiense DNA from detergent treated human blood spotted on 903 cards. August 2011 | Volume 5 | Issue 8 | e1249 8 www.plosntds.org References (2005) Evaluation of loop-mediated isothermal amplification (LAMP), PCR and parasitological tests for detection of Trypanosoma evansi in experimentally infected pigs. Vet Parasitol 130: 327–330. g 33. Nikolskaia OV, de ALAP, Kim YV, Lonsdale-Eccles JD, Fukuma T, et al. (2008) Erratum. J Clin Invest 118: 1974. 34. Radwanska M, Chamekh M, Vanhamme L, Claes F, Magez S, et al. (2002) The serum resistance-associated gene as a diagnostic tool for the detection of Trypanosoma brucei rhodesiense. Am J Trop Med Hyg 67: 684–690. 16. Njiru ZK, Mikosza AS, Matovu E, Enyaru JC, Ouma JO, et al. (2008) African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int J Parasitol 38: 589–599. 35. Radwanska M, Claes F, Magez S, Magnus E, Perez-Morga D, et al. (2002) Novel primer sequences for polymerase chain reaction-based detection of Trypanosoma brucei gambiense. Am J Trop Med Hyg 67: 289–295. 17. Njiru ZK, Mikosza AS, Armstrong T, Enyaru JC, Ndung’u JM, et al. (2008) Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense. PLoS Negl Trop Dis 2: e147. 36. Pays E, Dekerck P, Van Assel S, Babiker EA, Le Ray D, et al. (1983) Comparative analysis of a Trypanosoma brucei gambiense antigen gene family and its potential use in epidemiology of sleeping sickness. Mol Biochem Parasitol 7: 63–74. 18. Thekisoe OMM, Inoue N, Namangala B, Sugimoto C (2010) Loop-mediated isothermal amplification (LAMP) assays for specific detection of Trypanosoma vivax infections in livestock and tsetse flies. The 39th annual conference of the Parasitological Society of Southern Africa. Drakensville Resort, KwaZulu-Natal, South Africa. 37. Sambrook J, Russell DW (2001) Preparation and analysis of eukaryotic genomic DNA; Sambrook J, Russell DW, eds. New York, New York: Cold Spring Harbor Laboratory Press. 38. Stresman GH, Kamanga A, Moono P, Hamapumbu H, Mharakurwa S, et al. (2010) A method of active case detection to target reservoirs of asymptomatic malaria and gametocyte carriers in a rural area in Southern Province, Zambia. Malar J 9: 265. 19. Njiru ZK, Ouma JO, Enyaru JC, Dargantes AP (2010) Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B. Exp Parasitol 125: 196–201. www.plosntds.org August 2011 | Volume 5 | Issue 8 | e1249 August 2011 | Volume 5 | Issue 8 | e1249 9 www.plosntds.org LAMP for Detection of Live African Trypanosomes LAMP for Detection of Live African Trypanosomes 45. Hildebrandt F, Singh-Sawhney I (1999) Polymerase Chain Reaction. In: Hildebrandt F, Igarashi P, eds. Techniques in molecular medicine: Springer- Verlag. 39. Mlambo G, Vasquez Y, LeBlanc R, Sullivan D, Kumar N (2008) A filter paper method for the detection of Plasmodium falciparum gametocytes by reverse transcription polymerase chain reaction. Am J Trop Med Hyg 78: 114–116. 40. Buckton AJ (2008) New methods for the surveillance of HIV drug resistance in the resource poor world. Curr Opin Infect Dis 21: 653–658. 40. Buckton AJ (2008) New methods for the surveillance of HIV d 46. Deborggraeve S, Lejon V, Ekangu RA, Mumba Ngoyi D, Pati Pyana P, et al. (2011) Diagnostic Accuracy of PCR in gambiense Sleeping Sickness Diagnosis, Staging and Post-Treatment Follow-Up: A 2-year Longitudinal Study. PLoS Negl Trop Dis 5: e972. the resource poor world. Curr Opin Infect Dis 21: 653–658. 41. Johannessen A, Garrido C, Zahonero N, Sandvik L, Naman E, et al. (2009) Dried blood spots perform well in viral load monitoring of patients who receive antiretroviral treatment in rural Tanzania. Clin Infect Dis 49: 976–981. 47. Njiru ZK (2011) Rapid and sensitive detection of human African trypanoso- miasis by loop-mediated isothermal amplification combined with a lateral-flow dipstick. Diagn Microbiol Infect Dis 69: 205–209. 42. Garrido C, Zahonero N, Corral A, Arredondo M, Soriano V, et al. (2009) Correlation between human immunodeficiency virus type 1 (HIV-1) RNA measurements obtained with dried blood spots and those obtained with plasma by use of Nuclisens EasyQ HIV-1 and Abbott RealTime HIV load tests. J Clin Microbiol 47: 1031–1036. p g 48. Inverso JA, Uphoff TS, Johnson SC, Paulnock DM, Mansfield JM (2010) Biological variation among african trypanosomes: I. Clonal expression of virulence is not linked to the variant surface glycoprotein or the variant surface glycoprotein gene telomeric expression site. DNA Cell Biol 29: 215–227. 43. Recommended genotyping procedures (RGPs) to identify parasite populations; 2007 May 29-31; Amsterdam, The Netherlands. 49. Felgner P, Brinkmann U, Zillmann U, Mehlitz D, Abu-Ishira S (1981) Epidemiological studies on the animal reservoir of gambiense sleeping sickness. II. Parasitolgical and immunodiagnostic examination of the human population. Tropenmed Parasitol 32: 124–140. 44. Cox AP, Tosas O, Tilley A, Picozzi K, Coleman P, et al. (2010) Constraints to estimating the prevalence of trypanosome infections in East African zebu cattle. Parasit Vectors 3: 82. 47. Njiru ZK (2011) Rapid and sensitive detection of human African trypanoso- miasis by loop-mediated isothermal amplification combined with a lateral-flow dipstick. Diagn Microbiol Infect Dis 69: 205–209. 43. Recommended genotyping procedures (RGPs) to identify parasite populations; 2007 May 29-31; Amsterdam, The Netherlands. 44. Cox AP, Tosas O, Tilley A, Picozzi K, Coleman P, et al. (2010) Constraints to estimating the prevalence of trypanosome infections in East African zebu cattle. Parasit Vectors 3: 82. 45. Hildebrandt F, Singh-Sawhney I (1999) Polymerase Chain Reaction. In: Hildebrandt F, Igarashi P, eds. Techniques in molecular medicine: Springer- Verlag. LAMP for Detection of Live African Trypanosomes www.plosntds.org August 2011 | Volume 5 | Issue 8 | e1249 10 www.plosntds.org www.plosntds.org
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A Network-Based Method for Mechanistic Investigation and Neuroprotective Effect on Post-treatment of Senkyunolid-H Against Cerebral Ischemic Stroke in Mouse
Frontiers in neurology
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ORIGINAL RESEARCH published: 19 December 2019 doi: 10.3389/fneur.2019.01299 Jie Zhang 1†, Yunyao Jiang 2†, Nan Liu 3, Ting Shen 4, Hyo Won Jung 5,6, Jianxun Liu 7* and Bing Chun Yan 1,8,9* 1 Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Department of Traditional Chinese and Western Medicine, Yangzhou University, Yangzhou, China, 2 School of Pharmaceutical Sciences, Institute for Chinese Materia Medica, Tsinghua University, Beijing, China, 3 Beijing Increase Research for Drug Efficacy and Safety Co., Ltd., Beijing, China, 4 School of Life Sciences, Huaiyin Normal University, Huai’an, China, 5 Department of Herbology, College of Korean Medicine, Dongguk University, Gyeongju-si, South Korea, 6 Korean Medicine R&D Center, Dongguk University, Gyeongju-si, South Korea, 7 Beijing Key Laboratory of TCM Pharmacology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China, 8 Department of Neurology, Affiliated Hospital, Yangzhou University, Yangzhou, China, 9 Jiangsu Key Laboratory of Zoonosis, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China Edited by: Anthony McGoron, Florida International University, United States Reviewed by: Carina Rodrigues Boeck, UFN-Universidade Franciscana, Brazil Neil M. Fournier, Trent University, Canada Reviewed by: Carina Rodrigues Boeck, UFN-Universidade Franciscana, Brazil Neil M. Fournier, Trent University, Canada Senkyunolide-H (SEH), a major bioactive compound extracted from Ligusticum chuanxiong, has been reported to be effective in preventing cerebral ischemic stroke (CIS). In this study, we employed network pharmacology to reveal potential mechanism of SEH against CIS on a system level and confirmed the therapeutic effects of SEH on CIS by models of cerebral ischemia-reperfusion in vivo and in vitro. Through protein-protein interaction networks construction of SEH- and CIS-related targets, a total of 62 key targets were obtained by screening topological indices and analyzed for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Gene Ontology analysis indicated that SEH might have a role in treating CIS via regulating some biological processes including regulation of transcription from RNA polymerase II promoter, epidermal growth factor receptor signaling pathway, phosphatidylinositol-mediated signaling, and some molecular function, such as transcription factor and protein phosphatase binding and nitric oxide synthase regulator activity. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes analysis showed that phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was significantly enriched. In addition, our result showed that SEH posttreatment significantly decreased the neurological scores, infarct volume, and neuronal death in the middle cerebral artery occlusion mice. Moreover, the PI3K/Akt/nuclear factor kappa B signaling pathway was activated by intragastric administration of 40 mg/kg SEH, as verified by Western blot. In vitro, treatment of PC12 cells with 100 µM SEH markedly reduced cell death induced by oxygen-glucose deprivation through the *Correspondence: Bing Chun Yan bcyan@yzu.edu.cn Jianxun Liu jianxun_liu@163.com †These authors have contributed equally to this work †These authors have contributed equally to this work Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neurology Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neurology Received: 11 May 2019 Accepted: 25 November 2019 Published: 19 December 2019 December 2019 | Volume 10 | Article 1299 A Network-Based Method for Mechanistic Investigation and Neuroprotective Effect on Post-treatment of Senkyunolid-H Against Cerebral Ischemic Stroke in Mouse Jie Zhang 1†, Yunyao Jiang 2†, Nan Liu 3, Ting Shen 4, Hyo Won Jung 5,6, Jianxun Liu 7* and Bing Chun Yan 1,8,9* Jie Zhang 1†, Yunyao Jiang 2†, Nan Liu 3, Ting Shen 4, Hyo Won Jung 5,6, Jianxun Liu 7* and Bing Chun Yan 1,8,9* Citation: Zhang J, Jiang Y, Liu N, Shen T, Jung HW, Liu J and Yan BC (2019) A Network-Based Method for Mechanistic Investigation and Neuroprotective Effect on Post-treatment of Senkyunolid-H Against Cerebral Ischemic Stroke in Mouse. Front. Neurol. 10:1299. doi: 10.3389/fneur.2019.01299 December 2019 | Volume 10 | Article 1299 Frontiers in Neurology | www.frontiersin.org 1 Zhang et al. Neuroprotection of Senkyunolid-H on Ischemic-Stroke activation of PI3K/Akt/nuclear factor kappa B pathway, and the therapeutic effect of SEH was obviously inhibited by 10 µM LY294002. In summary, these results suggested that SEH carries a therapeutic potential in CIS involving multiple targets and pathways, and the most crucial mechanism might be through the activation of PI3K/Akt/nuclear factor kappa B (NF-κB) signaling pathway to inhibit inflammatory factor releases and increase the antiapoptosis capacity. Our study furnishes the future traditional Chinese medicine research with a network pharmacology framework. Keywords: senkyunolide-H, cerebral ischemic stroke, network pharmacology, PI3K/Akt/NF-κB pathway, neuroprotection Keywords: senkyunolide-H, cerebral ischemic stroke, network pharmacology, PI3K/Akt/NF-κB pathway, neuroprotection INTRODUCTION with Z-ligustilide (9, 13), no published information to date has been carried out to explore the possible mechanisms of SEH on CIS. Stroke is reaching epidemic proportions in the world, marked by high incidence, death, and disability rates (1). Responsible for ∼88% of global stroke subtypes, cerebral ischemic stroke (CIS) occurs in a cerebral vascular obstruction, which causes pathological processes such as metabolic disorders, inflammation, and cellular apoptosis, and leads to severe neurological symptoms (2). Rapid restoration of the cerebral blood flow and neuronal protection are the major therapeutic strategies for CIS (3). Until now, recombinant tissue plasminogen activator is the only thrombolytic drug approved by the US Food and Drug Administration for CIS treatment (4). However, simply 5% of patients benefit from recombinant tissue plasminogen activator therapy owing to the time window limitations (<4.5 h) and fatal side effects of reperfusion injury (5). Because of the huge burden of social and economic diseases, traditional Chinese medicine (TCM) serves as an important replacement or complementary therapy in many countries. As multicomponent and multitarget medicine, TCM basically achieves the therapeutic effect via conjointly regulating the molecular network of body system with its active ingredients (6). In recent years, greater attention has been paid to monomer pharmacological effects as the development of extract technology. Network pharmacology is emerging as a prospective strategy, one that combines systematic methods for the exploration of multichannel signaling pathways regulation (14, 15). Achieving an agent targets network from an overall and comprehensive angle is its greatest advantage (16). In addition, it helps to understand the polypharmacology of a drug, increase drug curative effect and success rate of clinical tests, and reduce the discovery costs. Until now, lots of researches on exploring the molecular mechanisms of TCM formulae and investigating effective components from traditional herbs have been published, such as Tian-Ma-Gou-Teng-Yin against Alzheimer’s disease (17), Tong Sheng tablets against cerebral ischemia reperfusion injury (18), and quercetin for cardiovascular disease treatment (19). Therefore, we are going to investigate the mechanisms of SEH treatment on CIS with the novel network pharmacology program, including herbal target prediction, disease target collection, protein-protein interaction (PPI) network construction, topological feature analysis, and key target functional characterization, and then to validate its therapeutic effect on in vivo and in vitro models of CIS (Figure 2). MATERIALS AND METHODS Senkyunolide-H (SEH, Figure 1A) is one of the major bioactive components of TCM Ligusticum chuanxiong Hort, which is widely used to treat migraine, anemia, and cardiovascular and cerebrovascular diseases in China (7, 8). It can enter the blood and cerebrospinal fluid quickly with its well fat and water solubility. Recent experimental studies have shown that SEH could alleviate neuroinflammatory injury induced by intracerebral hemorrhage and activate endogenous cytoprotective mechanism against oxidative damage in human liver HepG2 cells (8, 9). Intriguingly, SEH and its stereoisomer senkyunolide-I (Figure 1B) are primary metabolite of Z- ligustilide (Figure 1C), which holds antiapoptotic ameliorative effects on focal cerebral ischemia in vitro and in vivo via activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways (10). Furthermore, researches by Yangye Hu suggested that senkyunolide-I exhibited definite antiapoptotic biological activity on cerebral ischemia/reperfusion injury in vivo and anti-inflammatory effects against endotoxin insult in vitro (11, 12). Despite its higher stability and bioavailability compared Frontiers in Neurology | www.frontiersin.org Predicting SEH Potential Targets Predicting SEH Potential Targets The structure of SEH (PubChem CID: 10036567) was downloaded from the NCBI PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and input onto the PharmMapper server (http://lilab.ecust.edu.cn/pharmmapper/) in a MDL sdf. format file, which was designed for identifying possible targets of small molecules via a reverse pharmacophore matching approach (20). The species was limited to “Homo sapiens,” and the first 100 potential targets were ranked according to the fitted scores for subsequent research. Owing to the non-standard naming, we got corresponding official symbols through UniProtKB database (http://www.uniprot.org/), which is the central hub for collecting protein function information with exact, consistent, and abundant annotation (21). Mining Known CIS-Associated Targets CIS-related known targets were mined from the two available databases, OMIM database (Online Mendelian Inheritance in Man, http://www.omim.org) and DisGeNET December 2019 | Volume 10 | Article 1299 Frontiers in Neurology | www.frontiersin.org Frontiers in Neurology | www.frontiersin.org 2 Neuroprotection of Senkyunolid-H on Ischemic-Stroke Zhang et al. FIGURE 1 | Chemical structures of SEH (A), SEI (B), and LIG (C). SEH, senkyunolide-H; SEI, senkyunolide-I; LIG, Z-ligustilide. FIGURE 1 | Chemical structures of SEH (A), SEI (B), and LIG (C). SEH, senkyunolide-H; SEI, senkyunolide-I; LIG, Z-ligustilide. FIGURE 1 | Chemical structures of SEH (A), SEI (B), and LIG (C). SEH, senkyunolide-H; SEI, senkyunolide-I; LIG, Z-ligustilide. FIGURE 1 | Chemical structures of SEH (A), SEI (B), and LIG (C). SEH, senkyunolide-H; SEI, senkyunolide-I; LIG, Z-ligustilide. FIGURE 2 | The flowchart of this study based on an integration strategy of network pharmacology and experimental verification for deciphering pharmacological mechanisms of SEH acting on CIS. SEH, senkyunolide-H; CIS, cerebral ischemic stroke; OMIM, Online Mendelian Inheritance in Man; DisGeNET, a database of gene-disease associations; OGD, oxygen-glucose deprivation; MCA, middle cerebral artery. FIGURE 2 | The flowchart of this study based on an integration strategy of network pharmacology and experimental verification for deciphering pharmacological mechanisms of SEH acting on CIS. SEH, senkyunolide-H; CIS, cerebral ischemic stroke; OMIM, Online Mendelian Inheritance in Man; DisGeNET, a database of gene-disease associations; OGD, oxygen-glucose deprivation; MCA, middle cerebral artery. database (http://www.disgenet.org/). We used “cerebral infarction” and “cerebral ischemic stroke” as the keywords, respectively, for genes searching, respectively (22, 23). is more than two-fold the median degree of all the nodes as big hubs. Constructing PPI Networks and Analyzing Network Topological Features To provide the scientific and reasonable interpretation of the complex relationships between the targets of SEH and CIS, networks construction and analysis were carried out as previously described (24). PPI networks of SEH targets and CIS targets were constructed by Cytoscape3.6.0 software (National Institute of General Medical Sciences, United States) new plugin BisoGenet, which integrates six kinds of PPI databases: the IntAct Molecular Interaction Database (IntAct), Molecular Interaction Database (MINT), the Biological General Repository for the Database of Interacting Proteins (DIP), the Biomolecular Interaction Network Database (BIND), the Human Protein Reference Database (HPRD), and Interaction Datasets (BioGRID). Then, the two networks were merged to acquire important targets. After the network analysis, we chose the nodes the degree of which Predicting SEH Potential Targets Subsequently, the topological features were calculated by degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC) (DC values are twice larger than the median value, BC and CC value larger than the median value of all the network nodes) to screen the putative targets for key targets. Experimental Animals Comparative Medicine Center of Yangzhou University (Yangzhou, China) supplied 8-week-old male Institute of Cancer Research mice (25-30 g), and all mice were domesticated for 7 days at least. The environment was maintained at room temperature of 23◦C, humidity of 60%, and adequate free forage and water. To simulate a normal growing environment, we made a light-dark cycle that changes every 12 h. All experimental procedures were carried out in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Yangzhou University-Institutional Animal Care and Use Committee (YIACUC-14-0015) based on ethical procedures and scientific nursing. Gene Ontology and Pathway Enrichment Analysis y The Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/) were applied for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of key targets. This involves a two-step operation: first, the ID of gene was inputted and the species was specified as “Homo sapiens;” second, enrichment analysis was executed using functional annotation tool. Three GO enrichment categories of biological process, molecular function, and cellular component were selected to December 2019 | Volume 10 | Article 1299 Frontiers in Neurology | www.frontiersin.org 3 Neuroprotection of Senkyunolid-H on Ischemic-Stroke Zhang et al. draw bar charts. A bubble chart of top 20 pathways was made by OmicShare platform (http://www.omicshare.com/). draw bar charts. A bubble chart of top 20 pathways was made by OmicShare platform (http://www.omicshare.com/). lateral thrust, but no circling; and level 3, poor resistance to lateral thrust with circling. Blinded assessment was conducted at a specified time. The animal model is built successfully if the scores are greater than or equal to level 1. Groups and Drug Administration According to previous studies (11, 25, 26), mice were randomly divided into the following groups (n = 14 in each group): sham operation (sham group); sham operation with 40 mg/kg SEH treatment (sham-SEH group); MCAO treatment (MCAO group); MCAO with 20 mg/kg SEH treatment (20 SEH group); and MCAO with 40 mg/kg SEH treatment (40 SEH group). SEH was administered intragastrically at the start and end of ischemic surgery immediately. Equal volume of 0.9% saline was intragastrically injected into two groups of mice (sham and MCAO). Mice were killed at 6 and 24 h after reperfusion. SEH (Shanghai Standard technology Co., Ltd, Shanghai, China) was dissolved in 0.9% saline and 0.1% dimethyl sulfoxide, and the purity was above 98.32%. Immunohistochemistry was conducted according to our formerly published program (29). The slices were first treated with 0.3% hydrogen peroxide (H2O2) diluted in PBS for 20 min and 5% normal serum diluted in 0.01 M PBS for 30 min. Then, the slices were incubated successively with diluted rabbit antineuronal nuclei (anti-NeuN) (1:1,000, Cell Signaling Technology) overnight at 4◦C, biotinylated goat antirabbit IgG (1:250, Vector, Burlingame, CA), and streptavidin peroxidase complex (1:200, Vector). After that, the tissues were dyed with 3,3′-diaminobenzidine tetrahydrochloride in 0.01 M PBS and dehydrated on the adhesion microscope slides. Finally, we used neutral gum (Solarbio, Beijing, China) sealing piece. Digital images of CA1 region were observed with a computer-based microscope (Nikon, Chiyoda-Ku, Tokyo, Japan), which is equipped with an image-analyzing system. Cell counts were gained through averaging the counts from the sections of each animal. The staining intensity of NeuN immunoreactive structures was assessed on the basis of an optical density (OD), which was obtained after the transformation of the mean gray Tissue Processing for Histology We collected the tissues according to the method described in the previous study (29), and 10% chloral hydrate (Aladdin, China) was used to maintain anesthesia in the mice; subsequently, 0.1 M phosphate-buffered saline (PBS, pH 7.4) and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) were perfused transcardially. The brains of mice were removed in the same fixative solution and fixed for 4 h and cryoprotected by 30% sucrose infiltration overnight. Thereafter, frozen tissues were continuously sliced into 30-µm coronal sections via a cryostat (Leica, Wetzlar, Germany) and then collected into six porous plates containing PBS. Transient Cerebral Ischemia Model Transient Cerebral Ischemia Model Isoflurane (3-4%) (RWD Life Science, Guangdong Province, China) in 30% oxygen and 70% nitrous oxide was used to anesthetize the animal. Then, the right common carotid artery, vagus nerve, external carotid artery, and internal carotid artery were exposed and separated carefully after a median incision of the neck skin. A monofilament nylon filament was inserted into the internal carotid artery through an incision of external carotid artery until the middle cerebral artery (MCA) occluded and taken out to commence reperfusion after 45 min infraction. Sham-operated groups were given the same surgery without inserting the filament. The whole surgery would maintain the body temperature of mice at 37.0-37.5◦C by a rectal thermometer connected to an automated heating pad [i.e., transient MCA occlusion (MCAO)/reperfusion was induced by a filament insertion/removal in the carotid artery]. Thereafter, the mice were incubated in the thermal incubator to maintain the body temperature up to euthanasia. TTC Staining and Quantifying Infarct Volume The procedure is following that described above (28). The mice were killed with 10% chloral hydrate (Aladdin, Shanghai, China), and decapitation was performed immediately after full reperfusion (1 day). We took the brain very carefully, and then, the brain was weighed and cut into 2-mm thick slices. Two percent 2,3,5-triphenyl tetrazolium chloride (Sigma-Aldrich, St. Louis, MO) was used for staining for half an hour under dark condition. After staining, we transferred the brain slices into 4% formalin overnight. The availability of the slice was determined by the slice color; red color of the brain slice was available, while white color of the brain slice was inactivated. Image J software was used for measuring the infarct volume and whole area volume of brain slice. The infarct volume was calculated by multiplying the increased infarct size per slice by slice thickness (2 mm). The results were shown as (infarct volume/whole brain volume) × 100%. Neurological Score The neurological function of mice was assessed in accordance with the Bederson scale before killing. Four levels were divided in Bederson scale (27): level 0, no significant changes; grade 1, abnormal bending of the forelimbs; level 2, poor resistance to December 2019 | Volume 10 | Article 1299 Frontiers in Neurology | www.frontiersin.org 4 Neuroprotection of Senkyunolid-H on Ischemic-Stroke Zhang et al. modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) included penicillin (100 U/ml), 5% heat-inactivated fetal bovine serum (Gibco), streptomycin (100 mg/ml), and 10% horse serum (Gibco). To simulate the model of cerebral ischemia in vitro, cells were incubated in the serum/glucose-free DMEM after being seeded overnight and washed twice with PBS (pH 7.2) and then transferred to a hypoxic chamber (Thermo Fisher, Waltham, MA) containing 95% N2 and 5% CO2 for 6 h. After oxygen-glucose deprivation (OGD) exposure, the cells were incubated with conditioned DMEM at 37◦C in a damp atmosphere containing 5% CO2 for 24 h reperfusion. In addition, the cells were divided into different groups: the sham group, the OGD group, the OGD + SEH groups (25, 50, and 100 µM, respectively), and the OGD + SEH + LY294002 (10 µM) groups. The different concentrations of SEH and level using the formula: OD = log (256/mean gray level). The OD of the background was taken from areas near the measured area. After the background density was subtracted, a ratio of the OD of image file was calibrated as percent [relative OD (ROD)] through Adobe Photoshop version 8.0 and then analyzed by NIH Image 1.59 software. We normalized each sample against the level of vehicle-sham sample. All measurements were performed under the same conditions by two observers in blind conditions to ensure objectivity. PC12 Cell Culture and OGD Model PC12 cell line (rat pheochromocytoma) was purchased from the American Type Culture Collection, seeded at 1 × 106 cells/well in six-well plates and cultured at 37◦C in a damp atmosphere containing 5% CO2. Cells were grown in Dulbecco’s FIGURE 3 | The PPI networks construction and analysis for SEH against CIS. (A) PPI networks of SEH-related targets and CIS targets. (B) The interactive PPI network of SEH and CIS targets gained from (A). (C) The PPI network of key targets of SEH acting on CIS obtained from (B). Pink square node in (A,B) represent direct targets of SEH and CIS. Blue nodes represent indirect targets of SEH and CIS. Pink circular nodes in (C) represent key targets of SEH acting on CIS, while its size is proportional to the significance. Edges represent interactions among SEH and CIS targets. PPI, protein-protein interaction; SEH, senkyunolide-H; CIS, cerebral ischemic stroke. FIGURE 3 | The PPI networks construction and analysis for SEH against CIS. (A) PPI networks of SEH-related targets and CIS targets. (B) The interactive PPI network of SEH and CIS targets gained from (A). (C) The PPI network of key targets of SEH acting on CIS obtained from (B). Pink square node in (A,B) represent direct targets of SEH and CIS. Blue nodes represent indirect targets of SEH and CIS. Pink circular nodes in (C) represent key targets of SEH acting on CIS, while its size is proportional to the significance. Edges represent interactions among SEH and CIS targets. PPI, protein-protein interaction; SEH, senkyunolide-H; CIS, cerebral ischemic stroke. FIGURE 3 | The PPI networks construction and analysis for SEH against CIS. (A) PPI networks of SEH-related targets and CIS targets. (B) The interactive PPI network of SEH and CIS targets gained from (A). (C) The PPI network of key targets of SEH acting on CIS obtained from (B). Pink square node in (A,B) represent direct targets of SEH and CIS. Blue nodes represent indirect targets of SEH and CIS. Pink circular nodes in (C) represent key targets of SEH acting on CIS, while its size is proportional to the significance. Edges represent interactions among SEH and CIS targets. PPI, protein-protein interaction; SEH, senkyunolide-H; CIS, cerebral ischemic stroke. December 2019 | Volume 10 | Article 1299 5 Frontiers in Neurology | www.frontiersin.org Neuroprotection of Senkyunolid-H on Ischemic-Stroke Zhang et al. PC12 Cell Culture and OGD Model LY294002 (MCE MedChem Express, Monmouth, NJ) were added throughout the OGD-reperfusion (OGD/R) treatment within the standard medium. The vehicle group was subjected to the same experimental procedures without exposure to the serum/glucose-free DMEM medium and any drugs. Then, the cells were collected for Western blotting detection. Nanjing, China)/Total Protein Extraction Kit and measured the protein concentration with a Pierce BCA Protein Assay Kit (Thermo Scientific, USA). The same amount of protein (40 µg) was separated by suitable percentage sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, Bedford, USA). For a clear band, the membranes were cultured in Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20 for 60 min, then incubated in primary antibody overnight at 4◦C, and followed in the corresponding secondary antibody at room temperature for 2 h. Protein expression was detected by SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, USA). CCK8 Cell Viability Assay Cell Counting Kit-8 was used for measuring cell viability (CCK8; Dojindo Laboratories, Kumamoto, Japan). Cells were seeded into a 96-well plate at a density of 6 × 103 cells/100 µl. After stabilizing, cells were exposed to OGD/R in the absence or presence of indicated concentrations of SEH and LY294002 treatment. After 24-h reperfusion, 10 µl of CCK8 solution was included to each hole (100 µl medium), incubated at 37◦C for 2 h, and then, the absorbance was measured at 450 nm in a multimode plate reader (Espier, PerkinElmer, Singapore). Densitometric analysis on all scanned Western blot results was performed using Quantity One Analysis Software (Bio-Rad). Relative OD (ROD) calculations are mainly followed: the ratio of the calibrated ROD is expressed in percentage, and the sham group is defined as 100%. Three similar independent experiments were represented by each blot results at least. The primary and secondary antibodies were listed as follows: rabbit anti-PI3K (1:1,000, Cell Signaling Technology) and p-PI3K (1:1,000, Cell Signaling Technology), rabbit anti-Akt (1:1,000, Cell Signaling Technology) and p-akt (1:1,000, Cell Signaling Technology), rabbit anti-NF-κB (1:1,000, Abcam), rabbit anti-Bcl-xL (1:1,000, Cell Signaling Technology), rabbit anticleaved caspase 3 (1:1,000, Western Blot Analysis Western blot experiment was carried out on the basis of a procedure published before (28). The mice brains (n = 7 in each group) were cut into 400-µm thickness serially and transversely on a vibratome, and the hippocampus was carved with a scalpel. We preprocessed the tissues and the whole cell lysates from PC12 cell by Whole Cell Lysis Assay kit (KeyGEN, FIGURE 4 | GO analysis of candidate targets. The top 10 terms for biological processes, molecular function, and top 6 terms for cell component with p < 0.001 are shown. X-axis indicates p-values of GO enrichment for each subcategory. FIGURE 4 | GO analysis of candidate targets. The top 10 terms for biological processes, molecular function, and top 6 terms for cell component with p < 0.001 are shown. X-axis indicates p-values of GO enrichment for each subcategory. December 2019 | Volume 10 | Article 1299 Frontiers in Neurology | www.frontiersin.org 6 Neuroprotection of Senkyunolid-H on Ischemic-Stroke Zhang et al. Cell Signaling Technology), anti-β-actin (1:3,000, Abcam), and goat antirabbit IgG (Santa Cruz, USA). Cell Signaling Technology), anti-β-actin (1:3,000, Abcam), and goat antirabbit IgG (Santa Cruz, USA). detailed information after filtered overlapping protein targets from the above two available resources is described in Supplementary Table 2. PPI Networks Construction and Network Topological Parameters Analysis The data shown in the study represent the means ± SD and were plotted in histograms with GraphPad Prism 7.0. Differences in average values among groups were analyzed through one- way analysis of variance test by SPSS software. p < 0.05 was considered to have statistical significance. As the charts show (Figure 3A, Supplementary Tables 3, 4), SEH targets PPI network, and CIS-related target PPI network was constructed by BisoGenet to find out the genes with direct or indirect effects. There were 639 nodes and 7,724 edges in the SEH targets network. Meanwhile, network of CIS- related targets had 3,605 nodes and 89,245 edges. To reveal the relationship between SEH and CIS, a new network (Figure 3B) was merged from the two networks (Figure 3A) with the merge function of Cytoscape3.6.0, which included 352 overlapping targets and 4,754 edges. The common targets were not only the important goal of SEH treatment in CIS but also the vital targets screened for further research. Subsequently, the topological feature values of common targets in the network (Figure 3B, Supplementary Table 5) including DC, BC, and SEH Potential Targets Prediction and CIS-Associated Targets Collection g A total of 11 potential targets were derived and screened with a fit score value >4.5 from the top 100 potential human protein targets of SEH through PharmMapper server. Detailed information is described in Supplementary Table 1. By means of OMIM and DisGeNET databases, we obtained 11 and 130 ischemic-stroke-related targets, respectively. A A total of 11 potential targets were derived and screened with a fit score value >4.5 from the top 100 potential human protein targets of SEH through PharmMapper server. FIGURE 5 | The top 20 terms for KEGG signaling pathway enrichment of major targets. “Rich factor” indicates the ratio of the number of target genes belonging to a pathway and the number of the annotated genes located in the pathway. A higher rich factor reflects a higher level of enrichment. The size of the dot is proportional to FIGURE 5 | The top 20 terms for KEGG signaling pathway enrichment of major targets. “Rich factor” indicates the ratio of the number of target genes belonging to a pathway and the number of the annotated genes located in the pathway. A higher rich factor reflects a higher level of enrichment. The size of the dot is proportional to the number of key targets in the pathway, and the color of the dot refers to the p-value. FIGURE 5 | The top 20 terms for KEGG signaling pathway enrichment of major targets. “Rich factor” indicates the ratio of the number of target genes belonging to a pathway and the number of the annotated genes located in the pathway. A higher rich factor reflects a higher level of enrichment. The size of the dot is proportional to the number of key targets in the pathway, and the color of the dot refers to the p-value. December 2019 | Volume 10 | Article 1299 7 Frontiers in Neurology | www.frontiersin.org Neuroprotection of Senkyunolid-H on Ischemic-Stroke Zhang et al. CC were analyzed for the important key protein targets. Finally, a total of 62 nodes, which DC > 44, BC > 0.001, and CC > 0.470, were chosen as the key targets (Figure 3C, Supplementary Table 5). processes were related to the regulations of RNA polymerase II promoter transcription, DNA-templated transcription, NF-κB transcription factor activity, protein autophosphorylation, and signaling pathways of PI3K, ErbB2, epidermal growth factor receptor, and phosphatidylinositol mediated. GO Enrichment Analysis of Candidate Targets GO analysis of 62 candidate targets for SEH treating on CIS was performed via the DAVID database to investigate their functions and inherent significance in the biological system networks. The diagram (Figure 4, Supplementary Table 6) contained three parts, which were biological process, cellular component, and molecular function. We found that biological FIGURE 6 | The neuroprotective effects of SEH. (A) 2,3,5-Triphenyl tetrazolium chloride (TTC) staining in the sham, sham + SEH, MCAO, 20 and 40 mg/kg (intragastric administered) SEH-MCAO groups. (B) The infarct volume was expressed as the ratio of (infarct volume/the whole brain volume) × 100% (n = 7 per group; *p < 0.05: significantly different from the sham group; #p < 0.05: significantly different from the MCAO group). Bars indicate mean ± SD. (C) Bederson neurological score in the sham, MCAO, and 20 and 40 mg/kg (intragastric administered) SEH-MCAO groups (n = 7 per group; *p < 0.05: significantly different from the sham group; #p < 0.05: significantly different from the MCAO group). Bars indicate mean ± SD. (D) Immunohistochemistry for NeuN in the hippocampus CA1 region of the sham, MCAO, 40 mg/kg intragastric administered SEH-sham, and 40 mg/kg intragastric administered SEH-MCAO groups. (E) The ratio of NeuN immunoreactivity. Scale bar = 100 µm (n = 7 per group; *p < 0.05: significantly different from the corresponding sham groups; #p < 0.05: significantly different from the corresponding vehicle-MCAO groups; &p < 0.05: significantly different from the corresponding 6 h-MCAO groups). Bars indicate mean ± SD. FIGURE 6 | The neuroprotective effects of SEH. (A) 2,3,5-Triphenyl tetrazolium chloride (TTC) staining in the sham, sham + SEH, MCAO, 20 and 40 mg/kg (intragastric administered) SEH-MCAO groups. (B) The infarct volume was expressed as the ratio of (infarct volume/the whole brain volume) × 100% (n = 7 per group; *p < 0.05: significantly different from the sham group; #p < 0.05: significantly different from the MCAO group). Bars indicate mean ± SD. (C) Bederson neurological score in the sham, MCAO, and 20 and 40 mg/kg (intragastric administered) SEH-MCAO groups (n = 7 per group; *p < 0.05: significantly different from the sham group; #p < 0.05: significantly different from the MCAO group). Bars indicate mean ± SD. (D) Immunohistochemistry for NeuN in the hippocampus CA1 region of the sham, MCAO, 40 mg/kg intragastric administered SEH-sham, and 40 mg/kg intragastric administered SEH-MCAO groups. (E) The ratio of NeuN immunoreactivity. SEH Potential Targets Prediction and CIS-Associated Targets Collection The molecular function was related to the bindings of transcription factor, damaged DNA, protein phosphatase, transcription regulatory region DNA, ephrin receptor, ATP and insulin receptor, and activities of nitric oxide synthase regulator, kinase, protein tyrosine kinase, and cell proliferation. Finally, the cellular component was related to nuclear chromatin, transcription factor complex, receptor complex, cell-cell adherens junction, nuclear body, and basolateral plasma membrane. KEGG Pathway Enrichment Analysis for Key Targets function was 3) (Figure 6C). In addition, neuronal death in the hippocampus was observed by immunohistochemistry of NeuN. Most immunoreactions of NeuN cells were lost in the CA1 region of hippocampal in the reperfusion at 6 and 24 h after MCAO; however, in the 40 mg/kg SEH-MCAO group, many survival neurons were found in the CA1 region of hippocampal compared with that in the MCAO group (Figures 6D,E).These results indicated that SEH treatment reduced the infarct volume and improved neurological deficits after cerebral IR in mice, and the neuroprotection of SEH was especially embodied in the 40 mg/kg SEH-MCAO group. All 62 key targets were significantly enriched onto 49 pathways with the adjusted p < 0.01 by means of DAVID database (Supplementary Table 7). Top 20 KEGG pathways were picked and constructed in bubble diagram on the basis of p-value and number of key targets included. The picture (Figure 5) indicated the mechanisms that might be concerned with PI3K/Akt, ErbB, neurotrophin, FOXO, and estrogen signaling pathways. Detailed information was described in Supplementary Table 7. GO Enrichment Analysis of Candidate Targets Scale bar = 100 µm (n = 7 per group; *p < 0.05: significantly different from the corresponding sham groups; #p < 0.05: significantly different from the corresponding vehicle-MCAO groups; &p < 0.05: significantly different from the corresponding 6 h-MCAO groups). Bars indicate mean ± SD. December 2019 | Volume 10 | Article 1299 8 Frontiers in Neurology | www.frontiersin.org Zhang et al. Neuroprotection of Senkyunolid-H on Ischemic-Stroke FIGURE 7 | Effect of SEH treatment on changes of PI3K/Akt/NF-κB signaling pathway and antiapoptotic levels in the hippocampus region. (A) Protein bands of PI3K, p-PI3K, Akt, p-Akt, NF-κB, and Bcl-XL in each group. (B) The ratio of p-PI3K/PI3K. (C) The ratio of p-Akt/Akt. (D) NF-κB expression. (E) Bcl-XL expression. Relative optical density (ROD) as a percentage of the immunoblot band is presented (n = 7 per group; *p < 0.05: significantly different from the corresponding sham group; #p < 0.05: significantly different from the corresponding vehicle-MCAO group). Bars indicate mean ± SD. FIGURE 7 | Effect of SEH treatment on changes of PI3K/Akt/NF-κB signaling pathway and antiapoptotic levels in the hippocampus region. (A) Protein bands of PI3K, p-PI3K, Akt, p-Akt, NF-κB, and Bcl-XL in each group. (B) The ratio of p-PI3K/PI3K. (C) The ratio of p-Akt/Akt. (D) NF-κB expression. (E) Bcl-XL expression. Relative optical density (ROD) as a percentage of the immunoblot band is presented (n = 7 per group; *p < 0.05: significantly different from the corresponding sham group; #p < 0.05: significantly different from the corresponding vehicle-MCAO group). Bars indicate mean ± SD. Frontiers in Neurology | www.frontiersin.org Role of PI3K/Akt/NF-κB Signaling-Related Protein Levels in MCAO Mice After SEH Treatment (A) The cell viability was measured by CCK-8 assay in the PC12 cells after OGD/R. (B) Protein bands of cleaved caspase 3 and Bcl-XL in each group. (C) Cleaved caspase 3 expression. (D) Bcl-XL expression. (E) Protein bands of PI3K, p-PI3K, Akt, p-Akt, and NF-κB in each group. (F) The ratio of p-PI3K/PI3K. (G) The ratio of p-Akt/Akt. (H) NF-κB expression. Relative optical density (ROD) as a percentage of the immunoblot band is presented (n = 7 per group; *p < 0.05: significantly different from the sham group; #p < 0.05: significantly different from the OGD group; $p < 0.05: significantly different from the corresponding SEH treatment group). Bars indicate mean ± SD. SEH treatment protected against IR injury was related to the PI3K/Akt/NF-κB signaling pathway activation. SEH treatment protected against IR injury was related to the PI3K/Akt/NF-κB signaling pathway activation. compared with the sham group but gradually increased in the SEH-treated OGD/R groups with dose-dependent SEH concentration. In the groups of different SEH concentrations with 10 µM LY294002, the cell viability was significantly reduced compared with the only SEH treatment group. In addition, the levels of cleaved caspase 3 were markedly upregulated by OGD/R and downregulated by OGD/R with 100 µM SEH treatment. However, in the group including 10 µM LY294002 treatment, the level of cleaved caspase 3 was similar to that of only OGD/R treatment group (Figures 8B,C). Furthermore, we observed that the Bcl-XL expressions were opposite to the cleaved caspase 3 expressions in the PC12 cells under OGD/R. Besides, the ratios of p-PI3K/PI3K and p-Akt/Akt, as well as the ROD of Bcl-XL, were significantly increased in the group treated with Role of PI3K/Akt/NF-κB Signaling-Related Protein Levels in MCAO Mice After SEH Treatment The staining 2,3,5-triphenyl tetrazolium chloride was frequently used for the evaluation of infarct volume after brain ischemia. As shown in Figures 6A,B, in the 20 mg/kg SEH treatment ischemia group, the sizes of infarct regions were ∼23% of whole brain volume and markedly lower than those of MCAO group (accounting for 43% of whole brain volume). Meanwhile, only few infarct sizes (∼9% of the brain volume) were observed in the treatment of 40 mg/kg SEH ischemia group. Besides, we evaluated the neurological function of mice according to the Bederson scale (0-3) before killing. We observed that mice treated with 40 mg/kg SEH obtained better scores (the score of neurological function was 1), which was much lower than that in the MCAO group (the score of neurological To verify the mechanism by which SEH improved nerve damage after ischemic brain injury, Western blot analysis was used to measure relative protein expression levels. As shown in Figure 7, the PI3K expression was obviously decreased, and the p-PI3K level increased in the hippocampus region of the SEH treatment groups than that of the MCAO groups. Variation in the p-Akt/Akt protein ratio was consistent with the change in the p-PI3K/PI3K protein ratio. Yet, the SEH treatment groups exhibited markedly reduced expression of NF-κB while enhanced expression of Bcl-XL. The above data suggested that December 2019 | Volume 10 | Article 1299 Frontiers in Neurology | www.frontiersin.org 9 Zhang et al. Neuroprotection of Senkyunolid-H on Ischemic-Stroke FIGURE 8 | Effect of SEH and LY294002 treatment on cell viability, apoptotic levels, and PI3K/Akt/NF-κB signaling pathway protein of OGD/R-injured PC12 cells. (A) The cell viability was measured by CCK-8 assay in the PC12 cells after OGD/R. (B) Protein bands of cleaved caspase 3 and Bcl-XL in each group. (C) Cleaved caspase 3 expression. (D) Bcl-XL expression. (E) Protein bands of PI3K, p-PI3K, Akt, p-Akt, and NF-κB in each group. (F) The ratio of p-PI3K/PI3K. (G) The ratio of p-Akt/Akt. (H) NF-κB expression. Relative optical density (ROD) as a percentage of the immunoblot band is presented (n = 7 per group; *p < 0.05: significantly different from the sham group; #p < 0.05: significantly different from the OGD group; $p < 0.05: significantly different from the corresponding SEH treatment group). Bars indicate mean ± SD. FIGURE 8 | Effect of SEH and LY294002 treatment on cell viability, apoptotic levels, and PI3K/Akt/NF-κB signaling pathway protein of OGD/R-injured PC12 cells. DISCUSSION Ischemic stroke is a main cause of death and disability and a common central nervous system disease with poor prognosis. SEH is a bioactive compound extracted from the TCM Ligusticum chuanxiong Hort. In clinic, the treated duration of Ligusticum chuanxiong Hort is usually decided by different diseases and the patient’s condition. Previous studies reported that 14-60 days for chuanxiong intervention were used for treatment of acute ischemic stroke by hundreds of clinical studies (30). Meanwhile, the meta-analysis suggested that the Ligustrazine (a bioactive component contained in chuanxiong) treatment of 10 days−12 weeks had a significant therapeutic effect on diabetic nephropathy (31). In addition, we found that SEH could be used for many applications. Published researches suggested SEH as a new lead for the development of new antiatherosclerotic drug. For example, SEH showed strong antiproliferative activity in primary culture of mouse aorta smooth muscle cells (32). Recent studies indicated that the absorption of SEH was significantly increased in migrainous rats compared with normal rats (33), and SEH has shown potent neuroprotective effects on intracerebral hemorrhage (9). According to the latest research, SEH could attenuate MPP+-induced neurotoxicity and apoptosis in PC12 cells (34). However, as far as we know, the mechanisms responsible for the SEH function in CIS are not known. To elucidate the mechanism of SEH in CIS, we applied network pharmacology to have a comprehensive and systematic research. A total of 62 key targets of SEH against CIS were obtained and analyzed. GO and KEGG analysis suggested that multiple signaling pathways were involved in the potential mechanisms of SEH. Besides, we confirmed the therapeutic effects of SEH in the mouse MCAO model and PC12 cell OGD/R model. In KEGG analysis, PI3K/Akt, ErbB, neurotrophin, and FOXO signaling pathways were significantly enriched, and PI3K/Akt signaling pathway may be the most crucial mechanism. The PI3K/Akt pathway is the key to ErbB signal transduction, which controls protein homeostasis through boosting migration, angiogenesis, and cell proliferation. Zhou’s data showed that reactive oxygen species eliminated the inhibitory effect of PI3K/Akt signaling pathway on ERK activity during reperfusion, and the strong activation of ERK activity played a pivotal role in cell injury induced by reperfusion (42). In addition, nerve growth factor (NGF) is a member of the neurotrophin family, and NGF/tropomyosin-related kinase A signaling is related to neuronal survival, function, and differentiation (43). Cell Viability and the Protein Levels of Cleaved Caspase3, Bcl-XL, PI3K/Akt/NF-κB Signaling of OGD/R-Injured PC12 Cells After SEH and LY294002 Treatment The possible neuroprotective mechanisms of SEH on OGD/R injured PC12 cells were determined via the CCK8 assay and the Western blot analyses of apoptosis-related proteins and PI3K/Akt/NF-κB signaling expressions. As shown in Figure 8A, cell viability was obviously decreased in the OGD group December 2019 | Volume 10 | Article 1299 Frontiers in Neurology | www.frontiersin.org 10 Neuroprotection of Senkyunolid-H on Ischemic-Stroke Zhang et al. that polymorphism (rs145204276) in the promoter of long non-coding RNA growth arrest-specific 5 had a significant association with an increased CIS susceptibility by elevating the transcriptional activity of GAS530197169. EGFR signaling pathway plays critical roles in promoting neurogenesis (36). Autophosphorylation of EGFR activates ERK/MAPK signaling pathway and subsequently leads to cell proliferation and DNA synthesis (37). In Chen’s study, astragaloside VI could initiate EGFR/MAPK signaling cascades effectively, accelerate the proliferation of neural stem cells, strengthen motor function, and ameliorate the abilities of learning and memory in rats with transient cerebral ischemic (38). Besides, molecular functions mainly involve transcription factor binding, nitric oxide synthase regulator activity, protein phosphatase binding, and so on. Transcription factor ATF-3 is an appropriate marker for neurons injured by ischemia, and the synergistic effect of ATF-3 and c- Jun may be the key trigger factor for various transcriptional responses to ischemic injury (39). Protein phosphatase binding is closely related to CIS, for example, the insulin-like growth factor 1 receptor often facilitates cell survival, cell proliferation, metabolism, and stress resistance triggered by the tyrosine autophosphorylation of its β chains (40). In addition, our previous findings indicated that insulin-like growth factor 1 receptor may be an alternative target for preventing the cerebral ischemic injury (41). 100 µM SEH compared with the OGD group. However, these increases were inhibited by 10 µM LY294002 (Figures 8D–G). Moreover, the level of NF-κB was significantly upregulated by OGD/R treatment, which was blunted by 100 µM SEH treatment. After the treatment with 10 µM LY294002, the level of NF-κB was increased compared with the only SEH treatment group (Figure 8H). Together with the abovementioned data, these results suggested that a dose of 100 µM SEH is sufficient to markedly decrease OGD/R injury and that the mechanism of SEH neuroprotective effect on OGD/R-injured PC12 cells is related to the activation of the PI3K/Akt/NF-κB pathway and inhibited by LY294002. Frontiers in Neurology | www.frontiersin.org DISCUSSION Besides, LY294002, as a PI3K inhibitor that is widely used for the study of the characteristics of PI3K cell signaling pathways (51–53), inhibited the expressions of PI3K/Akt/NF-κB pathway and blocked the function of SEH treatment on PC12 cells injured by OGD/R. For instance, published study indicated that ginkgolides protected against ischemia-reperfusion damage in vivo and in vitro through the activation of Nrf2 and CREB via PI3K/Akt signaling, which could be reversed by cotreatment with LY294002 (54). At the same time, LY294002 is also a nonspecific inhibitor that may affect lots of other pathways (55), suggesting that, in addition to the PI3K/Akt pathway, SEH may also activate other pathways to exert therapeutic effects. For example, some studies reported that the neuroprotective effect of SEH was related to the MAPKs pathways (30) and Prx1/TLR4/NF-kB pathway (9). Therefore, our data also verified that SEH treatment could restrain neuronal apoptosis induced by IR through the activation of PI3K/Akt/NF-κB signaling pathway. each of the key targets, and targets that may be of most interest for the SEH treatment of CIS were not identified yet. Hence, future studies will focus more on providing insight into the specific cellular and molecular mechanisms of SEH therapeutic effect on CIS. ACKNOWLEDGMENTS We very much wish to thank all the individuals and families who generously participated in this research. We appreciate Project No. 81803772 supported by National Natural Science Foundation of China, National Basic Research Program of China (973 Program, 2015 CB554400), Yangzhou University International Academic Exchange Fund, Yangzhou University Graduate Training and Innovation Project (XKYCX19_155) and the University Natural Science Research major Project of Jiangsu Province (16KJA310006), Huai’an 2017 Annual Promotion Project for Science and Technology International Cooperation (HAC201702). To sum up, we explored multiple targets and pathways of SEH treatment against CIS through a network pharmacology approach and confirmed the therapeutic effects of SEH in vivo and in vitro. Our data indicated that SEH treatment on CIS may be through the activation of PI3K/Akt//NF-κB signaling pathway to inhibit inflammatory factor releases and increase the antiapoptosis capacity. However, owing to the incomplete information in these databases, some targets of SEH or CIS may be ignored and missed during the screening process. In addition, proteins and messenger RNAs validation was not conducted for SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fneur. 2019.01299/full#supplementary-material AUTHOR CONTRIBUTIONS JZ and YJ performed the experiments and drafted the manuscript. YJ, NL, TS, HJ, and JL analyzed the data. BY designed the experiments. All the authors discussed the results, reviewed the final manuscript, and approved it for the publication. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. ETHICS STATEMENT All experimental procedures were performed according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved based on ethical procedures and scientific care by the Yangzhou University-Institutional Animal Care and Use Committee (YIACUC-14-0015). 5. Grossman AW, Broderick JP. Advances and challenges in treatment and prevention of ischemic stroke. Annals Neurol. (2013) 74:363-72. doi: 10.1002/ana.23993 DISCUSSION Recent study showed that NGF-enhanced angiogenesis contributed to neurological functional recovery after ischemic stroke via the initiation of PI3K/Akt signaling pathway (44). Moreover, PI3K/Akt cascade inhibits the FOXO pathway for regulation of autophagy, metabolism, and oxidative stress. Previous research implicated that FOXO-4 could trigger apoptotic responses or cell cycle arrest via downregulation of Akt, which might be neuroprotective to drive the cells into a state of quiescence during a situation of reduced energy (45). Through the GO enrichment analysis of the predictive targets of SEH in the treatment of CIS, functions of the key targets and the related pathways information were obtained. It has notable significance for the biological processes such as regulation of transcription from RNA polymerase II promoter and phosphatidylinositol-mediated and epidermal growth factor receptor (EGFR) signaling pathways. Gene transcription regulation is a vital process in CIS. It is coordinated by transcription factors and other proteins and regulates gene functions via a variety of mechanisms, for instance, neurogenesis, inflammation, and angiogenesis (35). Recent study showed Lastly, our findings revealed that treatment of MCAO mice with 40 mg/kg SEH, as well as the treatment of OGD PC12 cells with 100 µM SEH, markedly increased p-PI3K, p-Akt, and Bcl-XL protein levels and decreased NF-κB protein expression. Meanwhile, the treatment of OGD/R-injured PC12 cells with 100 µM SEH also reduced cleaved caspase 3 protein expression, and the neuroprotective function of SEH was inhibited by 10 µM LY294002. These data indicated that SEH stimulated PI3K/Akt signaling pathway and inhibited NF-κB signaling pathway in CIS. Currently, it is reported that PI3K/Akt pathway plays a great role December 2019 | Volume 10 | Article 1299 11 Neuroprotection of Senkyunolid-H on Ischemic-Stroke Zhang et al. in the regulation of cell growth and neurons survival after brain ischemia, demonstrating that pharmacological upregulation of PI3K/Akt signaling could have therapeutic potential for the brain damage (46). Meanwhile, the activation of Akt can promote neuronal proliferation and survival through controlling multiple intracellular signals (47). As a downstream gene of Akt, NF-κB (p65) can be activated by phosphorylated Akt to phosphorylated p65, which causes NF-κB to enter the nucleus, and induces inflammatory response and apoptosis. A previous study has suggested that the NF-κB subunit activation leads to the response of lymphokine-6 and tumor necrosis factor α, which indicated that repressing NF-κB-induced neuroinflammation enhances functional outcomes and alleviates ischemic brain injury (48). 4. Kandadai MA, Meunier JM, Hart K, Holland CK, Shaw GJ. Plasmin-loaded echogenic liposomes for ultrasound-mediated thrombolysis. Transl Stroke Res. (2015) 6:78-87. doi: 10.1007/s12975-014-0376-4 3. Zhou Z, Lu J, Liu WW, Manaenko A, Hou X, Mei Q, et al. Advances in stroke pharmacology. Pharmacol Therap. (2018) 191:23-42. doi: 10.1016/j.pharmthera.2018.05.012 DISCUSSION Furthermore, recent studies have shown that inhibiting NF- κB could improve the prognosis of stroke, and the inhibitors of NF-κB activation may be possible targets for treatment (49, 50). In addition, Lv’s data indicated that CXCL8 gene silencing accelerated the activation of neuroglial cells while suppressing neuroinflammation via the PI3K/Akt/NF-κB pathway in the mice of CIS (50). Besides, LY294002, as a PI3K inhibitor that is widely used for the study of the characteristics of PI3K cell signaling pathways (51–53), inhibited the expressions of PI3K/Akt/NF-κB pathway and blocked the function of SEH treatment on PC12 cells injured by OGD/R. For instance, published study indicated that ginkgolides protected against ischemia-reperfusion damage in vivo and in vitro through the activation of Nrf2 and CREB via PI3K/Akt signaling, which could be reversed by cotreatment with LY294002 (54). At the same time, LY294002 is also a nonspecific inhibitor that may affect lots of other pathways (55), suggesting that, in addition to the PI3K/Akt pathway, SEH may also activate other pathways to exert therapeutic effects. For example, some studies reported that the neuroprotective effect of SEH was related to the MAPKs pathways (30) and Prx1/TLR4/NF-kB pathway (9). Therefore, our data also verified that SEH treatment could restrain neuronal apoptosis induced by IR through the activation of PI3K/Akt/NF-κB signaling pathway. in the regulation of cell growth and neurons survival after brain ischemia, demonstrating that pharmacological upregulation of PI3K/Akt signaling could have therapeutic potential for the brain damage (46). Meanwhile, the activation of Akt can promote neuronal proliferation and survival through controlling multiple intracellular signals (47). As a downstream gene of Akt, NF-κB (p65) can be activated by phosphorylated Akt to phosphorylated p65, which causes NF-κB to enter the nucleus, and induces inflammatory response and apoptosis. A previous study has suggested that the NF-κB subunit activation leads to the response of lymphokine-6 and tumor necrosis factor α, which indicated that repressing NF-κB-induced neuroinflammation enhances functional outcomes and alleviates ischemic brain injury (48). Furthermore, recent studies have shown that inhibiting NF- κB could improve the prognosis of stroke, and the inhibitors of NF-κB activation may be possible targets for treatment (49, 50). In addition, Lv’s data indicated that CXCL8 gene silencing accelerated the activation of neuroglial cells while suppressing neuroinflammation via the PI3K/Akt/NF-κB pathway in the mice of CIS (50). 3. Zhou Z, Lu J, Liu WW, Manaenko A, Hou X, Mei Q, et al. Advances in stroke pharmacology. Pharmacol Therap. (2018) 191:23-42. doi: 10.1016/j.pharmthera.2018.05.012 4. Kandadai MA, Meunier JM, Hart K, Holland CK, Shaw GJ. Plasmin-loaded echogenic liposomes for ultrasound-mediated thrombolysis. Transl Stroke Res. (2015) 6:78-87. doi: 10.1007/s12975-014-0376-4 5. Grossman AW, Broderick JP. Advances and challenges in treatment and prevention of ischemic stroke. Annals Neurol. (2013) 74:363-72. doi: 10.1002/ana.23993 REFERENCES doi: 10.1248/bpb.30.309 9. Han L, Liu DL, Zeng QK, Shi MQ, Zhao LX, He Q, et al. The neuroprotective effects and probable mechanisms of ligustilide and its degradative products on intracerebral hemorrhage in mice. Int Immunopharmacol. (2018) 63:43-57. doi: 10.1016/j.intimp.2018.06.045 27. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke. (1986) 17:472-6. doi: 10.1161/01.STR.17.3.472 10. Zhu H, Zhang Y, Shi Z, Lu D, Li T, Ding Y, et al. 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Editor's Page Editor's Page Upon assuming the duties of Editor of Names for this year, the undersigned wishes to pay tribute to Dr. Erwin G. Gudde, who started the journal, established a tradition, and served it faith- fully for five years, and to Dr. Madison S. Beeler, who continued it for three years and is also responsible for the present issue upon my suggestion. Change of editorship does not involve any basic changes in edi- torial policy for at least several years to come. The only possible changes have to be for improvement and these must be approved by the Editorial Board and, if necessary, by the Board of Managers of the American Name Society. The changes in the editorial ma- chinery the present writer has suggested are two: (a) the appoint- ment of a member of the Society, officer or other, as Associate Editor who would help in the editorial work and, in case of emergency, take over the duties of the elected Editor, and (b) the designation of a member as Book Review Editor. The Editorial Board, upon the Editor's suggestion, designated Mr. Elsdon C. Smith, one of its own members in the latter capacity. As soon as the Associate Editor is found, his appointment will be announced in the journal. An editorial policy at present will be formulated as to the kinds of contributions that are acceptable; likewise an explicit statement of book review policy will be offered. Not only members of the Editorial Board and of the Board of Managers but also all members of the Society are invited to send to the Editor a note of comment on each issue and their constructive suggestions. The policy for publications of Names at this time may be summarized, it seems to me, as follows: A. Although the A.N.S. is a society for the study of names in general, theoretical and specific, names of the Western Hemisphere claim a certain priority and this is particularly imposed by the limitations of space (ca. 256-270 pages in the annual volume) and the majority of the membership. However, worthy onomastic contributions in other fields are welcomed since a large number of the membership specialize in these other fields and promote the Society's scholarly cause. 63 64 Editor's Page Editor's Page B. Quality of the publications in the journal and certain sound basic principles that lead to. scholarly excellence, though approaches and methods in investigating names may vary, are imperative elements that mark higher standards. C. The broader circ~e of readers, who also are or may become members of the Society, has to find some attractive popular material in the journal, yet not to the disadvantage of standards. Beside solid scholarly papers, lighter material should be included, at least in some of the issues in each volume, designed for the information of the reader at large. Quality is to remain our foremost concern, for this will raise the standars of the journal, attract serious scholars to contribute, and uphold the reputation of the periodical at the international com- petitive level of onomastic scholarship. A fine organ with hundreds of onomastic contributions deposited in the journal's volumes will be a permanent achievement; even the popular material, needed for the general educated reader, must be of superior quality. We have to work toward that goal. Demetrius J. Georgacas
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Circular RNA circNRIP1 acts as a microRNA-149-5p sponge to promote gastric cancer progression via the AKT1/mTOR pathway
Molecular cancer
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Zhang et al. Molecular Cancer (2019) 18:20 https://doi.org/10.1186/s12943-018-0935-5 Zhang et al. Molecular Cancer (2019) 18:20 https://doi.org/10.1186/s12943-018-0935-5 Open Access Circular RNA circNRIP1 acts as a microRNA- 149-5p sponge to promote gastric cancer progression via the AKT1/mTOR pathway Xing Zhang1†, Sen Wang1†, Haixiao Wang4†, Jiacheng Cao1†, Xiaoxu Huang1, Zheng Chen2, Penghui Xu1, Guangli Sun1, Jianghao Xu1, Jialun Lv1 and Zekuan Xu1,3* * Correspondence: xuzekuan@njmu.edu.cn * Correspondence: xuzekuan@njmu.edu.cn †Xing Zhang, Sen Wang, Haixiao Wang and Jiacheng Cao contributed equally to this work. q y 1Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, No.300, Guangzhou Road, Nanjing, Jiangsu Province, China 3Collaborative Innovation Center For Cancer Personalized Medicine, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China Full list of author information is available at the end of the article Abstract Background: CircRNA has emerged as a new non-coding RNA that plays crucial roles in tumour initiation and development. ‘MiRNA sponge’ is the most reported role played by circRNAs in many tumours. The AKT/mTOR axis is a classic signalling pathway in cancers that sustains energy homeostasis through energy production activities, such as the Warburg effect, and blocks catabolic activities, such as autophagy. Additionally, the AKT/mTOR axis exerts a positive effect on EMT, which promotes tumour metastasis. Methods: We detected higher circNRIP1 expression in gastric cancer by performing RNA-seq analysis. We verified the tumour promotor role of circNRIP1 in gastric cancer cells through a series of biological function assays. We then used a pull-down assay and dual-luciferase reporter assay to identify the downstream miR-149-5p of circNRIP1. Western blot analysis and immunofluorescence assays were performed to demonstrate that the circNRIP1-miR-149- 5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development. We then adopted a co-culture system to trace circNRIP1 transmission via exosomal communication and RIP experiments to determine that quaking regulates circNRIP1 expression. Finally, we confirmed the tumour suppressor role of microRNA-133a-3p in vivo in PDX mouse models. Results: We discovered that knockdown of circNRIP1 successfully blocked proliferation, migration, invasion and the expression level of AKT1 in GC cells. MiR-149-5p inhibition phenocopied the overexpression of circNRIP1 in GC cells, and overexpression of miR-149-5p blocked the malignant behaviours of circNRIP1. Moreover, it was proven that circNRIP1 can be transmitted by exosomal communication between GC cells, and exosomal circNRIP1 promoted tumour metastasis in vivo. We also demonstrated that quaking can promote circNRIP1 transcription. In the final step, the tumour promotor role of circNRIP1 was verified in PDX models. Conclusions: We proved that circNRIP1 sponges miR-149-5p to affect the expression level of AKT1 and eventually acts as a tumour promotor in GC. Keywords: Gastric cancer, circRNA, miRNA, ceRNA, AKT1, Organoid, PDX model Background However, some researchers still argue that not all miR- NAs targeted by circRNAs are suppressed, and current ceRNA validation methods are limited [11]. g Gastric cancer (GC), also called stomach adenocarcin- oma, has raised societal concerns worldwide, especially in East Asian countries in recent years. GC ranks as the fourth most frequent cancer and the third leading cause of cancer mortality worldwide according to the GLOBO- CAN database [1–3]. Although many advancements have been achieved in terms of diagnostic methods and surgical procedures, the overall survival of GC patients has remained largely unsatisfactory in recent years. The 5-year overall survival is less than 30% in most countries [4]. The complexity of GC treatment lies in its hetero- geneity within tumour tissues, not only due to genetic but also epigenetic alterations. Therefore, there is an ur- gent need to gain an in-depth understanding of the mo- lecular mechanisms explaining GC initiation and development. It has been reported that mTOR acts as downstream molecule of AKT1, and the AKT/mTOR pathway is one of the classic signalling pathways to mediate tumour metabolic homeostasis, which is beneficial for tumour growth and metastasis [27]. The AKT/mTOR axis sustains energy homeostasis through energy pro- duction activities, such as the Warburg effect, to satisfy the proliferation needs of GC cells [28]. mTORC1 (mTOR complex consisting of mTORC1 and mTORC2) is reported to accelerate tumour growth by promoting a shift to the Warburg effect, which likely facilitates the incorporation of nutrients into new bio-products to sustain the highly proliferative charac- ter of GC cells. mTORC1 facilitates the expression level of the transcription factor HIF1α, which increases the translation of several glycolytic enzymes, such as phospho-fructo kinase (PFK)]. Additionally, the AKT/ mTOR axis enables anabolic metabolism, such as pro- tein synthesis, and blocks catabolic activities, such as autophagy, to ultimately favour cell growth in GC [29, 30]. mTORC1 promotes cell growth by suppressing catabolic activities, including autophagy, to slow the aging of GC cells. mTORC1 phosphorylates ULK1 to prevent it from being activated by AMPK, a key activa- tor of autophagy. It has been reported that non-coding RNAs regulate the initiation and development of GC. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are two major non-coding RNAs in tumour biology that have mainly been investigated due to their crucial roles [5–7]. CircRNA has emerged as a new non-coding RNA that is important in tumour initiation and development [8–10]. Background CircRNAs within eukaryotic cells were discov- ered 40 years ago. However, circRNAs are not properly understood and were identified as incorrect gene rear- rangements and splicing errors until recently [11]. More functional circRNAs have been discovered thanks to high-throughput sequencing analysis and bioinformatic methods. CircRNAs are derived basically from the exons of their parental genes, although intronic and exonic-intronic circRNAs might sometimes occur [12, 13]. Exonic circRNAs are covalently connected and are structured in a heat-to-tail closed loop [14]. This circular structure is responsible for its stable existence and abun- dance in GC tissues, which makes circular RNAs them- selves hallmarks for the diagnosis and prognosis of GC. Moreover, circRNAs have been reported to have many important roles, including as protein sponges [15, 16], in translation [17, 18] and as miRNA sponges [19]. ‘miRNA sponge’ is the most commonly reported role of cir- cRNAs in many tumours [20, 21]. Many RNA transcripts share binding sites with miRNAs, and they compete with one another to act as competing endogenous RNAs (ceRNAs) to further regulate tumour development [22, 23]. CircRNAs assume the role of ceRNAs by acting as miRNA sponges in many tumours [24]. For example, circHIPK3 sponges miR-558 to suppress heparanase ex- pression in bladder cancer cells [25]; additionally, the circular RNA profile of circPVT1 identifies it as a prolif- erative factor and prognostic marker in gastric cancer, and circular RNA MTO1 acts as the sponge of miR-9 to suppress hepatocellular carcinoma progression [26]. Autophagy inhibition is also of great importance for the well vascularized condition of GC tumours. Additionally, the AKT/mTOR axis is also reported to exert a positive role in EMT, promoting tumour metastasis. Exosomes are reported to mediate the molecular com- munication and material transfer between primary tumour sites and distant metastasis sites [31, 32]. Exoso- mal communication maintains a very close relationship with tumour cell migration and invasion by regulating a series of cellular activities, including EMT [33, 34]. Many miRNAs, lncRNAs and circRNAs are reported to promote or suppress tumours via exosomes. In our study, we proved that circNRIP1 sponges miR-149-5p to affect the expression level of AKT1 and eventually acts as a tumour promotor in GC. CircNRIP1 can alter metabolism and autophagy through the AKT1/ mTOR axis and promote tumour metastasis through exosome communication. In conclusion, targeting cir- cNRIP1 to explore therapeutic methods is very promis- ing for future research. © 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. Page 2 of 24 Page 2 of 24 Page 2 of 24 Zhang et al. Molecular Cancer (2019) 18:20 Zhang et al. Molecular Cancer (2019) 18:20 Plasmid construction and stable transfection We synthesized human circNRIP1 cDNA and subse- quently cloned it into luciferase-labelled pcDNA3.1 vec- tor (Thermo Fisher, USA). Wild-type and mutant QKI cDNAs were synthesized and cloned into pZW1 vectors (Shanghai Institutes for Biological Sciences). In addition, MKN-45 and BGC-823 cells were transfected with the aforementioned plasmids. RNA-seq analysis q y TRIzol reagent (Life Technologies, Carlsbad, CA, USA) was used for total RNA isolation. Approximately 3 mg of total RNA from each sample was subjected to the Ribo- Minus Eukaryote Kit (Qiagen, Valencia, CA) to remove ribosomal RNA before the construction of RNA-seq li- braries. The NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Beverly, MA, USA) was used for the preparation of strand-specific RNA-seq libraries. Briefly, approximately 50 ng of ribosome-depleted RNA sample was fragmented and then used for first- and second-strand cDNA synthesis with random hexamer primers. Second-strand cDNA synthesis was performed by adding a dUTP mix. An End-It DNA End Repair Kit was used to repair the ends of the double-stranded cDNA fragments, which were then modified by the Kle- now fragment so that an A was added to the 3′ end of the DNA fragments; the fragments were finally ligated to adapters. The ligated products were purified and treated with uracil DNA glycosylase (UDG) to remove second-strand cDNA. Purified first-strand cDNA was subjected to 13–15 cycles of PCR amplification, followed by library analysis with a Bioanalyser 2100 (Agilent, Santa Clara, CA, USA); the cDNA was then sequenced in a HiSeq 2000 system (Illumina, San Diego, CA, USA) on a 100-bp paired-end run. RNA preparation, treatment with RNAse R, and PCR All RNAs were isolated by TRIzol reagent (Invitrogen) according to the manufacturer’s protocol and then were incubated with 3 U/mg RNAse R (Epicentre Technolo- gies, USA) at 37 °C for 20 mins. These RNAs were then reverse-transcribed into cDNA by PrimeScript RT Re- agent (TaKaRa, RR036A, Japan). Quantitative real-time reverse transcription polymerase chain reactions were performed on a 7500 Real-time PCR System (Applied Biosystems, Carlsbad, CA,USA) with Universal SYBR Green Master Mix (4,913,914,001, Roche, Shanghai, China). Meanwhile, we used GAPDH, β-actin and RNU6–1 as the internal references for circRNA, mRNA and miRNA, respectively. Western blot Protein extraction from stable transfected cells was per- formed by using a protein extraction kit (Key Gene, China) following the manufacturer’s protocol. We mea- sured the protein concentration by using a BCA kit (Pierce, Rockford, IL), separated protein samples via electrophoresis by using SDS-containing polyacrylamide gels, and then transferred the separated protein samples onto a polyvinylidene fluoride (PVDF) membrane (Milli- pore, Billerica, MA, USA). After blocking with 5% BSA in TBST buffer for 2 h, the membrane was incubated at 4 °C overnight with the primary antibody mentioned previously. Afterwards, the membrane was washed 3 times with TBST buffer for 10 min. We used a corre- sponding HRP-labelled secondary antibody to incubate the membrane for 2 h at room temperature and then washed 3 times with TBST buffer. Finally, blot signals were visualized by an enhanced chemiluminescence de- tection system with Chemiluminescence HRP Substrate (Millipore, WBKL0100). Oligonucleotide transfection The human gastric cell lines MKN-45 and BGC-823 were seeded in a 6-well plate and incubated at 37 °C in humidified 5% CO2 atmosphere overnight. CircNRIP1 siRNA, miRNA mimics and inhibitors (GenePharma, Shanghai, China) were transfected by Lipofectamine RNAiMax (Life Technologies) according to the manufac- turer’s protocol. Tissue specimens Tumour tissues and adjacent normal stomach mucosa tissues were collected from GC patients who received Page 3 of 24 Zhang et al. Molecular Cancer (2019) 18:20 Zhang et al. Molecular Cancer (2019) 18:20 Page 3 of 24 radical gastrectomy at the Department of Gastrointes- tinal Surgery, the First Affiliated Hospital of Nanjing Medical University, from 2013 to 2017. All patients did not receive radiotherapy and chemotherapy before sur- gery. All specimens were collected under the guidance of the HIPAA protocol and supervised by the ethics committee. TNM stage classification complied with the TNM classification system of the International Union Against Cancer (7th edition). We used Kaplan Meier method to draw the overall survival curve and disease-free survival curve according to the relative ex- pression of circNRIP1 (or miR-149-5p) and the cut-off value (Median of the expression) for circNRIP1 (or miR-149-5p). Fluorescence in situ hybridization We seeded cells on glass coverslips in 12-well plates and transfected them with circNRIP1 OE and miR-149-5p mimics using Lipofectamine 2000 in MKN-45 and BGC-823 cells. Then, we washed cells with PBS and fixed them in 4% paraformaldehyde for 15 min. After cells were transfected for 48 h, we permeabilized them overnight in 70% ethanol. Next, we washed cells twice in PBS containing 5 mM MgCl2 and rehydrated them for 10 min in 50% formamide and 2 × SSC. We compounded a solution with 50% formamide, 0.25 mg/mL Escherichia coli transfer RNA, 2 × SSC, 0.25 mg/mL salmon sperm DNA, 2.5 mg/mL BSA, and 0.5 ng/mL fluorescently la- belled circNRIP1 and miR-149-5p probes, and cells were incubated in this solution at 37 °C. Three hours later, we washed cells twice for 20 min at 37 °C in 50% formamide Wound-healing assay All exosome isolation (from cell culture media) re- agents (4,478,359,Invitrogen) were purchased from Invitrogen (USA). The manufacturer guided us through the exosome isolation procedures. After exosomes were isolated, total RNA and protein were purified by the Total Exosome RNA and Protein Isolation Kit (4,478,545, Invitrogen). g y We seeded cells in a 6-well plate and transfected and cultured them at 37 °C. We created thin scratches with a constant width along the centre of each well with a ster- ile pipette tip when the cells adhered to the bottom of the plate. Then, we used an inverted microscope (Olym- pus Optical Co., Ltd., Tokyo, Japan) to photograph im- ages instantly (0 h) and marked the 6-well plate so that we could position the same field again. After the cells were incubated at 37 °C for 24 h, we removed the culture medium and washed the cells 3 times with PBS to re- move surrounding cellular debris. CCK-8 The proliferation rate of MKN-45/BGC-823 cells was detected by the Cell Counting Kit-8 assay (Dojindo Laboratories, Kumamoto, Japan). Ten thousand cells were seeded in a 96-well plate, and 10 μL of CCK-8 solution were added to each well at the same time every day. After a 2-h incubation, the absorbance at 450 nm of the experimental wells was measured with an automatic microplate reader (BioTek, Winooski, VT, USA). Cell culture and treatment The human GC cell lines BGC-823, SGC-7901, MGC-803, MKN-45, HGC-27, and AGS were used. Normal GES-1 stomach mucosa epithelium cells were purchased from the Cell Center of Shanghai Institutes for Biological Sciences. The human gastric cell line AGS was cultured in F12K medium, while the rest of the cells were cultured in RPMI 1640 medium. Both media were supplemented with 10% foetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Gibco, USA). Cells were incubated at 37 °C in a hu- midified atmosphere of 5% CO2. Zhang et al. Molecular Cancer (2019) 18:20 Page 4 of 24 Page 4 of 24 Zhang et al. Molecular Cancer (2019) 18:20 Transwell assay y y y ( ) y We utilized an EdU assay kit (RiboBio, China) to detect DNA synthesis and cell proliferation. We seeded 10,000 treated GC cells in a 96-well plate for one night. The next day, we added Edu solution (25 μM) into the 96-well plate and waited for 24 h. Afterwards, we applied 4% formalin to fix the GC cells at room temperature for 2 h. In the following step, we used 0.5% TritonX-100 to permeabilize the GC cells for 10 mins and then added Apollo reaction solution (200 μL) to stain the EdU for 30 mins and Hoechst 33342 (200 μL) to stain the nuclei. Fi- nally, we used a Nikon microscope (Nikon, Japan) to ob- serve DNA synthesis and cell proliferation, reflected by red and blue signals, respectively. The methods used for the migration assay and invasion assay were similar. We placed transwell assay inserts (Millipore, Billerica, MA, USA) in a 24-well plate. How- ever, the difference between the methods lies in the type of membrane: The membrane in the upper transwell chamber for the invasion assay was a Matrigel-coated membrane (BD Biosciences), while that for the migration assay was a normal membrane. In the experiment, first, we placed 500 μl of serum-free RPMI 1640 with 10% FBS in the bottom chamber. Following this, we seeded 10,000 cells in 200 μl of RPMI 1640 in the upper cham- ber. After 24 to 48 h, we used methanol to fix the cells within the membrane and stained them with crystal vio- let. Finally, the cells were observed by microscope. GC organoid model GC organoid model Human GC organoids were constructed as described previously to simulate the microenvironment of the hu- man body [35]. A circNRIP1 overexpression plasmid and circNRIP1 siRNA were transfected into the organoids to facilitate our investigation of the novel role played by circNRIP1 in gastric cancer progression. Human GC organoid growth was observed daily by microscope. Transmission electron microscopy (TEM) We fixed the exosomes purified from MKN-45 and BGC-823 GC cells with 2.5% glutaraldehyde at 4 °C over- night. Subsequently, 1% OsO4 was used to further fix the exosome samples. After the samples were fixed, we used ethanol and propylene oxide to dehydrate the exosome samples. Samples were cut into slide sections and stained with 0.3% lead citrate before we finally utilized a JEM-1010 electron microscope (JEOL, Tokyo, Japan, 2500x or 8800x magnification) to detect the exosomes. Immunofluorescence analysis The GC cell lines were seeded on collagen-coated glass and incubated in RPMI 1640 medium at 37 °C in a hu- midified atmosphere of 5% CO2 for one night. The cells were washed with PBS twice before being fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100. After being blocked with 1% BSA for 30 mins, the cells were incubated with a specific primary antibody at 4 °C for one night. The secondary antibody Cy™ 3-Goat Anti-Rabbit IgG (Jackson, 1:100) and DAPI were successively added in a specially designed dish. After the final treatment, the cells were observed with a confocal microscope (Nikon, Japan). Patient-derived xenograft models (PDX models) First, we kept the tissues in iced RPMI 1640 with 10% foetal bovine serum, cut them into 2*2*3-mm3 pieces and then used fresh RPMI 1640 to wash the tissues twice. Be- fore subsequent procedures, we kept the tissues in PRMI 1640 supplemented with penicillin and streptomycin. NOD/SCID mice were chosen to be the first-generation PDX mice that carried patient tissues. We used 10% chloral hydrate (0.004 mL/g) to anesthetize the mice. In a sterile operation, we buried tumour tissues in mouse backs subcutaneously while simultaneously supplementing with penicillin and streptomycin. Subsequent generations of PDX mice were BALB/c-nude mice. When each xeno- grafted tumour tissue grew to 1–2 cm3, we followed the aforementioned protocols to harvest the tissues and im- mediately transplanted them into next-generation mice four times. We injected the circNRIP1 plasmids and cholesterol-conjugated circNRIP1 siRNA into tumour tis- sues continuously from day 0 to day 20 and then har- vested the tumour tissues for further analysis on day 40. Clonogenic assay For the clonogenic assay, we seeded LV-miR-149-5p, LV-miR-149-5p-IN and LV-miR-NC GC cells in a 6-well plate (500 cells per well) for 2 weeks. All aforementioned cells in plates were then fixed in 2 mL of methanol for 30 mins and stained with crystal violet for 20 mins. Page 5 of 24 Zhang et al. Molecular Cancer (2019) 18:20 Zhang et al. Molecular Cancer (2019) 18:20 and 2 × SSC. The following step consisted of four 5-min washes in PBS. The penultimate wash contained 4′,6′-dia- midino-2-phenylindole (DAPI). Finally, we washed the cells briefly with nuclease-free water. In vivo metastasis assay The firefly luciferase gene was stably transduced into cir- cNRIP1 pcDNA3.1 vectors. We injected exosomes con- taining circNRIP1 with a luciferase label via the tail vein into BALB/c nude mice. Finally, we observed the bio- luminescent signal after injecting 100 mg/kg D-luciferin (Xenogen, Hopkinton, MA) into mice by using an IVIS 100 Imaging System (Xenogen). ECAR measurements We used a Seahorse XF24 analyzer (Seahorse Biosciences) to determine the glycolytic capacity according to the man- ufacturer’s instructions. Lactate,Glucose and ATP assay For lactate assay, we used a lactate assay kit (K627, BioVision) to detect the lactate concentration in the whole-cell lysis according to the manufacturer’s instructions. For glucose uptake assay,the indicated cells were incu- bated with 100 μM 2-NBDG (11,046, Cayman) 30 mins before they were washed by iced-PBS.Subsequently,we recorded the FL-1 fluorescence according to the manu- facturer’s instructions. Immunohistochemical (IHC) analysis The GC tissues were fixed with 10% formalin and em- bedded in paraffin before the sections were treated with specific primary antibodies. After being incubated at 4 ° C for one night, the sections were washed twice and subsequently incubated with HRP-polymer-conjugated secondary antibody (Abcam, UK) at room temperature. These samples were then stained with 3,3-diaminobenzi- dine solution and haematoxylin. Finally, we observed the slides through a microscope. Haematoxylin and eosin staining of tissue A total of 1 × 107 gastric cancer cells were harvested, lysed and sonicated. The circNRIP1 probe was used for incuba- tion with C-1 magnetic beads (Life Technologies) at 25 °C for 2 h to generate probe-coated beads. Cell lysate with circNRIP1 probe or oligo probe was incubated at 4 °C for one night. After washing with wash buffer, the RNA mix bound to the beads was eluted and extracted with an RNeasy Mini Kit (QIAGEN) for RT–PCR or real-time PCR. First, we used microscope slides to rehydrate the tissue samples fixed in alcohol. Subsequently, we agitated the slides for 30 s in deionized water to hydrate the tissues. The slides were then placed into a bottle filled with haematoxylin, agitated for 30 s and washed in deionized water for 30 s. After the previous steps, we used 1% eosin Y solution to stain the slides and rehydrated the samples with 95% alcohol followed by 100% alcohol. We then used xylene to extract the alcohol. In the final step, we covered the slides and observed them with a microscope. Dual-luciferase reporter assay A wild-type or mut-circNRIP1 fragment was constructed and inserted downstream of the luciferase reporter gene of the pMIR-REPORT plasmid (H306, Obio Technology, Shanghai, China). We used Lipofectamine 3000 to transfect the reporter plasmid into GC cells. We then co-transfected the miR-149-5p mimic with the reporter gene into For ATP assay,we took an ATP assay kit (S0026,Beyo- time) to detect intracellular ATP in whole-crll extracts by detecting the luciferase activity. Page 6 of 24 Page 6 of 24 Zhang et al. Molecular Cancer (2019) 18:20 BGC-823 and MKN-45 cells. For the last step, we used the DualLuciferase Reporter System Kit (E1910, Promega, USA) to detect firefly and renilla luciferase activity. which the most highly upregulated circRNA attracted our attention. To further verify whether the expression level of circNRIP1 was high in GC tumours according to the RNA-seq data, we detected higher circNRIP1 expression in 80 GC samples relative to adjacent normal samples via qRT-PCR, which was consistent with the RNA-seq data (Fig. 1d). We next confirmed the higher levels of cir- cNRIP1 in the SGC-7901, BGC-823, MGC-803, AGS, the MKN-45 and HGC-27 GC cell lines relative to GES-1 cells (Fig. 1e). BGC-823 cells showed the highest expres- sion of circNRIP1, and MKN-45 cells showed the second highest expression of circNRIP. Thus, we selected BGC-823 cells and MKN-45 GC cells to investigate the downstream regulatory pathway of circNRIP1. BGC-823 and MKN-45 cells. For the last step, we used the DualLuciferase Reporter System Kit (E1910, Promega, USA) to detect firefly and renilla luciferase activity. Identification of the circular structure and clinical features of circNRIP1 CircNRIP1 arises from the NRIP gene, which is located at chromosome 21 and consists of the head-to-tail spli- cing of exon 2 and exon 3 (16386664–16,415,895) (Fig. 2a).To rule out the possibility that such head-to-tail splicing products may also derive from genomic rearrangements and trans-splicing, we first performed a qRT-PCR assay with specially designed di- vergent and convergent primers and discovered that circNRIP1, rather than linear-NRIP1 or actin, could re- sist digestion by RNAse R (Fig. 2b). Moreover, we de- tected the expression level of back-spliced or canonical forms of NRIP1 with or without RNAse R supplemen- tation in the cDNA and gDNA of GC cells by PCR and an agarose gel electrophoresis assay (Fig. 2c). We de- tected circNRIP1 in cDNA with divergent primers, even under RNAse R treatment. The opposite result was ob- served in gDNA PCR products. Additionally, the linear form of NRIP1 could not be amplified by convergent primers, demonstrating that circNRIP1 was not attrib- utable to genomic rearrangements and PCR artefacts. Subsequently, we confirmed the head-to-tail splicing of circNRIP1 in the RT–PCR product of circHIPK3 by Sanger sequencing and also determined its genomic size and sequence as reported in the CircBase database (Fig. 2a). Furthermore, we found that circNRIP1 was more stable than linear NRIP1 in GC cells under treat- ment with actinomycin D, a transcription inhibitor. This result suggested that circNRIPP1 can be a suitable diagnostic or prognosis marker (Fig. 2d). Furthermore, when we collected the clinical data on the aforemen- tioned patients, we found that the expression level of circNRIP1 significantly correlated with GC tumour size and lymphatic invasion (Table 1). Additionally, we drew overall survival (OS) and disease-free survival (DFS) curves using the Kaplan-Meier method and obtained survival information for the patients we followed up previously. Patients who had high levels of circNRIP1 RNA-binding protein immunoprecipitation (RIP) An RNA-Binding Protein Immunoprecipitation Kit (17–700, Merck, Millipore) was purchased to perform a RIP assay to determine the binding between the QKI protein and pre-mNRIP1. The procedure complied with the guidance of the manufacturer. Finally, we performed qRT-PCR on the QKI-associated RNA mixture absorbed by the magnetic beads. Reagents and antibodies Regarding the primary antibodies used in the study, anti-PFK (ab181861) and anti-QKI (ab126742) were purchased from Abcam (Cambridge, UK); anti-GDH was purchased from Shybio (Shanghai, China); anti- LC3A/B (12741), anti-SQSTM/p62 (8025), anti-mTOR (2983), anti-phospho-mTOR (5536,1230), anti-Akt (4685), anti-phospho-Akt (4060), anti-E-cadherin (3195), anti-N- cadherin (13116), anti-Slug (9585), anti-Snail (3879) and anti-TWSIT1 (46072) were purchased from Cell Signaling Technology (Danvers, PA USA). Statistics We performed our experiments in triplicate, and the re- sults are presented as the mean value ± standard devi- ation. We statistically analysed the data with Student’s t-test using SPSS statistical software, and p < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001. CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues p g We performed RNAseq analyses of ribosomal RNA-de- pleted total RNA obtained from 3 clinical gastric cancer tissues and their paired adjacent normal tissues and con- structed a circRNA profiling database. We detected 35,156 distinct circRNAs in all. Among them, 5528 cir- cRNAs have been reported in circBase. The length of most circRNAs was less than 1000 nucleotides (Fig. 1). More- over, we analysed the composition of the circRNAs in terms of gene distribution (Fig. 1b). A cluster heat map was used to show the expression variations of these cir- cRNA transcripts in cancerous tissues relative to matched normal tissues (Additional file 4: Figure S4a). Among the 49 differentially expressed circRNAs, 33 were upregulated and 16 were downregulated in GC tissues relative to nor- mal tissues (Fig. 1c, see Additional file 1). We focused on the most highly upregulated and downregulated circRNAs and matched them with circRNADb.Circ_0004771 (termed circNRIP1 in the remainder of the article), among Zhang et al. Molecular Cancer (2019) 18:20 Page 7 of 24 Zhang et al. Molecular Cancer Fig. 1 CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues. a. The length of most circRNAs was less than 1000 nucleotides. b. We analysed circRNA composition in terms of gene distribution. c. A cluster heat map was used to show the expression variations of these circRNA transcripts in cancerous tissues relative to matched normal tissues. d. Volcano plots illustrated that among differentially expressed circRNAs, 33 were upregulated and 16 were downregulated in GC tissues relative to normal tissues. e. We detected higher circNRIP1 expression in 80 GC samples relative to adjacent normal samples via qRT-PCR. f. We confirmed the higher levels of circNRIP1 in the SGC-7901, BGC-823, MGC-803, AGS, MKN-45 and HGC-27 GC cell lines relative to GES-1 cells. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 Fig. 1 CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues. a. The length of most circRNAs was less than 1000 nucleotides. b. We analysed circRNA composition in terms of gene distribution. c. A cluster heat map was used to show the expression variations of these circRNA transcripts in cancerous tissues relative to matched normal tissues. d. Volcano plots illustrated that among differentially expressed circRNAs, 33 were upregulated and 16 were downregulated in GC tissues relative to normal tissues. e. CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues We detected higher circNRIP1 expression in 80 GC samples relative to adjacent normal samples via qRT-PCR. f. We confirmed the higher levels of circNRIP1 in the SGC-7901, BGC-823, MGC-803, AGS, MKN-45 and HGC-27 GC cell lines relative to GES-1 cells. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 diagnostic and prognosis marker that is worthy of further exploration. within their GC tissues had significantly shorter overall survival (median survival of 21 months vs 51 months; P = 0.0019, log-rank test; Fig. 2e) and disease-free survival (me- dian survival of 18 months vs 49 months; P = 0.0004, log-rank test; Fig. 2f).To summarize, circNRIP1 was con- firmed to be a circular RNA and is a stable and significant CircNRIP1 serves as a miRNA sponge of miR-149-5p Many researchers have reported that circRNA assumes the role of a miRNA sponge in tumour development Zhang et al. Molecular Cancer (2019) 18:20 Page 8 of 24 Zhang et al. Molecular Cancer Fig. 2 (See legend on next page.) l d Zhang et al. Molecular Cancer (2019) 18:20 Page 9 of 24 (See figure on previous page.) Fig. 2 Identification of the circular structure and clinical features of circNRIP1. a. We confirmed the head-to-tail splicing of circNRIP1 in the circHIPK3 RT–PCR product by Sanger sequencing and also determined its genomic size and sequence as reported in the CircBase database. b. We performed a qRT-PCR assay with specially designed divergent and convergent primers and discovered that circNRIP1, rather than linear-NRIP1 or actin, could resist digestion by RNase R. c We detected the expression levels of the back-spliced and canonical forms of NRIP1 in the presence or absence of RNase R supplementation in cDNA and gDNA from GC cells by PCR and an agarose gel electrophoresis assay. d We found that circNRIP1 was more stable than linear NRIP1 in GC cells under treatment with actinomycin D (a transcription inhibitor). (e, f Patients who had high levels of circNRIP1 within their GC tissues had a significantly shorter overall survival (median survival of 21 months vs 51 months; P = 0.0019, log-rank test) and disease-free survival (median survival of 18 months vs 49 months; P = 0.0004, log-rank test). All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 regulation. In addition, circRNA can further regulate downstream gene expression. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues We predicted the target miRNAs of circNRIP1 by cross-analysing three predic- tion databases: CircNet, RNA22 and RegRNA. We then selected the top 10 common miRNAs based on conjuga- tion scores, which might indicate the potentially func- tional miRNAs absorbed by circNRIP1 in GC cells (Fig. 3a). A pull-down assay was used to further deter- mine cirNRIP1-associated miRNAs via a specific biotin-labelled circNRIP1 probe. We first verified the significantly upregulated pull-down efficiency of the cir- cNRIP1 probe in a circNRIP1 overexpression plasmid (pcDNA3.1) transfected into MKN-45 and BGC-823 cells (Additional file 1: Figure S1a). We then used a qRT-PCR assay to analyse the expression levels of 10 common miRNAs predicted by 3 websites, namely hsa-miR-29b-1-5p, hsa-miR-148a-5p, hsa-miR-204-5p, hsa-miR-214-5p, hsa-miR-146a-3p, hsa-miR-149-5p, hsa-miR-148b-5p, hsa-miR-339-5p, hsa-miR-511 and hsa-miR-516b-5p, in the sponge complex from the cir- cNRIP1 pull-down experiments in MKN-45 and BGC-823 cells. In this case, we confirmed the rich en- richment of circNRIP1 and miR-149-5p in both GC cell lines (Fig. 3b), while the other miRNAs did not show such close interactions with circNRIP1. To further verify the absorption of miR-149-5p and circNRIP1, we used a specific biotin-labelled miR-149-5p probe to successfully capture circNRIP1 relative to the NC group (Fig. 3c). Apart from the pull-down assay, we performed a dual-luciferase reporter assay to determine the direct binding between circNRIP1 and miR-149-5p based on their complementary sequences. A circNRIP1 fragment was constructed and inserted downstream of the lucifer- ase reporter gene. Then, we co-transfected a miR-149-5p mimic with the reporter gene into BGC-823 and MKN-45 cells. A significant reduction in luciferase re- porter activity was observed relative to co-transfection with control RNA (Fig. 3d). In the next phase, we regulation. In addition, circRNA can further regulate downstream gene expression. We predicted the target miRNAs of circNRIP1 by cross-analysing three predic- tion databases: CircNet, RNA22 and RegRNA. We then selected the top 10 common miRNAs based on conjuga- tion scores, which might indicate the potentially func- tional miRNAs absorbed by circNRIP1 in GC cells (Fig. 3a). A pull-down assay was used to further deter- mine cirNRIP1-associated miRNAs via a specific biotin-labelled circNRIP1 probe. We first verified the significantly upregulated pull-down efficiency of the cir- cNRIP1 probe in a circNRIP1 overexpression plasmid (pcDNA3.1) transfected into MKN-45 and BGC-823 cells (Additional file 1: Figure S1a). CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues We then used a qRT-PCR assay to analyse the expression levels of 10 common miRNAs predicted by 3 websites, namely hsa-miR-29b-1-5p, hsa-miR-148a-5p, hsa-miR-204-5p, hsa-miR-214-5p, hsa-miR-146a-3p, hsa-miR-149-5p, hsa-miR-148b-5p, hsa-miR-339-5p, hsa-miR-511 and Table 1 The expression level of circNRIP1 is significantly correlated with the GC tumour size and lymphatic invasion, and the expression level of miR-149-5p is negatively correlated with the GC tumour size Parameters Group circNRIP1 expression miR-149-5p expression Cases Low High P-value Cases Low High P-value Gender female 24 11 13 0.6256 15 7 8 0.6212 male 56 29 27 35 19 16 Age at surgery ≥55 64 34 30 0.2636 33 17 16 0.7653 < 55 16 6 10 17 8 9 T grade T1 + T2 40 22 18 0.3711 24 11 13 0.5713 T3 + T4 40 18 22 26 14 12 Lymphatic invasion Negative (N0) 14 11 3 0.0186* 15 5 10 0.1228 Positive (N1-N3) 66 29 37 35 20 15 Tumor site Cardiac 30 21 9 0.1689 18 7 11 0.2386 Non-cardiac 50 19 31 32 18 14 Stage I-II 43 24 19 0.2622 23 11 12 0.7766 III-IV 37 16 21 27 14 13 Size(cm) < 3 37 23 14 0.0436* 24 8 16 0.0235* ≥3 43 17 26 26 17 9 Histology grade Well-moderately 28 13 15 0.6392 16 7 9 0.5443 Poorly-signet 52 27 25 34 18 16 All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 evel of circNRIP1 is significantly correlated with the GC tumour size and lymphatic invasion, and the 49-5p is negatively correlated with the GC tumour size Table 1 The expression level of circNRIP1 is significantly correlated with the GC tumour size and lymphatic i expression level of miR-149-5p is negatively correlated with the GC tumour size Zhang et al. Molecular Cancer (2019) 18:20 Page 10 of 24 Fig. 3 CircNRIP1 serves as a miRNA sponge of miR-149-5p. a We selected the top 10 most common miRNAs based on their conjugation scores, which might indicate potentially functional miRNAs absorbed by circNRIP1 in GC cells. b. We confirmed the enrichment of circNRIP1 and miR- 149-5p in MKN-45 and BGC-823 cells. c. We used a specific biotin-labelled miR-149-5p probe to successfully capture circNRIP1 relative to the NC group. d. We performed a dual-luciferase reporter assay to determine the direct binding between circNRIP1 and miR-149-5p based on their complementary sequences. e. CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues We found that miR-149-5p co-localized with circNRIP1 in the cytoplasm by FISH analysis, scale bar = 100 μm. f. We determined the negative correlation between the expression levels of circNRIP1 and miR-149-5p by performing qRT-PCR on 80 paired GC tumour tissues and normal tissues. (g, h). We confirmed that the low level of miR-149-5p was positively correlated with the OS (median survival of 58 months vs 21 months; P = 0.0007, log-rank test) and DFS (median survival of 56 months vs 19 months; P = 0.0002, log-rank test) of GC patients. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 Fig. 3 CircNRIP1 serves as a miRNA sponge of miR-149-5p. a We selected the top 10 most common miRNAs based on their conjugation scores, which might indicate potentially functional miRNAs absorbed by circNRIP1 in GC cells. b. We confirmed the enrichment of circNRIP1 and miR- 149-5p in MKN-45 and BGC-823 cells. c. We used a specific biotin-labelled miR-149-5p probe to successfully capture circNRIP1 relative to the NC group. d. We performed a dual-luciferase reporter assay to determine the direct binding between circNRIP1 and miR-149-5p based on their complementary sequences. e. We found that miR-149-5p co-localized with circNRIP1 in the cytoplasm by FISH analysis, scale bar = 100 μm. f. We determined the negative correlation between the expression levels of circNRIP1 and miR-149-5p by performing qRT-PCR on 80 paired GC tumour tissues and normal tissues. (g, h). We confirmed that the low level of miR-149-5p was positively correlated with the OS (median survival of 58 months vs 21 months; P = 0.0007, log-rank test) and DFS (median survival of 56 months vs 19 months; P = 0.0002, log-rank test) of GC patients. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 Fig. 3 CircNRIP1 serves as a miRNA sponge of miR-149-5p. a We selected the top 10 most common miRNAs based on their conjugation scores, which might indicate potentially functional miRNAs absorbed by circNRIP1 in GC cells. b. We confirmed the enrichment of circNRIP1 and miR- 149-5p in MKN-45 and BGC-823 cells. c. We used a specific biotin-labelled miR-149-5p probe to successfully capture circNRIP1 relative to the NC group. d. We performed a dual-luciferase reporter assay to determine the direct binding between circNRIP1 and miR-149-5p based on their complementary sequences. e. CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues We found that AKT1 was significantly upregulated in GC tissues (415 GC tissues vs 34 normal tissues), and patients with high levels of AKT1 (298 GC tissues vs 94 normal tissues) had lower OS based on an analysis of the TCGA database. c, d. We subsequently detected higher levels of AKT1 in GC tissues than in adjacent normal tissues by performing immunochemistry analysis on 12 paired GC tumour tissues and normal stomach tissues, scale bar = 200 μm. e. qRT-PCR further confirmed the expression level of AKT1 and showed that it was upregulated in 80 GC tissues relative to adjacent normal tissues. f. We detected the circNRIP1 level in both groups and determined that the level of circNRIP1 in the high AKT1 group(n = 40) was significantly higher than that in the low AKT1 group (n = 40). g. Two circNRIP1 small interfering RNAs (siRNAs) specifically targeting the backsplice junction sequences at different binding sites in circNRIP1 were designed. h. Immunofluorescence assay showed that circNRIP1 knockdown successfully inhibited the expression level of AKT1, scale bar = 50 μm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 Fig. 4 Knockdown of circNRIP1 reduces the expression of AKT1. a. We performed a dual-luciferase reporter assay to determine the direct binding between 149-5p and AKT1 in GC cells. b. We found that AKT1 was significantly upregulated in GC tissues (415 GC tissues vs 34 normal tissues), and patients with high levels of AKT1 (298 GC tissues vs 94 normal tissues) had lower OS based on an analysis of the TCGA database. c, d. We subsequently detected higher levels of AKT1 in GC tissues than in adjacent normal tissues by performing immunochemistry analysis on 12 paired GC tumour tissues and normal stomach tissues, scale bar = 200 μm. e. qRT-PCR further confirmed the expression level of AKT1 and showed that it was upregulated in 80 GC tissues relative to adjacent normal tissues. f. We detected the circNRIP1 level in both groups and determined that the level of circNRIP1 in the high AKT1 group(n = 40) was significantly higher than that in the low AKT1 group (n = 40). g. Two circNRIP1 small interfering RNAs (siRNAs) specifically targeting the backsplice junction sequences at different binding sites in circNRIP1 were designed. h. CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues We found that miR-149-5p co-localized with circNRIP1 in the cytoplasm by FISH analysis, scale bar = 100 μm. f. We determined the negative correlation between the expression levels of circNRIP1 and miR-149-5p by performing qRT-PCR on 80 paired GC tumour tissues and normal tissues. (g, h). We confirmed that the low level of miR-149-5p was positively correlated with the OS (median survival of 58 months vs 21 months; P = 0.0007, log-rank test) and DFS (median survival of 56 months vs 19 months; P = 0.0002, log-rank test) of GC patients. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 Zhang et al. Molecular Cancer (2019) 18:20 Page 11 of 24 Zhang et al. Molecular Cancer Fig. 4 Knockdown of circNRIP1 reduces the expression of AKT1. a. We performed a dual-luciferase reporter assay to determine the direct binding between 149-5p and AKT1 in GC cells. b. We found that AKT1 was significantly upregulated in GC tissues (415 GC tissues vs 34 normal tissues), and patients with high levels of AKT1 (298 GC tissues vs 94 normal tissues) had lower OS based on an analysis of the TCGA database. c, d. We subsequently detected higher levels of AKT1 in GC tissues than in adjacent normal tissues by performing immunochemistry analysis on 12 paired GC tumour tissues and normal stomach tissues, scale bar = 200 μm. e. qRT-PCR further confirmed the expression level of AKT1 and showed that it was upregulated in 80 GC tissues relative to adjacent normal tissues. f. We detected the circNRIP1 level in both groups and determined that the level of circNRIP1 in the high AKT1 group(n = 40) was significantly higher than that in the low AKT1 group (n = 40). g. Two circNRIP1 small interfering RNAs (siRNAs) specifically targeting the backsplice junction sequences at different binding sites in circNRIP1 were designed. h. Immunofluorescence assay showed that circNRIP1 knockdown successfully inhibited the expression level of AKT1, scale bar = 50 μm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 Fig. 4 Knockdown of circNRIP1 reduces the expression of AKT1. a. We performed a dual-luciferase reporter assay to determine the direct binding between 149-5p and AKT1 in GC cells. b. CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues To further analyse the regulatory role of circNRIP1 on AKT1, we designed two circNRIP1 small interfering RNAs (siR- NAs) to specifically target the backsplice junction sequence at different binding sites of circNRIP1 (Fig. 4f). We knocked down circNRIP1 expression in MKN-45 and BGC-823 cells by using circNRIP1 siRNA. The efficiency of circNRIP1 silencing was confirmed by qRT-PCR (Add- itional file : Figure S2a). To rule out the possibility that cir- cNRIP1 siRNA might exerts effects on the mRNA level of NRIP1 (linear NRIP1), we analysed the NRIP1 mRNA level and found no significant changes (Additional file 2: Figure S2a). Then, we chose si-circNRIP1–1 to perform further experiments based on its higher knockdown efficiency. We detected lower expression levels of AKT1 by qRT-PCR after knocking down circNRIP1 in MKN-45 and BGC-823 GC cells (Additional file 2: Figure S2b). Finally, an immuno- fluorescence assay was utilized to observe AKT1 expression in human GC organoid models. The outcomes showed that circNRIP1 knockdown successfully inhibited the expression level of AKT1 (Fig. 4g). Thus, we proved the positive regu- lation the ceRNA AKT1 by circNRIP1. negative correlation between the expression level of cir- cNRIP1 and miR-149-5p by performing qRT-PCR on 80 paired GC tumour tissues (Fig. 3f). Many reports have described the tumour suppressor role of miR-149-5p in various cancers [36, 37], which allowed us to determine the role of miR-149-5p in gastric cancer. We detected that overexpression of miR-149-5p in MKN-45 and BGC-823 cells blocked the proliferation rate (Additional file 1: Figure S1b), DNA synthesis (Additional file 1: Fig- ure S1c), migration and invasion (Additional file 1: Fig- ure S1d) abilities of GC cells, while miR-149-5p reduction had the opposite effects on GC cells. Further- more, we collected clinical data from the aforemen- tioned patients and found that the expression level of miR-149-5p was negatively correlated with GC tumour size (Table 1). We then used GC tissues from patients who were followed up for a period of time to verify the expression of miR-149-5p and used the Kaplan-Meier Plotter method to draw overall survival (OS) and disease-free survival (DFS) curves based on the median expression of miR-149-5p (Additional file 5: Figure S5b). CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues Immunofluorescence assay showed that circNRIP1 knockdown successfully inhibited the expression level of AKT1, scale bar = 50 μm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 mutated two binding sites in the 3’UTR of the cir- cNRIP1 sequence. We found that co-transfection of miR-149-5p and mut-circNRIP1, with either one of the two binding sites or both mutated, did not induce a significant reduction in the luciferase signal (Fig. 3d, Additional file 5: Figure S5a). Therefore, the direct interaction of circNRIP1 and miR-149-5p was con- firmed. FISH analysis was performed in BGC-823 and MKN-45 cells, and we found that miR-149-5p was co-localized with circNRIP1 in the cytoplasm, which fur- ther suggested an interaction between circNRIP1 and miR-149-5p (Fig. 3e). Furthermore, we determined the Page 12 of 24 Page 12 of 24 Zhang et al. Molecular Cancer (2019) 18:20 the luciferase signal (Fig. 4a). To validate the ceRNA analysis, we made an effort to verify the specific tumour expression and prognostic role of AKT1 in gastric can- cer among a large number of patients by analysing the TCGA debase. We found that AKT1 was significantly upregulated in GC tissues (415 GC tissues vs 34 normal tissues), and patients with high levels of AKT1 (298 GC tissues vs 94 normal tissues) had a lower OS (Additional file 5: Figure S5c). To verify the aforementioned expres- sion pattern of AKT1 in GC tissues, we subsequently de- tected higher levels of AKT1 in GC tissues than in adjacent normal tissues by performing immunochemis- try analysis on 12 paired GC tumour tissues and paired normal stomach tissues (Fig. 4b, c). qRT-PCR further confirmed the expression level of AKT1 and showed that it was upregulated in 80 GC tissues relative to adjacent normal tissues (Fig. 4d). Next, we wanted to determine whether circNRIP1 is co-expressed with AKT1, and we sorted the 80 GC tissue samples into a low AKT1 group (n = 40) and a high AKT1 expression group (n = 40) based on the median expression of AKT1. Then, we de- tected the circNRIP1 level in two groups and found that the level of circNRIP1 in the high AKT1 group was signifi- cantly higher than that in the low AKT1 group (Fig. 4e). CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues To rule out the possibility that cir- cNRIP1 siRNA might exerts effects on the mRNA level of NRIP1 (linear NRIP1), we analysed the NRIP1 mRNA level and found no significant changes (Additional file 2: Figure S2a). Then, we chose si-circNRIP1–1 to perform further experiments based on its higher knockdown efficiency. We detected lower expression levels of AKT1 by qRT-PCR after knocking down circNRIP1 in MKN-45 and BGC-823 GC cells (Additional file 2: Figure S2b). Finally, an immuno- fluorescence assay was utilized to observe AKT1 expression in human GC organoid models. The outcomes showed that circNRIP1 knockdown successfully inhibited the expression level of AKT1 (Fig. 4g). Thus, we proved the positive regu- lation the ceRNA AKT1 by circNRIP1. Knockdown of circNRIP1 reduces the expression of AKT1 It has been reported that miR-149-5p can inhibit cell proliferation, invasion and migration in human hepato- cellular carcinoma by targeting AKT1 and further facili- tating the mTOR pathway [38]. The interaction between miR-149-5p and AKT1 is also reported in colon carcin- oma and glioblastoma [39, 40]. Based on the assumption that circNRIP1 plays a tumour promotor role in GC through the classic ceRNA mechanism, we tried to iden- tify the ceRNA of circNRIP1, which should also be the downstream target gene of miR-149-5p. Herein, we per- formed a dual-luciferase reporter assay to determine the direct binding between 149-5p and AKT1 in GC cells. The wild-type (wild-AKT1) and mutant AKT1 (mut-AKT1) reporter genes were constructed as described in the aforementioned reports. Then, we co-transfected a miR-149-5p mimic with the reporter genes into BGC-823 and MKN-45 cells. A significant re- duction in luciferase reporter activity was observed rela- tive to co-transfection with control RNA (Fig. 4a). Moreover, we found that co-transfection of miR-149-5p and mut-AKT1 did not induce a significant reduction in CircRNA expression profiles in gastric cancer tissues and paired normal gastric tissues Thus, we confirmed that a high level of miR-149-5p was positively correlated with the OS (median survival of 58 months vs 21 months; P = 0.0007, log-rank test; Add- itional file 5: Figure S5b) and DFS (median survival of 56 months vs 19 months; P = 0.0002, log-rank test; Add- itional file 5: Figure S5b) of GC patients. In conclusion, it is suggested that circNRIP1 can serve as a sponge of miR-149-5p in the cytoplasm within GC cells, and the high level of miR-149-5p is postively correlated with the survival of GC patients. the luciferase signal (Fig. 4a). To validate the ceRNA analysis, we made an effort to verify the specific tumour expression and prognostic role of AKT1 in gastric can- cer among a large number of patients by analysing the TCGA debase. We found that AKT1 was significantly upregulated in GC tissues (415 GC tissues vs 34 normal tissues), and patients with high levels of AKT1 (298 GC tissues vs 94 normal tissues) had a lower OS (Additional file 5: Figure S5c). To verify the aforementioned expres- sion pattern of AKT1 in GC tissues, we subsequently de- tected higher levels of AKT1 in GC tissues than in adjacent normal tissues by performing immunochemis- try analysis on 12 paired GC tumour tissues and paired normal stomach tissues (Fig. 4b, c). qRT-PCR further confirmed the expression level of AKT1 and showed that it was upregulated in 80 GC tissues relative to adjacent normal tissues (Fig. 4d). Next, we wanted to determine whether circNRIP1 is co-expressed with AKT1, and we sorted the 80 GC tissue samples into a low AKT1 group (n = 40) and a high AKT1 expression group (n = 40) based on the median expression of AKT1. Then, we de- tected the circNRIP1 level in two groups and found that the level of circNRIP1 in the high AKT1 group was signifi- cantly higher than that in the low AKT1 group (Fig. 4e). To further analyse the regulatory role of circNRIP1 on AKT1, we designed two circNRIP1 small interfering RNAs (siR- NAs) to specifically target the backsplice junction sequence at different binding sites of circNRIP1 (Fig. 4f). We knocked down circNRIP1 expression in MKN-45 and BGC-823 cells by using circNRIP1 siRNA. The efficiency of circNRIP1 silencing was confirmed by qRT-PCR (Add- itional file : Figure S2a). Knockdown of circNRIP1 inhibits the proliferation, migration and invasion of GC cells in vitro As we previously proved that knockdown of circNRIP1 significantly inhibits AKT1 expression in GC cells, we hypothesized that blocking the expression of circNRIP1 might reduce the survival abilities of GC cells in terms of proliferation and metastasis. Based on this hypothesis, we transfected the circNRIP1 siRNA and circNRIP1 Zhang et al. Molecular Cancer (2019) 18:20 Page 13 of 24 Zhang et al. Molecular Cancer (2019) 18:20 Page 13 of 24 Zhang et al. Molecular Cancer Fig. 5 (See legend on next page.) Fig. 5 (See legend on next page.) Fig. 5 (See legend on next page.) Page 14 of 24 Zhang et al. Molecular Cancer (2019) 18:20 (See figure on previous page.) Fig. 5 Knockdown of circNRIP1 inhibits the proliferation, migration and invasion of GC cells in vitro. a. We observed that circNRIP1 silencing significantly inhibited the cell proliferation rate, as indicated by the CCK8 assay, and overexpression of circNRIP1 exerted the opposite effect on the GC cell proliferation rate. b. We observed that circNRIP1 silencing significantly inhibited DNA synthesis, as determined by the Edu assay, and overexpression of circNRIP1 exerted the opposite effect on GC cell DNA synthesis as indicated by the EDU assay, scale bar = 100 μm. c. We observed that circNRIP1 silencing significantly inhibited colony formation ability, and overexpression of circNRIP1 exerted the opposite effect on colony formation. d. Knockdown of circNRIP1 successfully reduced the migration and invasion ability of GC cells, and overexpression of circNRIP1 exerted the opposite effect on metastasis, as indicated by the transwell assay, scale bar = 200 μm. e. We observed that both circNRIP1 knockdown and miR-149-5p overexpression significantly inhibited the growth of the organoid models, while circNRIP1 overexpression promoted organoid model survival. f. We detected downregulation of the mesenchymal cell markers TWIST, snail, slug, and N-cadherin and upregulation of the epithelial cell marker E-cadherin in human GC organoid models when we knocked down circNRIP1 or overexpressed miR-149-5p. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 upregulation of the expression of the transcription fac- tor TWIST and the DNA-binding proteins snail and slug [43, 44]. We detected the downregulation of the mesenchymal cell markers TWIST, snail, slug, and N-cadherin and the upregulation of the epithelial cell marker E-cadherin in human GC organoid models when we knocked down circNRIP1 or overexpressed miR-149-5p (Fig. 5f). Knockdown of circNRIP1 inhibits the proliferation, migration and invasion of GC cells in vitro Conversely, circNRIP1 overex- pression increased the expression levels of mesenchy- mal cell markers and reduced the expression level of the epithelial cell marker (Fig. 5f). Thus, the miRNA sponge role of circNRIP1 was further confirmed be- cause miR-149-5p inhibitors could phenocopy the bio- logical function of circNRIP1 overexpression. In summary, we proved that circNRIP1 knockdown could inhibit the proliferation, migration and invasion of GC cells and block the growth of human GC organoid models. CircNRIP1 functions as a tumour suppressor by sponging miR-149-5p, which remains to be explored in depth. overexpression plasmid into MKN-45 and BGC-823 cells. In the following steps, CCK8, Edu and colony for- mation assays were performed to determine the prolifer- ation ability of GC cells, and the metastasis ability of GC cells was measured by a transwell assay and wound heal- ing assay. We observed that circNRIP1 silencing signifi- cantly inhibited the cell proliferation rate as indicated by the CCK8 assay (Fig. 5a, Additional file 6: Figure S6b), DNA synthesis as determined by the Edu assay (Fig. 5b, Additional file 6: Figure S6c), and the colony formation ability (Fig. 5c, Additional file 6: Figure S6a) of MKN-45 and BGC-823 cells. In addition, knockdown of cir- cNRIP1 successfully reduced the migration and invasion of GC cells (Fig. 5d, Additional file 2: Figure S2c, Add- itional file 6: Figure S6d). However, overexpression of circNRIP1 exerted the opposite effect on GC cell proliferation and metastasis (Fig. 5a, b, c, d, Additional file 2: Figure S2c, Additional file 6: Figure S6a-d). We as- sumed that miR-149-5p overexpression phenocopied the tumour-suppressor effects of circNRIP1 silencing in MKN-45 and BGC-823 cells. To verify our assumption, we then transfected GC cells with a miR-149-5p mimic, and both the sequence and transfection efficiency were confirmed by qRT-PCR (Additional file 2: Figure S2d). The circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development y q ( g ) We found that miR-149-5p overexpression almost phe- nocopied the biological effects of circNRIP1 knockdown on GC cell proliferation and metastasis (Fig. 5a, b, c, d, Additional file 2: Figure S2c, Additional file 6: Figure S6a-d). Finally, we built human GC organoid models to investigate the biological functions of circNRIP1 and miR-149-5p. We observed that both circNRIP1 knock- down and miR-149-5p overexpression significantly inhibited the growth of the organoid models, while circNRIP1 overexpression promote its growth (Fig. 5e, Additional file 6: Figure S6e). Epithelial–mesenchymal transition (EMT) is recognized as an important mech- anism mediating the initiation and promotion of me- tastasis in GC [41, 42]. We already determined the relation between circNRIP1 and GC metastasis in vitro and the positive regulation of AKT1 by cir- cNRIP1. Many reports have described the promoting role of the PI3K/AKT signalling pathway on EMT via p To verify whether circNRIP1 plays a tumour promotor role via the AKT/mTOR pathway by sponging miR-149-5p, we attempted to detect the expression level of AKT1, the target of miR-149-5p, in GC cells. MiR-149-5p inhibitors were used to examine whether the therapeutic effects of circNRIP1 knockdown could be reversed by knocking down miR-149-5p. The se- quence and transfection efficiency of miR-149-5p inhibi- tors were confirmed by qRT-PCR (Additional file 2: Figure S2d). Subsequently, we found that knockdown of circNRIP1 and miR-149-5p significantly reversed the downregulated expression levels of AKT1, p-AKT1, mTOR and p-mTOR in MKN-45 and BGC-823 cells relative to circNRIP1 inhibition (Fig. 6a, Additional file 7: Figure S7a). We attempted to determine whether the biological function of circNRIP1 in GC cells could also be reversed by miR-149-5p inhibitors. We observed that the reduced proliferation indicated by the Edu assay Zhang et al. Molecular Cancer (2019) 18:20 Page 15 of 24 Zhang et al. Molecular Cancer Fig. 6 The circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development. a. Knockdown of circNRIP1 and miR-149-5p significantly reversed the expression levels of AKT1/mTOR pathway molecules, certain metabolism markers and EMT markers achieved by knocking down only circNRIP1 in MKN-45 and BGC-823 cells. b. We observed that the reduction of GC cell proliferation mediated by circNRIP1 knockdown was successfully blocked by miR-149-5p inhibition, scale bar = 100 μm. c. The circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development We observed that the reduction of metastasis of GC cells mediated by circNRIP1 knockdown was successfully blocked by miR-149-5p inhibition, scale bar = 100 μm. d. Knocking down both miR-149-5p and circNRIP1 reversed the inhibition of GC organoid growth achieved by only knocking down circNRIP1, scale bar = 50 nm. e. We found that knockdown of circNRIP1 and miR-149-5p significantly reversed the downregulated expression levels of AKT1 in the GC organoid models relative to the circNRIP1 inhibition group, scale bar = 50 μm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 Fig. 6 The circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development. a. Knockdown of circNRIP1 and miR-149-5p significantly reversed the expression levels of AKT1/mTOR pathway molecules, certain metabolism markers and EMT markers achieved by knocking down only circNRIP1 in MKN-45 and BGC-823 cells. b. We observed that the reduction of GC cell proliferation mediated by circNRIP1 knockdown was successfully blocked by miR-149-5p inhibition, scale bar = 100 μm. c. We observed that the reduction of metastasis of GC cells mediated by circNRIP1 knockdown was successfully blocked by miR-149-5p inhibition, scale bar = 100 μm. d. Knocking down both miR-149-5p and circNRIP1 reversed the inhibition of GC organoid growth achieved by only knocking down circNRIP1, scale bar = 50 nm e We found that knockdown of circNRIP1 and miR 149 5p significantly reversed the downregulated expression levels of AKT1 in the Fig. 6 The circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development. a. Knockdown of circNRIP1 and miR-149-5p significantly reversed the expression levels of AKT1/mTOR pathway molecules, certain metabolism markers and EMT markers achieved by knocking down only circNRIP1 in MKN-45 and BGC-823 cells. b. We observed that the reduction of GC cell proliferation mediated by circNRIP1 knockdown was successfully blocked by miR-149-5p inhibition, scale bar = 100 μm. c. We observed that the reduction of metastasis of GC cells mediated by circNRIP1 knockdown was successfully blocked by miR-149-5p inhibition, scale bar = 100 μm. d. Knocking down both miR-149-5p and circNRIP1 reversed the inhibition of GC organoid growth achieved by only knocking down circNRIP1, scale bar = 50 nm. e. The circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development We found that knockdown of circNRIP1 and miR-149-5p significantly reversed the downregulated expression levels of AKT1 in the GC organoid models relative to the circNRIP1 inhibition group, scale bar = 50 μm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Fig. 6b, Additional file 7: Figure S7b) and reduced me- tastasis indicated by the transwell assay (Fig. 6c, Add- itional file 7: Figure S7c) in GC cells mediated by circNRIP1 knockdown were successfully restored by miR-149-5p inhibition. Additionally, the GC organoid models were further used to investigate the cir- cNRIP1/miR-149-5p regulatory role in tumour growth. We found that knocking down miR-149-5p and circNRIP1 could reverse the inhibited growth of the GC organoids achieved by only knocking down circNRIP1 (Fig. 6d). In the next step, we tried to understand the deeper tumour promotor mechanism underlying the cir- cNRIP1/miR-149-5p/AKT/mTOR axis. Thus, we Zhang et al. Molecular Cancer (2019) 18:20 Page 16 of 24 Zhang et al. Molecular Cancer Fig. 7 Exosomal circNRIP1 regulates AKT1 expression as well as EMT in vitro and promotes metastasis in vivo. a. We determined the significantly higher expression level of circNRIP1 in plasmatic exosomes from GC patients by qRT-PCR. b. We used a transmission electron microscope (TEM) to determine the existence and morphology of exosomes purified from GC cell medium (exosome-free FBS), scale bar = 25 μm. We further confirmed the exosomes by detecting protein markers, including CD63 and CD81, by western blot. c. Red exosome signals were found in the cytoplasm of GFP-labelled tumour cells when exo-RFP GC cells were mixed with the same amount of GFP-labelled GC cells for 72 h, scale bar = 50 μm. d. We than purified exosomes and added them into GFP-labelled MKN-45 or BGC-823 GC cells. The red signal of circNRIP1 similarly appeared in the cytoplasm of GFP-labelled GC cells after 72 h, scale bar = 50 μm. e. We performed qRT-PCR and detected higher circNRIP1 expression in exosomes purified from circNRIP1-overexpressing GC cells relative to those from NC cells. f. We detected upregulated AKT1, mTOR and EMT markers in GC cells by co-culturing them with exosomes of OV circNRIP1 GC cells (OV exosomes) for 72 h via western blot. g. According to the luciferase Intensities detected in the thoracic cavity, we found that GC cells treated with OV exosomes showed higher metastasis potential. h. The circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development We harvested lung tissues for H&E staining to characterize the cancerous nodes. Cancerous node size was consistent with luciferase intensity, scale bar = 200 μm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 Fig. 7 Exosomal circNRIP1 regulates AKT1 expression as well as EMT in vitro and promotes metastasis in vivo. a. We determined the significantly higher expression level of circNRIP1 in plasmatic exosomes from GC patients by qRT-PCR. b. We used a transmission electron microscope (TEM) to determine the existence and morphology of exosomes purified from GC cell medium (exosome-free FBS), scale bar = 25 μm. We further confirmed the exosomes by detecting protein markers, including CD63 and CD81, by western blot. c. Red exosome signals were found in the cytoplasm of GFP-labelled tumour cells when exo-RFP GC cells were mixed with the same amount of GFP-labelled GC cells for 72 h, scale bar = 50 μm. d. We than purified exosomes and added them into GFP-labelled MKN-45 or BGC-823 GC cells. The red signal of circNRIP1 similarly appeared in the cytoplasm of GFP-labelled GC cells after 72 h, scale bar = 50 μm. e. We performed qRT-PCR and detected higher circNRIP1 expression in exosomes purified from circNRIP1-overexpressing GC cells relative to those from NC cells. f. We detected upregulated AKT1, mTOR and EMT markers in GC cells by co-culturing them with exosomes of OV circNRIP1 GC cells (OV exosomes) for 72 h via western blot. g. According to the luciferase Intensities detected in the thoracic cavity, we found that GC cells treated with OV exosomes showed higher metastasis potential. h. We harvested lung tissues for H&E staining to characterize the cancerous nodes. Cancerous node size was consistent with luciferase intensity, scale bar = 200 μm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 detected the expression levels of mTORC1, HIF1α, PFK, p-ULK, LC3, and P62 in GC cells. To test our hypothesis that circNRIP1 could modulate the Warburg effect via miR-149-5p, we performed a series of glycolysis detec- tion experiments. We found that knockdown of circNRIP1 reduced the lactate contents, glucose uptake and ATP production in MKN-45 and BGC-823 cells. However, the reduction in glycolysis activity could be re- stored when we knocked down both circNRIP1 and miR-149-5p (Additional file 3: Figure S3a, b, c). The circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development Fur- thermore, we found that knockdown of circNRIP1 could upregulate autophagy and epithelial cell markers, while co-transfection with a miR-149-5p inhibitor could abolish the aforementioned results (Fig. 6a, Additional file 7: Fig- ure S7a). In the next step, we performed an immunofluor- escence assay to detect the AKT1 expression level in GC organoid models. We found that knockdown of both cir- cNRIP1 and miR-149-5p significantly reversed the down- regulated expression levels of AKT1 in the GC organoid models relative to circNRIP1 inhibition (Fig. 6e). g g To trace the exosome communication between GC cells under more realistic conditions, we established a co-cultivation system and transfected an RFP-tagged CD63 plasmid into MKN-45 and BGC-823 cells (exo-RFP-MKN-45 and exo-RFP-BGC-823) to label the exosomes within tumour cells. Red exosome signal was found in the cytoplasm of GFP-labelled tumour cells when exo-RFP GC cells were mixed with the same amount of GFP-labelled GC cells for 72 h (Fig. 7c, Add- itional file 8: Figure S8b). These results prove that exo- somes could be used as a communication tool between GC cells. To further validate the ability of circNRIP1 to assemble into exosomes, we transfected an RFP-tagged circNRIP1-overexpressing plasmid into MKN-45 and BGC-823 cells and then purified and added exosomes into GFP-labelled MKN-45 or BGC-823 GC cells. The red signal of circNRIP1 similarly appeared in the cyto- plasm of GFP-labelled GC cells after 72 h (Fig. 7d, Add- itional file 8: Figure S8c). We detected higher circNRIP1 expression in exosomes purified from circNRIP1-overexpressing GC cells relative to those from NC cells (Fig. 7e, Additional file 8: Figure S8d). These results all proved that circNRIP1 could enter into exosomes from GC cells. Subsequently, we detected upregulated AKT1, mTOR and EMT markers in GC cells by co-culturing them with exosomes of OV circNRIP1 GC cells (OV exosomes) for 72 h via western blotting (Fig. 7f, Additional file 8: Figure S8e). We considered recent reports describing the posi- tive correlation between exosomal communication and metastasis. Additionally, we proved that circNRIP1 func- tions as a promoter of EMT. We attempted to detect the role of exosomal circNRIP1 in distant metastasis via tail vein injection of GC cells co-cultured with OV exo- somes and NC exosomes into BALB/c nude mice. We observed mostly lung metastases and a few peritoneal metastases by detecting luciferase intensities every week for four weeks. The circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development According to the luciferase Intensities detected in the thoracic cavity, we found that GC cells treated with OV exosomes showed higher metastasis po- tential (Fig. 7g, Additional file 8: Figure S8f). Next, we harvested lung tissues for H&E staining (Fig. 7h, Together, we proved that circNRIP1 promotes GC progression as a sponge of miR-149-5p through AKT/ mTOR-mediated metabolism and the EMT pathway. The circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development To Zhang et al. Molecular Cancer (2019) 18:20 Page 17 of 24 Zhang et al. Molecular Cancer Fig. 8 (See legend on next page.) Fig. 8 (See legend on next page.) Fig. 8 (See legend on next page.) Page 18 of 24 Zhang et al. Molecular Cancer (2019) 18:20 (See figure on previous page.) Fig. 8 Expression of circNRIP1 in GC can be regulated by QKI. a. We constructed a series of mutation plasmids (pZW1) consisting of either mutated I1QB or I3QB (#1, #2) or both I1QB and I3QB (#3), and we also constructed a wild-type plasmid spanning intron 1 to intron 3 (#4). b. We observed that only the wild-type plasmid (#4), and neither the I1QB nor I3QB deletion constructs (#1–3), could overexpress circNRIP1 according to northern blotting of GC cells.c. We observed that only the wild-type plasmid (#4), and neither the I1QB nor I3QB deletion constructs (#1–3), could overexpress circNRIP1 according to qRT-PCR in GC cells. d. We knocked down QKI and observed a significant reduction in circNRIP1 but not pre- mNRIP1 or mNRIP1. e. Enrichment of I1QB and I3QB was observed when we used an antibody against QKI. f. We detected higher QKI expression level in GC tumour tissues relative to adjacent normal stomach tissues among 40 patients by immunohistochemistry, scale bar = 200 μm. g. We performed qRT-PCR on these 40 patients and discovered that the expression level of circNRIP1 was positively related to the QKI histochemistry score. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 markers, including CD63 and CD81, through western blotting (Fig. 7b). further verify the regulatory role of circNRIP1 in glycoly- sis via miR-149-5p, the extracellular acidification rate (ECAR) was measured. As expected, knockdown of both miR-149-5p and circNRIP1 rescued the reduced glycoly- sis rate and glycolytic capacity achieved when knocking down only circNRIP1 (Additional file 3: Figure S3d). The aforementioned results indicate that circNRIP1 could regulate cellular metabolism, particularly glycolysis, via miR-149-5p. Additionally, we previously proved that cir- cNRIP1 could promote the metastasis of GC cells via EMT. Thus, we also investigated the aforementioned EMT markers under treatment with miR-149-5p inhibi- tors and circNRIP1 siRNA. We found that knockdown of circNRIP1 could downregulate the above anabolism tar- gets and mesenchymal cell markers, while co-transfection with a miR-149-5p inhibitor could abolish the aforemen- tioned results (Fig. 6a, Additional file 7: Figure S7a). Exosomal circNRIP1 regulates AKT1 expression as well as EMT in vitro and promotes metastasis in vivo First, we hypothesized that there is exosomal communi- cation between GC cells. To verify our assumption, we purified exosomes in plasma from 40 GC patients and 40 normal subjects, and we determined the significantly higher expression level of circNRIP1 in plasmatic exo- somes from GC patients (Fig. 7a). Next, we used a trans- mission electron microscope (TEM) to determine the existence and morphology of exosomes purified from GC cell medium (exosome-free FBS) (Fig. 7b). We fur- ther confirmed the exosomes by detecting protein Zhang et al. Molecular Cancer (2019) 18:20 Page 19 of 24 Zhang et al. Molecular Cancer Fig 9 (See legend on next page ) Fig. 9 (See legend on next page.) Fig. 9 (See legend on next page.) Page 20 of 24 Zhang et al. Molecular Cancer (2019) 18:20 (See figure on previous page.) Fig. 9 CircNRIP1 plays a tumour promotor role during GC growth in vivo. a. The circNRIP1 overexpression plasmid and cholesterol-conjugated circNRIP1 siRNA were continuously injected intratumorally into PDX mice. b. The donor patients were clinically characterized. c. The engrafted tumours were histopathologically analysed, scale bar = 100 μm. d. We found that circNRIP knockdown in vivo significantly blocked tumour growth in terms of tumour weight and volume relative to the negative control group, whereas overexpression of circNRIP1 promoted the growth of xenografted tumours. e. qRT-PCR was performed in PDX tumours, and we found increased and persistent levels of circNRIP1 in the circNRIP1 overexpression plasmid-treated groups relative to the NC group and lower levels of circNRIP1 in the long-term circNRIP1 siRNA-treated groups. f. The expression levels of p-AKT1, p-mTOR, PFK, LC3 and EMT markers were observed in PDX tumour tissues by western blotting. g. The expression levels of AKT1 and mTOR were again observed in PDX tumours by IHC, scale bar = 100 μm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 I3QB are indispensable for the production of circNRIP1. In the next step, we attempted to determine whether QKI could regulate circNRIP1 formation post-transcriptionally during GC development. We knocked down QKI and observed a significant reduction in circNRIP1 but not pre-mNRIP1 or mNRIP1 (Fig. 8d, Additional file 9: Figure S9c). In addition, we performed an RNA immunoprecipitation (RIP) experiment to ob- serve whether QKI could bind I1QB or I3QB. The expression of circNRIP1 in GC can be regulated by QKI The majority of circRNAs originate from exons flanked with introns. CircRNA formation was reported to be regu- lated by several RNA binding proteins targeting specific se- quence motifs within the flanking introns. When bound to RNA-binding proteins, the aforementioned sequences are entwined into RNA duplexes and then undergo back-splicing editing to finally become circRNAs [45, 46]. Thus, we assumed that circNRIP1 formation is controlled under a similar mechanism. Quaking (QKI) is one of the RNA binding proteins reported to be a major regulator of circRNA biogenesis in EMT [47]. Considering the promo- tor role of circNRIP1 in EMT, we attempted to determine whether QKI could target specific binding sequences within NRIP1 pre-mRNA to promote circNRIP1 formation. In summary, the upregulation of circNRIP1 was attrib- uted at least in part to the promotion of QKI in GC tissues. p p With circNRIP1 derived from exon 2 and exon 3, we first aligned flanking intron 1 and intron 3 of the NRIP1 gene to the known QKI binding motif and found four canonical QKI binding sequences. Two were located up- stream, while the other two were located downstream of the circNRIP1-forming splice sites (Fig. 8a). We desig- nated these binding sequences as I1QB (intron 1 QKI binding sequences), encompassing the two upstream QKI binding sites, and I3QB (intron 3 QKI binding se- quences), including the two downstream QKI binding sites. Subsequently, we constructed a series of mutation plasmids (pZW1) by either mutating I1QB or I3QB (#2,#1) or mutating both I1QB and I3QB (#3), and we also constructed a wild-type plasmid spanning intron 1 to intron 3 (#4) to verify whether circNRIP1 was pro- moted by I1QB and I3QB (Fig. 8a). We observed that only the wild-type plasmid (#4), not the I1QB and I3QB deletion constructs (#1–3), could overexpress circNRIP1 by northern blot (Fig. 8b, Additional file 9: Figure S9a) and qRT-PCR (Fig. 8c, Additional file 9: Figure S9b) in GC cells. This result illustrates that both I1QB and Exosomal circNRIP1 regulates AKT1 expression as well as EMT in vitro and promotes metastasis in vivo Subse- quently, the enrichment of I1QB and I3QB was observed when we used an antibody against QKI (Fig. 8e, Additional file 9: Figure S9d). In the next step, we detected higher QKI expression levels in GC tumour tissues relative to adjacent normal stomach tissues among 40 patients by immunohistochemistry (Fig. 8f). We also performed qRT-PCR on these 40 patients and discovered that the expression level of circNRIP1 was positively correlated with the QKI histochemistry score (Fig. 8g). Additional file 8: Figure S8g) to characterize the cancer- ous nodes in lung tissues. Cancerous node size was con- sistent with luciferase intensity. In summary, we proved the role of circNRIP1 in pro- moting EMT and metastasis in vivo via exosomal communication. CircNRIP1 plays a tumour promotor role in GC growth in vivo Patient-derived xenograft (PDX) mouse models were fur- ther used to assess the effects of circNRIP1 on GC growth in vivo. The circNRIP1 overexpression plasmid and cholesterol-conjugated circNRIP1 siRNA were con- tinuously injected intratumorally into PDX mice (Fig. 9a). First, the donor patients were clinically characterized (Additional file 9: Figure S9e), and the engrafted tu- mours were histopathologically analysed (Additional file 9: Figure S9f). The expression level of circNRIP1 was higher, and the level of miR-149-5p was lower in these 3 GC clinical specimens relative to the adjacent normal tissues (Additional file 9: Figure S9f). Additionally, we found that circNRIP knockdown in vivo significantly blocked tumour growth in terms of tumour weight and volume relative to the negative control group, whereas the overexpression of circNRIP1 promoted the growth of xenografted tumours (Fig. 9b, Additional file 9: Figure S9g). Furthermore, qRT-PCR was performed in PDX tu- mours, and we found increased and persistent levels of Page 21 of 24 Page 21 of 24 Zhang et al. Molecular Cancer (2019) 18:20 circNRIP1 in the circNRIP1 overexpression plasmid-treated groups relative to the NC group and lower levels of circNRIP1 in long-term circNRIP1 siRNA-treated groups (Fig. 9c). The expression levels of p-AKT1, p-mTOR, PFK and LC3 were observed in PDX tumours (Fig. 9d). Moreover, AKT1 and mTOR expression levels were also determined by immuno- histochemistry (Fig. 9e). The aforementioned results suggested that circNRIP1 promoted energy production activities, such as the Warburg effect, and inhibited catabolic activities, such as autophagy, by activating the AKT1/mTOR signalling pathway to ultimately favour GC tumour growth in vivo. Considering that circNRIP1 could successfully upregulate EMT markers in GC cells and be transmitted by exosomes to pro- mote GC metastasis via EMT, we again verified the positive interaction between circNRIP1 and EMT in the aforementioned PDX tumour tissues (Fig. 9f). MiR-149-5p inhibition phenocopied circNRIP1 overex- pression in GC cells, and miR-149-5p overexpression blocked the malignant behaviours of circNRIP1. More- over, we proved that circNRIP1 could be transmitted by exosomal communication between GC cells, and exoso- mal circNRIP1 could further promote tumour metastasis in vivo. We also demonstrated that the RNA binding protein QKI could promote the circular transcription of the NRIP1 gene. In the final step, the tumour promotor role of circNRIP1 was verified in PDX models. Discussion In our study, we performed next-generation sequencing to detect differentially expressed circRNAs within GC tissues relative to adjacent normal stomach tissues. We selected circNRIP1, an upregulated circRNA with a high fold-change and significant P value in tumour tissues, to investigate its regulatory role in GC progression. First, we applied bioinformatic prediction and RNA immuno- precipitation to verify whether miR-149-5p is the bind- ing miRNA of circNRIP1. Subsequently, we utilized a dual-luciferase assay to verify their complementary bind- ing. Many researchers have reported the regulatory role of miR-149-5p on AKT1 in other cancers [48, 49]. In the next phase, the expression levels of circNRIP1 and AKT1, which is a ceRNA of circNRIP1, were both veri- fied by polymerase chain reaction (qRT-PCR) in GC tis- sues and GC cell lines. Moreover, the AKT/mTOR axis is a classic signalling pathway that sustains energy homeostasis through energy production activities, such as the Warburg, effect to satisfy the proliferation needs of GC cells [50, 51]. Additionally, AKT/mTOR enables anabolic metabolism, such as protein synthesis, and blocks catabolic activities, such as autophagy, to ultim- ately favour cell growth in GC [52]. Additionally, the AKT/mTOR axis exerts a positive role on EMT, which promotes tumour metastasis [53, 54]. Thus, we sought to determine the interactions between the aforemen- tioned metabolic activities and circNRIP1. With the development of our knowledge of cir- cRNAs, many researchers have recognized that cir- cRNAs do not only act as miRNA sponges, and some circRNAs can sponge trans-acting elements to pro- mote or block the transcription of parental genes. Moreover, certain exonic-intronic circRNAs (EIciR- NAs) gather in the nucleus and bind to the linear transcripts of their parental genes to mediate mRNA translation. Additionally, some circRNAs are even translated into proteins to fulfil crucial biological functions, whereas several circRNAs are wrapped into exosomes to promote tumour metastasis. CircNRIP1 plays a tumour promotor role in GC growth in vivo Although the mechanisms explaining how circRNA acts as a regulator during carcinogenesis and cancer pro- gression have not been fully elucidated, many re- searchers have stated that circRNAs can function as ceRNAs in tumour biology [55, 56]. The ceRNA hypoth- esis is derived from many evidence-based reports and il- lustrates interactions between non-coding RNAs and coding RNAs via miRNA mediation, as well as how non-coding RNAs compete with each other to absorb miRNA and finally affect tumour development at the posttranscriptional level. The biogenesis of circRNAs is now generally believed to occur via non-linear reverse splicing [57]. CircRNAs are stable relative to miRNAs because their 5′ cap and 3′ tail are buried in the loop [14]. Many researchers have reported that circRNAs act as miRNA sponges to modulate the expression of tumour suppressor or promotor genes through the circRNA-miRNA-mRNA axis [58]. In our study, we showed that circNRIP1 acts as a sponge absorbing miR-149-5p to modulate AKT1 expression in GC. g Collectively, intratumoral knockdown of circNRIP1 ef- fectively reduced PDX tumour growth in BALB/c nude mice via downstream metabolism alterations mediated by the AKT/mTOR signalling pathway. Additional files We used qRT-PCR to verify the circNRIP1 silencing efficiency and rule out the possibility that circNRIP1 siRNA might exert effects on the mRNA level of NRIP1 (linear NRIP1). (B). We detected lower expression levels of AKT1 by qRT-PCR after knocking down circNRIP1 in MKN-45 and BGC-823 GC cells. (C). The knockdown of circNRIP1 successfully reduced the migration ability of GC cells, and overexpression of circNRIP1 exerted the opposite effect on migration as indicated by the wound healing assay, scale bar = 100 µm. (D). The sequences and transfection efficiencies of miR-149-5p mimics and inhibitors were confirmed by qRT-PCR. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 1463 kb) Additional file 3: Figure S3. (A). We performed a series of glycolysis detection experiments. We found that knockdown of circNRIP1 reduced lactate contents, glucose uptake and ATP production in MKN-45 and BGC-823 cells. However, the reduction in glycolysis activity was restored when we knocked down both circNRIP1 and miR-149-5p. (D). The extracellular acidification rate (ECAR) was measured. Knockdown of both miR-149-5p and circNRIP1 rescued the reduced glycolysis rate and glycolytic capacity observed when knocking down only circNRIP1. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 350 kb) Additional file 3: Figure S3. (A). We performed a series of glycolysis detection experiments. We found that knockdown of circNRIP1 reduced lactate contents, glucose uptake and ATP production in MKN-45 and BGC-823 cells. However, the reduction in glycolysis activity was restored when we knocked down both circNRIP1 and miR-149-5p. (D). The extracellular acidification rate (ECAR) was measured. Knockdown of both miR-149-5p and circNRIP1 rescued the reduced glycolysis rate and glycolytic capacity observed when knocking down only circNRIP1. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 350 kb) Additional file 9: Figure S9. (A). We observed that only the wild-type plasmid (#4), and neither the I1QB nor I3QB deletion constructs (#1-3), could overexpress circNRIP1 according to northern blotting in BGC-823 cells. (B). We observed that only the wild-type plasmid (#4), and neither the I1QB nor I3QB deletion constructs (#1-3), could overexpress circNRIP1 according to qRT-PCR in BGC-823 cells. (C). We knocked down QKI and observed a significant reduction in circNRIP1 but not pre-mNRIP1 or mNRIP1 in BGC-823 cells. (D). Conclusions We demonstrated that circNRIP1 is significantly upregu- lated in human gastric cancer tissues and can success- fully sponge miR-149-5p to promote the proliferation, migration and invasion of GC cells. We also found that inhibition of circNRIP1 can block the malignant behav- iour of GC cells through the AKT1/mTOR signalling pathway. We proved that circNRIP1 assumes the role of a miRNA sponge and that circNRIP1 inhibition will be a promising therapeutic target in GC in the years to come. According to our experiments and analysis, circNRIP1 knockdown successfully blocked the proliferation, migra- tion, invasion and AKT1 expression levels of GC cells. Page 22 of 24 Zhang et al. Molecular Cancer (2019) 18:20 Zhang et al. Molecular Cancer Abbreviations ′ R ′ Additional file 6: Figure S6. (A). We observed that circNRIP1 silencing significantly inhibited colony formation ability, and overexpression of circNRIP1 exerted the opposite effect on colony formation in BGC-823 cells. (B). We observed that circNRIP1 silencing significantly inhibited the cell proliferation rate, as indicated by the CCK8 assay, and overexpression of circNRIP1 exerted the opposite effect on the GC cell proliferation ratein BGC-823 cells. (C). We observed that circNRIP1 silencing significantly inhibited DNA synthesis, as determined by the Edu assay, and overexpression of circNRIP1 exerted the opposite effect on GC cell DNA synthesis as indicated by the EDU assayin BGC-823 cells, scale bar = 100 µm. (D). Knockdown of circNRIP1 successfully reduced the migration and invasion ability of GC cells, and overexpression of circNRIP1 exerted the opposite effect on metastasis, as indicated by the transwell assayin BGC-823 cells, scale bar = 200 µm. (E). We observed that both circNRIP1 knockdown and miR-149-5p overexpression significantly inhibited the growth of the organoid models, while circNRIP1 overexpression Additional file 6: Figure S6. (A). We observed that circNRIP1 silencing significantly inhibited colony formation ability, and overexpression of circNRIP1 exerted the opposite effect on colony formation in BGC-823 cells. (B). We observed that circNRIP1 silencing significantly inhibited the cell proliferation rate, as indicated by the CCK8 assay, and overexpression of circNRIP1 exerted the opposite effect on the GC cell proliferation ratein BGC-823 cells. (C). We observed that circNRIP1 silencing significantly inhibited DNA synthesis, as determined by the Edu assay, and overexpression of circNRIP1 exerted the opposite effect on GC cell DNA synthesis as indicated by the EDU assayin BGC-823 cells, scale bar = 100 µm. (D). Knockdown of circNRIP1 successfully reduced the migration and invasion ability of GC cells, and overexpression of circNRIP1 exerted the opposite effect on metastasis, as indicated by the transwell assayin BGC-823 cells, scale bar = 200 µm. (E). We observed that both circNRIP1 knockdown and miR-149-5p overexpression significantly inhibited the growth of the organoid models, while circNRIP1 overexpression 3′-UTR: 3′-Untranslated region; DFS: Disease-free survival; EMT: Epithelial- mesenchymal transition; GC: Gastric cancer; I1QB: Intron 1 QKI binding sequences; I3QB: Intron 3 QKI binding sequences; mTOR: Mammalian target of rapamycin; OS: Overall survival; PDX: Patient-derived xenograft; QKI: Quaking; qRT-PCR: Quantitative real-time PCR,; siRNA: Short interfering RNA Additional files Enrichment of I1QB and I3QB was observed when we used an antibody against QKI in BGC-823 cells. (E). The donor patients were clinically characterized. (F). The engrafted tumours were histopathologically analysed, scale bar = 100 µm. (G). We found that circNRIP knockdown in vivo significantly blocked tumour growth in terms of tumour weight and volume relative to the negative control group, whereas overexpression of circNRIP1 promoted the growth of xenografted tumours All data are presented as the mean ± SEM *p < Additional file 4: Figure S4. (A). A cluster heat map was used to show the expression variations of these circRNA transcripts in cancerous tissues relative to matched normal tissues. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 1364 kb) Additional file 4: Figure S4. (A). A cluster heat map was used to show the expression variations of these circRNA transcripts in cancerous tissues relative to matched normal tissues. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 1364 kb) Additional file 5: Figure S5. (A). Potential binding between circNRIP1 and miR-149-5p based on their complementary sequences. (B). We confirmed that the low level of miR-149-5p was positively correlated with the OS (median survival of 58 months vs 21 months; P= 0.0007, log-rank test) and DFS (median survival of 56 months vs 19 months; P= 0.0002, log-rank test) of GC patients. (C). We found that AKT1 was significantly upregulated in GC tissues (415 GC tissues vs 34 normal tissues), and patients with high levels of AKT1 (298 GC tissues vs 94 normal tissues) had lower OS based on an analysis of the TCGA database. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 2223 kb) xenografted tumours. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 6448 kb) Additional files (TIF 3849 kb) Additional file 8: Figure S8. (A). We used a transmission electron microscope (TEM) to determine the existence and morphology of exosomes purified from GC cell medium (exosome-free FBS) in the BGC- 823 cells, scale bar = 25 µm. (B). Red exosome signals were found in the cytoplasm of GFP-labelled tumour cells when exo-RFP GC cells were mixed with the same amount of GFP-labelled GC cells for 72 hours in the BGC-823 cells, scale bar = 50 µm. (C). We than purified exosomes and added them into GFP-labelled MKN-45 or BGC-823 GC cells. The red signal of circNRIP1 similarly appeared in the cytoplasm of GFP-labelled GC cells after 72 hours in the BGC-823 cells, scale bar = 50 µm. (D). We performed qRT-PCR and detected higher circNRIP1 expression in exosomes purified from circNRIP1-overexpressing GC cells relative to those from NC cells in the BGC-823 cells. (E). We detected upregulated AKT1, mTOR and EMT markers in GC cells by co-culturing them with exosomes of OV circNRIP1 GC cells (OV exosomes) for 72 h via western blot in the BGC-823 cells. (F). According to the luciferase Intensities detected in the thoracic cavity, we found that GC cells treated with OV exosomes showed higher metastasis potential in the BGC-823 cells. (G). We harvested lung tissues for H&E staining to characterize the cancerous nodes. Cancerous node size was consistent with luciferase intensity, scale bar = 200 µm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 7218 kb) Additional file 2: Figure S2. (A). We used qRT-PCR to verify the circNRIP1 silencing efficiency and rule out the possibility that circNRIP1 siRNA might exert effects on the mRNA level of NRIP1 (linear NRIP1). (B). We detected lower expression levels of AKT1 by qRT-PCR after knocking down circNRIP1 in MKN-45 and BGC-823 GC cells. (C). The knockdown of circNRIP1 successfully reduced the migration ability of GC cells, and overexpression of circNRIP1 exerted the opposite effect on migration as indicated by the wound healing assay, scale bar = 100 µm. (D). The sequences and transfection efficiencies of miR-149-5p mimics and inhibitors were confirmed by qRT-PCR. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 1463 kb) Additional file 2: Figure S2. (A). Additional files promoted organoid model survival. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 4931 kb) Additional file 1: Figure S1. (A). We verified the significantly upregulated pull-down efficiency of the circNRIP1 probe in MKN-45 and BGC-823 cells transfected with the circNRIP1 overexpression plasmid (pcDNA3.1). (B). We observed that miR-149-5p silencing promoted the cell proliferation rate as indicated by the CCK8 assay, and overexpression of miR-149-5p exerted the opposite effect on the GC cell proliferation rate. (C). We observed that miR-149-5p silencing significantly promoted DNA synthesis as determined by the Edu assay, and overexpression of miR-149-5p exerted the opposite effect on GC cell DNA synthesis, scale bar = 100 µm. (D). The knockdown of miR-149-5p successfully promoted the migration and invasion ability of GC cells, and overexpression of miR- 149-5p exerted the opposite effect on metastasis, scale bar = 200 µm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 2828 kb) Additional file 7: Figure S7. (A). Knockdown of circNRIP1 and miR-149- 5p significantly reversed the expression levels of AKT1/mTOR pathway molecules, certain metabolism markers and EMT markers achieved by knocking down only circNRIP1 in BGC-823 cells. (B). We observed that the reduction of GC cell proliferation mediated by circNRIP1 knockdown was successfully blocked by miR-149-5p inhibition in BGC-823 cells, scale bar = 100 µm. (C). We observed that the reduction of metastasis of GC cells mediated by circNRIP1 knockdown was successfully blocked by miR-149- 5p inhibition in BGC-823 cells, scale bar = 100 µm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (TIF 3849 kb) Additional file 7: Figure S7. (A). Knockdown of circNRIP1 and miR-149- 5p significantly reversed the expression levels of AKT1/mTOR pathway molecules, certain metabolism markers and EMT markers achieved by knocking down only circNRIP1 in BGC-823 cells. (B). We observed that the reduction of GC cell proliferation mediated by circNRIP1 knockdown was successfully blocked by miR-149-5p inhibition in BGC-823 cells, scale bar = 100 µm. (C). We observed that the reduction of metastasis of GC cells mediated by circNRIP1 knockdown was successfully blocked by miR-149- 5p inhibition in BGC-823 cells, scale bar = 100 µm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. 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Funding g This work was partially supported by the National Natural Science Foundation of China (81572362); the National Natural Science Foundation Project of International Cooperation (NSFC-NIH, 81361120398); the Primary Research & Development Plan of Jiangsu Province (BE2016786); the Program for Development of Innovative Research Team in the First Affiliated Hospital of NJMU; the Priority Academic Program Development of Jiangsu Higher Page 23 of 24 Zhang et al. Molecular Cancer (2019) 18:20 Zhang et al. Molecular Cancer (2019) 18:20 Zhang et al. Molecular Cancer (2019) 18:20 Education Institutions (PAPD, JX10231801); the 333 Project of Jiangsu Province (BRA2015474); Jiangsu Key Medical Discipline (General Surgery) (ZDXKA2016005); and the Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University. 11. Thomson DW, Dinger ME. Endogenous microRNA sponges: evidence and controversy. Nat Rev Genet. 2016;17(5):272–83. 12. Zhang Y, Zhang XO, Chen T, Xiang JF, Yin QF, Xing YH, et al. Circular intronic long noncoding RNAs. Mol Cell. 2013;51:792–806. 13. Li Z, Huang C, Bao C, Chen L, Lin M, Wang X, et al. Exon-intron circular RNAs regulate transcription in the nucleus. Nat Struct Mol Biol. 2015;22:256–64. Availability of data and materials 14. Jeck WR, Sharpless NE. Detecting and characterizing circular RNAs. Nat Biotechnol. 2014;32:453–461. The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. 15. Qu S, Zhong Y, Shang R, Zhang X, Song W, Kjems J, et al. The emerging landscape of circular RNA in life processes. RNA Biol. 2016;11:1–8. Authors’ contributions Qu S, Yang X, Li X, Wang J, Gao Y, Shang R, Sun W, Dou K, Li H. Circ 16. Qu S, Yang X, Li X, Wang J, Gao Y, Shang R, Sun W, Dou K, Li H. Circular RNA: a new star of noncoding RNAs. Cancer Lett. 2015;365(2):141–8. SW and HW participated in the experimental design and data analysis. JC performed some of the experiments. XZ conceived of the project, performed most of the experiments and was a major contributor in writing the manuscript. All of the authors read and approved the final manuscript. 17. Yang Y, et al. Extensive translation of circular RNAs driven by N(6)- methyladenosine. Cell Res. 2017;27(5):626–41. 17. Yang Y, et al. Extensive translation of circular RNAs driven by N 18. Legnini I, et al. Circ-ZNF609 is a circular RNA that can be translated and functions in Myogenesis. Mol Cell. 2017;66(1):22–37.e9. Received: 16 October 2018 Accepted: 27 December 2018 30. Polivka JJ, Janku F. Molecular targets for cancer therapy in the PI3K/AKT/ mTOR pathway. Pharmacol Ther. 2014;142(2):164–75. mTOR pathway. Pharmacol Ther. 2014;142(2):164–75. Author details 1D f 1Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, No.300, Guangzhou Road, Nanjing, Jiangsu Province, China. 2Department of Surgical Oncology, University of Miami, Miami, USA. 3Collaborative Innovation Center For Cancer Personalized Medicine, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China. 4Department of General Surgery, The Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University, Huaian 223300, Jiangsu, China. g gy 27. Xia P, Xu XY. PI3K/Akt/mTOR signaling pathway in cancer stem cells: from basic research to clinical application. Am J Cancer Res. 2015;5(5):1602–9. y 27. Xia P, Xu XY. PI3K/Akt/mTOR signaling pathway in cancer stem cells: from basic research to clinical application. Am J Cancer Res. 2015;5(5):1602–9. 28. Su MA, et al. An invertebrate Warburg effect: a shrimp virus achieves successful replication by altering the host metabolome via the PI3K-Akt- mTOR pathway. PLoS Pathog. 2014;10(6):e1004196. 29. Lu CL, et al. Tumor cells switch to mitochondrial oxidative phosphorylation under radiation via mTOR-mediated hexokinase II inhibition--a Warburg- reversing effect. PLoS One. 2015;10(3):e0121046. Received: 16 October 2018 Accepted: 27 December 2018 Received: 16 October 2018 Accepted: 27 December 2018 Consent for publication Not applicable. 23. Zhong Y, Du Y, Yang X, Mo Y, Fan C, Xiong F, Ren D, Ye X, Li C, Wang Y, Wei F, Guo C, Wu X, Li X, Li Y, Li G, Zeng Z, Xiong W. Circular RNAs function as ceRNAs to regulate and control human cancer progression. Mol Canc. 2018;17:79. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate 19. Hansen TB, et al. Natural RNA circles function as efficient microRNA sponges. Nature. 2013;495(7441):384–8. We obtained human GC tissues and adjacent normal tissues from GC patients at the First Affiliated Hospital of Nanjing Medical University. The ethics committee of Nanjing Medical University approved all of our experiments. BALB/c-null mice and SCID mice were purchased from the animal centre of Nanjing Medical University (Nanjing, China) and bred under special pathogen-free (SPF) conditions. We followed protocols approved by the Institutional Committee on Animal Care of Nanjing Medical University. sponges. Nature. 2013;495(7441):384–8. 20. Liu W, et al. Circular RNA hsa_circRNA_103809 promotes lung cancer progression via facilitating ZNF121-dependent MYC expression by sequestering miR-4302. Biochem Biophys Res Commun. 2018;500(4):846–5 21. He JH, et al. The CircRNA-ACAP2/Hsa-miR-21-5p/ Tiam1 regulatory feedback circuit affects the proliferation, migration, and invasion of colon cancer SW480 cells. Cell Physiol Biochem. 2018;49(4):1539–50. 22. Dong Y, He D, Peng Z, Peng W, Shi W, Wang J, et al. Circular RNAs in cancer: an emerging key player. J Hematol Oncol. 2017;10:2. Consent for publication Not applicable. 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“Not just dogs, but rabid dogs”: tensions and conflicts amongst research volunteers in Malawi
Global bioethics
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“Not just dogs, but rabid dogs”: tensions and conflicts amongst research volunteers in Malawi Mackwellings Phiri, Kate Gooding, Deborah Nyirenda, Rodrick Sambakuns Moses Kelly Kumwenda & Nicola Desmond Global Bioethics ISSN: 1128-7462 (Print) 1591-7398 (Online) Journal homepage: http://www.tandfonline.com/loi/rgbe20 Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=rgbe20 ABSTRACT ABSTRACT Building trust between researchers and communities involved in research is one goal of community engagement. This paper examines the implications of community engagement for trust within communities, including trust among community volunteers who assist with research and between these volunteers and other community members. We describe the experiences of two groups of community volunteers recruited as part of an HIV and TB intervention trial in Malawi: cluster representatives, recruited both to act as key informants for TB suspects and mortality reporting and to identify and report community concerns, and community counsellors, recruited to provide semi-supervised HIV self-testing. We examine tensions experienced due to playing multiple roles, and the implications of volunteer responsibilities for short- and long-term community relationships. Data was collected through a workshop, in-depth interviews and focus group discussions with volunteers and community members. While the volunteer system initially enhanced trust among volunteers and with the community, relationships deteriorated when cluster representatives assumed an additional supervisory role part- way through the trial. Combined with challenging recruitment targets and unequal power relations between volunteers, this new role damaged trust, with implications for volunteer well- being and social relationships. These experiences suggest researchers should consider potential social implications when designing community engagement systems. Mackwellings Phiri, Kate Gooding, Deborah Nyirenda, Rodrick Sambakunsi, Moses Kelly Kumwenda & Nicola Desmond To cite this article: Mackwellings Phiri, Kate Gooding, Deborah Nyirenda, Rodrick Sambakunsi, Moses Kelly Kumwenda & Nicola Desmond (2018) “Not just dogs, but rabid dogs”: tensions and conflicts amongst research volunteers in Malawi, Global Bioethics, 29:1, 65-80, DOI: 10.1080/11287462.2018.1509925 To link to this article: https://doi.org/10.1080/11287462.2018.1509925 © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 03 Sep 2018. Submit your article to this journal Article views: 118 View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=rgbe20 GLOBAL BIOETHICS 2018, VOL. 29, NO. 1, 65–80 https://doi.org/10.1080/11287462.2018.1509925 © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 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. CONTACT Mackwellings Phiri mackmaganizo@hotmail.com, mmphiri@mlw.mw Malawi Liverpool Wellcome Trust Clinical Research Programme, Chichiri, P.O. Box 30096, Chichiri, Blantyre, Malawi “Not just dogs, but rabid dogs”: tensions and conflicts amongst research volunteers in Malawi Mackwellings Phiri a, Kate Gooding a,b, Deborah Nyirenda a, Rodrick Sambakunsia, Moses Kelly Kumwenda a and Nicola Desmonda,b Mackwellings Phiri a, Kate Gooding a,b, Deborah Nyirenda a, Rodrick Sambakunsia, Moses Kelly Kumwenda a and Nicola Desmonda,b aMalawi Liverpool Welcome Trust Clinical Research Programme, Blantyre, Malawi; bLiverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK ARTICLE HISTORY Received 23 October 2017 Accepted 5 August 2018 KEYWORDS Trust; community representative; community counsellor; community engagement; HIV self-testing ARTICLE HISTORY Received 23 October 2017 Accepted 5 August 2018 CONTACT Mackwellings Phiri mackmaganizo@hotmail.com, mmphiri@mlw.mw Malawi Liverpool Wellc Clinical Research Programme, Chichiri, P.O. Box 30096, Chichiri, Blantyre, Malawi © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecom licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is pro Introduction Community engagement (CE) is increasingly seen as important in biomedical research. There is no universal understanding of the terms community or community engagement. For instance, a community may refer to a group of people residing within a specific geo- graphical location, a group with similar characteristics, or a group served by one health facility (Gbadegesin & Wendler, 2006; Marsh, Kamuya, Rowa, Gikonyo, & Molyneux, M. PHIRI ET AL. 66 2008; Tindana et al., 2007). We define community as a group of people living within a specified geographical location, and CE as the practice of relevant partners working together to deliver shared goals and interests (Tindana et al., 2007). The attention to community engagement is based largely on ethical goals (Simon & Mosavel, 2010). These goals include ensuring safety of research subjects through under- standing community views on the research and identifying potential risks and means of averting those dangers (Anderson & Solomon, 2013; Dickert & Sugarman, 2005), ensuring that study procedures meet community needs (Boga et al., 2011), and protecting commu- nities from exploitation, particularly in developing country settings (Chantler et al., 2013; Gbadegesin & Wendler, 2006). Community participation in research decision-making can help to ensure that research responds to community needs, and ultimately that research benefits are shared equitably between researchers and participating communities (Pratt et al., 2013; Wallerstein & Duran, 2010). Supporting these aims, development of trust between communities and researchers is often understood as a key function of engage- ment (Anderson & Solomon, 2013; Dunn, 2011; Kamuya, Marsh, Kombe, Geissler, & Molyneux, 2013; Molyneux, Peshu, & Marsh, 2005). One approach to community engagement is the use of community volunteers as an interface between researchers and communities (Marsh et al., 2008). Terminologies for community members engaged in research vary, and include community health workers, community health helpers, village reporters (Chantler et al., 2013), commu- nity volunteers (Sambakunsi, Kumwenda, Choko, Corbett, & Desmond, 2015), or key informants (Jahn et al., 2007). The way that community volunteers are engaged in research takes different forms, for example, it may be as individuals or as members of community advisory groups or boards. The roles undertaken by commu- nity volunteers also vary. Introduction For example, they may be asked to act as representatives who share feedback from communities (Clodagh, 2015), a role more closely aligned to the ethical goals discussed above, or to play roles focused on undertaking or facilitating research, such as helping to recruit participants (Simon & Mosavel, 2010) or collect research data (Jahn et al., 2007) In such diverse roles, community volunteers, especially those working as recruiters or collecting data, may experience challenges, including tensions in meeting the needs of the research study whilst continuing to serve their communities. They may face incompatible requirements of meeting performance expectations while adhering to ethical practice (Kamuya, Theobald, et al., 2013), for example, ensuring high participation rates while respecting voluntary participation (Sambakunsi et al., 2015). Managing personal obli- gations alongside research needs can also create pressures for community volunteers and limit their ability to work effectively (Sambakunsi et al., 2015). These and other press- ures can lead to loss of resources and energy for community volunteers engaged in research, and cause distress and demotivation (Attree et al., 2011). Fieldworkers, who have more formal contracts with research institutions than research volunteers (Molyneux et al., 2013), may play similar roles and face similar challenges to those described for volunteers. A key issue highlighted in the literature on fieldworkers is the role of trust, both in enabling their work and as an outcome for their activities. For example, it is easier for fieldworkers to obtain consent if community members have confidence in them (Kamuya, Theobald, et al., 2013). However, closeness of fieldworkers to the community may also hinder trust in the fieldworkers (Chantler et al., 2013), because GLOBAL BIOETHICS 67 67 participants might be concerned about privacy and confidentiality (Molyneux et al., 2013). Furthermore, fieldworkers may also find it hard to manage pressure linked to set recruit- ment targets and to maintain respectful relationships with communities, and are some- times compelled to adopt seditious practices such as data forgery in order to meet performance expectations (Brown, Long, Weitz, & Milliken, 2001; Kingori & Gerrets, 2016). While there is growing discussion about the perspectives of community volunteers in developing countries, there is limited research focused specifically on their views about the inherent tensions around their roles. There is also limited evidence on the unintended and longer-term consequences of engagement initiatives for the engaged persons (Attree et al., 2011), including implications for trust. Introduction Trust is an interpersonal concept, centred between people, people and organizations, or people and events (Chantler et al., 2013). Trust can be voluntary or involuntary (Gilson, 2003). Voluntary trust is based on expectations of how others will behave in relation to yourself in the future. These expectations may be disappointed and, if so, will generate negative outcomes. Forms of conduct that usually underlie voluntary trust include being competent, open and dependable. Involuntary trust may exist in relationships that result from lack of choice or occur in the context of inequality, for example, relation- ships between health care providers and patients may appear as a form of dependency (Gilson, 2003). For the purpose of this article, we focus on voluntary trust between indi- viduals embedded within communities. This paper describes the experiences of two groups of community volunteers recruited to work within their own communities participating within an HIV/TB intervention trial in Malawi: cluster representatives (CRs) recruited as key informants for TB outcomes and mortality and concurrently as representing and reporting community concerns, and com- munity counsellors (CCs) recruited more formally to provide semi-supervised HIV self- testing. We examine the tensions experienced as a result of being asked to play multiple roles, and the implications of their volunteer responsibilities for short- and long-term community relationships and trust. Research methods Study setting: the HitTB intervention as a case study Community-driven selection of CCs and CRs CCs and CRs were identified and selected concurrently through an elective process led by the community. The selection took place during community sensitization meetings orga- nized by the research team. Before selection, the research team provided community members with basic nomination criteria, which specified that a CC/CR be: willing to vol- unteer, a regular cluster resident (defined as sleeping in the area for more than a fortnight in every month), honest and able to maintain confidentiality. A counsellor was addition- ally required to have at least a Junior Secondary Certificate (GCSE equivalent) since they would be required to attend national HIV Testing and Counselling (HTC) training in order to deliver semi-supervised HST. When individuals had been put forward and votes taken, local leaders and other stakeholders counted the votes and consolidated results publicly. Study setting: the HitTB intervention as a case study The study presented here took place between March 2015 and December 2015, in urban Blantyre, Malawi. It emerged out of a community-based intervention trial that ran from 2012 to 2015 investigating whether community-level active TB case finding and intensified TB prevention through regular semi-supervised, HIV self-testing (HST) and access to treatment could reduce TB incidence (Choko et al., 2015; Kumwenda et al., 2014; Kum- wenda et al., 2018; Sambakunsi et al., 2015). This trial was implemented in 28 clusters (14 intervention and 14 control) of high density and low-resource urban settlements with relatively high rates of social migration and many residents reliant on a range of informal and unreliable income-generating activities. The HST study was implemented in the intervention clusters only, whereas all clusters received the standard of care which included active TB case-finding and extended routine monitoring and evaluation, enumeration of households and survey to document if participants know their HIV M. PHIRI ET AL. 68 68 status and whether they are receiving care and support for increased Isoniazid Preventive Therapy through routine HIV care services during the course of the trial. py g g Map showing intervention and control clusters within high density and low-resource urban communities in Blantyre, Malawi. To support the trial, a community liaison system was established as a key component of an integrated community engagement strategy, which aimed at ensuring there was regular and sustained dialogue between the community and the research team. A total of 112 vol- unteers (56 men and 56 women) were recruited as cluster representatives (CRs), four within each intervention and control cluster. The HST intervention was driven by another set of volunteers in intervention clusters only; community counsellors (CCs), 28 in total, two representing each intervention cluster (14 men and 14 women). So, half To support the trial, a community liaison system was established as a key component of an integrated community engagement strategy, which aimed at ensuring there was regular and sustained dialogue between the community and the research team. A total of 112 vol- unteers (56 men and 56 women) were recruited as cluster representatives (CRs), four within each intervention and control cluster. The HST intervention was driven by another set of volunteers in intervention clusters only; community counsellors (CCs), 28 in total, two representing each intervention cluster (14 men and 14 women). So, half the number of CRs worked in the same intervention clusters as CCs. Official functions of CCs and CRs CCs were entrusted with the delivery of home-based HST services. Initially based at their own home with clients coming to them but eventually moving door-to-door to meet HST GLOBAL BIOETHICS 69 targets (Sambakunsi et al., 2015), they distributed HST test kits (OraQuick ADVANCE HIV I/II), counselled clients before and after HST, screened for TB among self-testing clients, and referred clients for appropriate health services. In contrast to the CCs, CRs had a number of roles: they were responsible for providing community feedback on the intervention, such as fears and concerns related to participation and conduct of the trial. They also helped with community sensitization through community meetings and door-to-door distribution of study brochures. Further, CRs collected and reported infor- mation on mortality and potential cases of TB occurring in their cluster. If someone had prolonged cough for example, CRs reported the individual to CCs for TB screening; and if someone died, they reported to study nurses who then attended to conduct a verbal autopsy. This was part of a key informant system designed to monitor mortality and TB incidence both in the intervention and control clusters. This gave CRs two potentially conflicting roles; one as key informants for the research team and the other of representing the interests and concerns of the community. Though CCs and CRs served on an officially voluntary basis, they received some mon- etary benefits for their work, offered as a token of appreciation for their time but dispa- rately apportioned: CCs received MWK 14,000 (approx. 56 USD in 2012) whereas CRs received |MWK 2,000 (approx. 11 USD) monthly. Data collection Data collection began with a one-day participatory workshop (PW), conducted to solicit broad ideas about how community representatives understood and experienced their dual role as key informants and representatives of the community. MP (research assistant) and RS (senior community liaison officer at the time) co-facilitated the work- shop, designed in such a way that issues could be explored freely and in the partici- pants’ own terms. MP previously worked on a qualitative longitudinal study that had recruited participants from HitTB and was familiar with the intervention trial. During this time, he had frequently interacted with the study communities during interviews and focus group discussions, and particularly with both CCs and CRs, who had always helped him with the recruitment of study participants. The rapport previously built between MP and the communities prior to his role in this study proved invaluable since participants felt free to discuss their experiences of the inter- vention trial openly with him. Seventeen CRs (9 men and 8 women) participated in the workshop, recruited from across intervention and control clusters. They were conveniently selected based on avail- ability and willingness to attend, rather than purposively according to specific predefined criteria. The workshop involved three sessions. First, participants sat in three smaller groups to discuss their roles and experiences, each group recording their ideas on a flipchart. Second, using the flipchart notes, each group presented their discussions. Third, the three groups merged for a single group discussion in which the two facilitators examined in-depth the issues that groups had separately raised. Each session lasted approximately 1 hour. We conducted further focus groups and interviews to complement findings of the PW. We conducted five focus groups: one with cluster residents, one with community counsellors, and three with cluster representatives. There were 8-12 participants per focus group, and the discussions lasted on average two hours. Because the topics being investigated were not gender sensitive, we combined men and women. We also conducted seven interviews with a sample of key stakeholders: one with a female cluster resident, three with CCs (one man and two women), and two with CRs (one man and one woman). The interviews lasted on average 30 minutes. Some of the workshop participants were purposively sampled for the focus groups and inter- views in order to follow up on issues reported from specific clusters. Additional duties for CRs following changes in procedures for recruitment by CCs CRs took on additional roles when the research team introduced recruitment targets for CCs. The intervention aimed to reach at least 80% HIV self-test uptake amongst adults aged 16 and above, resident in intervention clusters within a two-year timeframe. However, after one year of the study, the research team realized that uptake figures were lower than anticipated. Instead of 10 clients a week, CCs were on average recruiting 4 clients. This was largely due to a passive recruitment approach reliant on HST clients self-presenting at CCs homes. The community engagement team then held consultations with the principal investigator, field supervisors and CC representatives to identify strat- egies for improving recruitment through a door-to-door testing approach. Following these consultations, and given indications that CCs were deliberately delaying recruit- ment because of unhappiness with the amount of compensation they were getting, the research team decided to reimburse CCs based on performance. If a CC reached 100 clients in a month for example, they would receive an extra MWK40, 000 (approx. 240 USD in 2012). While the expectation was that improved compensation for CCs would increase testing uptake rates, the research team also wanted CCs to remain adherent to the study protocol when recruiting, and thus entrusted CRs with another role as watchdogs for quality assur- ance. To support this role and as a result of suspicions of false reporting by CCs, study nurses provided CRs with names of clients who had been reported to have self-tested, selected randomly from enrolment registers to check eligibility: residency within the desig- nated intervention clusters and aged 16 years or above. The research team informed CCs about quality assurance. However, CCs and communities were not informed that CRs were to play this role, and CRs were instructed not to share with CCs whatever they found. The possibility of this arrangement causing tensions and distrust between CCs and CRs was not foreseen at the time. 70 M. PHIRI ET AL. 70 M. PHIRI ET AL. Additional duties for CRs following changes in procedures for recruitment by CCs As a result of suspicions of developing conflict within the community-based interven- tion teams, we explored the experiences of the CRs and their multiple and often confl- icting roles as key informants, watchdogs and representatives of the community, interrogating the concept of simultaneous representation of two institutions; the com- munity and the research institution and the impact of this on their relationship in both the short and long term with two different social groups: community peers and CCs. Results Community misconceptions about the trial influenced their attitude towards community volunteers working for the trial (i.e. CRs and CCs). Alterations made to trial procedures to boost recruitment and safeguard data quality damaged the relationship between CCs and CRs and increased pressure for the CCs to perform. Data quality checks performed by CRs on self-tested clients diluted community trust in the CCs because of a perceived breach of confidentiality that CCs had initially assured clients during an informed consenting process. We discuss these findings in detail in this section. Data collection All interviews, focus groups and workshop discussions were conducted in Chichewa, the main local language within the study context, and audio recorded after obtaining written consent (Table 1). GLOBAL BIOETHICS 71 GLOBAL BIOETHICS 71 71 Table 1. Summary of study participants. Table 1. Summary of study participants. Participant type Gender Male Female IDI FGD PW IDI FGD PW CRs 1 15 9 1 10 8 CCs 1 6 2 6 Community members 4 1 4 Data analysis Data analysis and data collection ran concurrently and iteratively so that emerging findings could be followed up through later data collection. Flipchart notes from the work- shop were typed up and saved as data scripts. Audio recordings were transcribed verbatim in Chichewa, and all data scripts were imported into NVIVO 10 software (QSR, Mel- bourne, Australia) for better organization and analysis. MP and RS independently read the same four Chichewa transcripts and coded them inductively. Each author came up with a coding frame. The two separate coding frames were compared and discussed, and then merged to generate a single coding structure. After comparing the two coding frames, some of the codes were maintained and others were merged. The coded data were analysed using a thematic approach to identify key themes emerging from the data that were then discussed with co-authors. Representative quotes were extracted to represent each of these themes and translated into English. Study authorization Permission to conduct this study was obtained from The Malawi College of Medicine and The Liverpool School of Tropical Medicine research ethics committees. Initial distrust in the study concept transitions to trust through the roles of CRs and CCs Relations between CCs and CRs and the community they served changed over time, and oscillated between trust and distrust across each group. At the outset, the community reacted to the intervention with distrust due to unfamiliarity with HIV self-testing, an approach that residents viewed as peculiar and risky. 72 72 72 M. PHIRI ET AL. HIV self-testing was new. People had been used to finger pricking and were questioning: “Oral test kits, what kind of test kits are these? We’ve never seen this before.” Today, if something new is introduced, you are going to hear, “This is satanic, they want to collect blood from you, that thing will suck blood from you.” [FGD, female community member] This failure to comprehend how the procedure worked influenced ideas about the study being satanic, and resulted in the CRs being viewed with suspicion and rejected. People did not welcome us in their homes if we wanted to talk to them about the study. Some chased us away: “Go back! I don’t want to see you around my house! You might invite to my home the devils you worship in your organization!” Some would send their dogs after us. [PW, male CR] People did not welcome us in their homes if we wanted to talk to them about the study. Some chased us away: “Go back! I don’t want to see you around my house! You might invite to my home the devils you worship in your organization!” Some would send their dogs after us. [PW, male CR] Proximity to the community was another reason why people formerly did not trust CCs and CRs. Residents feared that their privacy regarding HIV status might be publicly dis- closed and were hesitant to approach the volunteers, as one community member explains: … we’re worried about confidentiality because they [CC] were coming from the same area as us. We would ask them, “How can we prove that you are going to protect our privacy?” … if you were going out, you were thinking that you might hear people talk about what you discussed with them, but a month went by without hearing anything from anyone. That’s when we proved that they were confidential. Initial distrust in the study concept transitions to trust through the roles of CRs and CCs [IDI, female community member] However, through the community mobilization that CRs performed, people got more informed about the study and assured about safety of their privacy, and community dis- trust in the CCs reversed: … if they gave you the paper [study brochure] to read, they would also tell you: “our colleagues will also stop by with oral test kits, everything will be confidential, and, if your test result is out, it’s your choice whether you want to tell them or not.” [FGD, male community member] Testimonies of demonstrated ability to safeguard clients’ privacy from those already self- tested also contributed to increasing community approval of the CRs: Testimonies of demonstrated ability to safeguard clients’ privacy from those already self- tested also contributed to increasing community approval of the CRs: We were handing out flyers and a woman asked her landlord: “Can I talk to these people about my issue? Can I trust them with my privacy?” The landlord said yes, and then she let us into her house and told us about her marriage problems including information about being initiated on HIV/AIDS treatment. [FGD, male CR] Community confidence in the volunteers was further negotiated through the role of local authorities. Village chiefs often requested their communities to welcome and listen to the researchers. As the subsequent quote illustrates, this indicated project authorization by the chiefs, and in turn increased trust and acceptability of the role of the CCs and CRs: Chiefs helped the study a lot because they informed people about what was happening in their areas. They announced at funerals: “People will be visiting you, they are from HitTB. Help them with the information they need because they are conducting a study.” When people heard this, they knew that the chief had allowed us to work in the area, and you would easily be accepted when you visited them. [FGD, male CR] Changes in study procedures amplify teamworking relations between CC d CR From the outset community-based CCs and CRs worked together as a unified team, mutually supporting each other’s roles. Initial passive self-presentation for HST meant 7 GLOBAL BIOETHICS 73 that CCs delivered the service directly from their homes and individuals were able to collect the kit and take it away with them. However, this initial approach faced challenges since firstly it relied on individuals taking the initiative to self-present and secondly, even when willing, residents often had problems locating where the CCs lived and thus in col- lecting the kit. This led to a change in approach with the encouragement of door-to-door sensitization to encourage residents to visit CCs, and required an additional role for the CRs to perform. If someone wanted HIV self-testing, CRs would escort them to or simply show them where the CCs lived. In return, CCs adopted an increased role in sup- porting the key informant component of CR activities, passing on information to CRs fol- lowing any deaths occurring within the cluster in which they were operating. This reciprocity developed into a mutual reliance between CCs and CRs and fostered trusting partnerships, as one CC explains: We considered CRs as colleagues. If they identified a TB case suspect, or came across someone critically ill, they passed the information on to us. [FGD, male CC] We considered CRs as colleagues. If they identified a TB case suspect, or came across someone critically ill, they passed the information on to us. [FGD, male CC] Disruption of trust between CRs and CCs through recruitment targets and watchdog roles … he invited me to his house and told me to mention names of all my relatives, dead and alive, using fake dates … he said they had given him a target to meet within 3 or so days … he gave Disruption of trust between CRs and CCs through recruitment targets and watchdog roles We don’t see each other eye to eye. They still blame us because one of them got sacked. [IDI, male CR] Over time and as the increase in incentives extended the pay differential between CCs and CRs, resentment built and increased tensions between those performing each role, exacer- bating growing social discord within study clusters which has endured beyond the com- pletion of the study. CCs looked upon themselves as superior to CRs due to higher compensation and the HTC training they received and, combined with the “watchdog” role of the CRs over their work ethic they often suspected CRs of wanting to steal their CC position. Counsellors were above us and reporting them seemed like you were interested in the counsellor position. [FGD, male CR] Counsellors were above us and reporting them seemed like you were interested in the counsellor osition. [FGD, male CR] The [HTC] training gave us job skills and they [CRs] were so bitter about it. They made false reports to the office, so that counsellors should look incompetent and not be trusted, because they were after the same position. [FGD, male CC] Though compensation and HTC training seemingly placed CCs above CRs, the watch- dog role implicitly gave the latter more social power over the former, especially since CRs’ reports on CCs’ performance now determined whether CCs remained in their role. More- over, the research team had instructed CRs not to share their findings with CCs, increasing suspicions of underlying subterfuge within communities. This disrupted the previously successful, existing social norm of mutual support between CCs and CRs and, as the quotes below illustrate, created mutual animosity: Our relationship with counsellors was like that of a cat and a rat … we were told not to disclose to the counsellors if we found that someone was recruited from outside the cluster. [FGD, male CR] They put in a dog [CR] ready to bite … [FGD, male CC] … and it was not just an ordinary dog, but a rabid one. [FGD, female CC] They put in a dog [CR] ready to bite … [FGD, male CC] … and it was not just an ordinary dog, but a rabid one. Disruption of trust between CRs and CCs through recruitment targets and watchdog roles As a result of insufficient coverage of HIV self-testing and the study requirement to achieve 80% uptake across each cluster a system of targets was introduced after a year of the intervention with incentivization for CCs. In order to facilitate the meeting of these targets and particularly to ensure reporting of numbers tested was accurate and reliable (those testing were actual cluster residents and were above 16 years of age), CRs were requested by the research staffto take on a further role. They became “watch- dogs” over the CCs, colleagues with whom they had previously worked hand in hand and naturally tensions emerged and previous mutual support between CCs and CRs was dra- matically reduced and in some cases stopped altogether. Cooperation stopped when CRs were given the responsibility of checking if study participants self-tested … [IDI, male CR] … especially when they had introduced Quality Assurance and used cluster representatives to enforce it, the enmity that was there [between us and CRs] was as if we were not working for the same organization. [FGD, male CC] For CCs the recruitment incentive linked to targets was significant in the context of daily livelihood challenges and a desire to increase income, whilst working within the study. On the one hand, it encouraged the CCs to work harder and spend more time recruiting. Targets made us work harder so that we can be rewarded. The allowance plus your usual monthly compensation meant that your income was improved. [FGD, female CC] On the other, the targets exerted immense pressure on the CCs as documented elsewhere (Sambakunsi et al., 2015), and created an incentive for the CCs to fabricate results such as the example below of a client describing how a CC made her self-test several times in order to meet a target: 74 74 M. PHIRI ET AL. 74 me test kits enough for the [ten] people I had mentioned and I used them on myself … he said he would give me money. [IDI, female community member] The CR watchdog role was meant to minimize such behaviours amongst CCs. In some cases, this monitoring role led to problems for the CCs. One CC was dismissed on account of making up results, and both the community and CCs blamed CRs for this dismissal: Some of the counsellors cheated and hated us for reporting them. Most of us are hated even today. Communities lose trust in CRs and CCs due to their multiple roles reigniting concerns around confidentiality Multiple allegiances were the primary reason for a shift in community perceptions towards CRs over time. In the event of death requiring verbal autopsy for example, CRs, as key informants charged with reporting the death, located the bereaved household for study nurses to conduct autopsies. Consequently, whilst they had been considered as represen- tatives of the community and routes to having their concerns heard, if a death occurred, CRs would be seen escorting members of the research team, and their previous role as community support would be replaced by the perception that they were on the side of the researchers representing their interests rather than those of the community. This led to a decrease in trust previously established between CRs and community members. When we had just the CR role, people knew we were on their side, and that we would stand up for them if anything bad happened concerning the study. They knew we weren’t employed … but when we were given the [key informant] role, we appeared to have been more on the side of the researchers. That’s when they stopped trusting us. [IDI, male CR] Further, the growing tensions previously described between CCs and CRs also had conse- quential effects on relationships between CRs and community members. For example, in the case where one CC had been sacked on data falsification grounds due to the CR having reported malpractice, community members criticized CRs as spying on the CCs. These guarded perceptions persisted even after the trial finished, demonstrating a longer-term impact of study dynamics, as illustrated in the quote below: Further, the growing tensions previously described between CCs and CRs also had conse- quential effects on relationships between CRs and community members. For example, in the case where one CC had been sacked on data falsification grounds due to the CR having reported malpractice, community members criticized CRs as spying on the CCs. These guarded perceptions persisted even after the trial finished, demonstrating a longer-term impact of study dynamics, as illustrated in the quote below: … checking counsellors’ work made the community doubt us. Even today some of the people we used to spend time with now avoid us, suspecting that we’re going to report what they say. They see us as journalists, coming to nose around. One household said to us, “You still want to betray your friends? Disruption of trust between CRs and CCs through recruitment targets and watchdog roles [FGD, female CC] These ideas of CRs as chasing CCs and ferociously reporting on CCs’ work show how this system eroded trust and friendship; CCs’ description of CRs as rabid dogs reflects their feeling that CRs were viciously seeking to ruin their relationship with the researchers. CCs’ distrust in CRs emerged largely in the context of CRs’ covert reporting to the researchers, behaviour that was in complete contrast to the former collaborative and open team working relationships. This led to fear amongst CCs that they might be reported by CRs as having cheated. The tensions that arose in relation to the redefined roles surfaced in all study sites, and were particularly a recurrent theme in the participatory workshop, where CRs universally 7 GLOBAL BIOETHICS 75 discussed issues of distrust between them and CCs, as one male CR said, “There was so much distrust between [cluster] representatives and [community] counsellors. They [CCs] felt we were intruding and looking to discredit their work … ” The majority of the CRs that attended the workshop were from the same clusters as CCs, and their reports of a difficult relationship with CCs in their respective clusters were identical. This perhaps confirms that conflicts between CCs and CRs were common to the clusters. Communities lose trust in CRs and CCs due to their multiple roles reigniting concerns around confidentiality Communities lose trust in CRs and CCs due to their multiple roles reigniting concerns around confidentiality You want to report your friends so that they get sacked?” [IDI, male CR] Community distrust in CCs re-emerged because of a perceived breach of secrecy linked to the watchdog role that CRs performed. When recruitment quotas were set and CRs were dispatched to check eligibility of clients, cluster residents were concerned that their privacy had been disclosed, and suspected CCs of sharing details of the clients with CRs, as demonstrated below: Clients would ask you: “Somebody [CR] came to my house asking about my test result. You said my test result was confidential, so how come he [CR] wants to know the outcome of my testing?” [IDI, female CC] Despite this periodic disbelief in the CRs amongst the communities they represented, and some longer-term distrust as a result, that lasted beyond the study, there were also many demonstrations of positive trust sustained after study completion. Many community M. PHIRI ET AL. 76 76 members continued to ask CRs for advice on health even when the study had closed, demonstrating a positive short-term residual impact of CR activities. When discussing post-trial roles, one of the things that CRs emphasized was the experience of frequently referring people to the hospital, and their experiences indicated some degree of commu- nity members depending on them for linkage into health services. This was reported in all study sites. There was death and members of the family came to us: “we are worried that our relative may have died of TB and that we might have been infected.” When I asked them to go to the hos- pital, they said, “Give us a referral letter so that they can attend to us quickly.” [IDI, female CR] People got used to coming to us for advice; if they had TB or HIV, they would come to us for guidance. Even today they are still coming to us for advice. This time we ask them to go to the hospital because the study closed. [IDI, male CR] Discussion For example, in circumstances where resources necessary for supporting recruitment are limited recruiters face extra burdens in meeting research goals (Brown et al., 2001). Simi- larly to our findings, other studies have shown that multiple roles of community volun- teers as representing community interests and advancing goals of the study may lead to tensions in fulfilling these dual roles (Molyneux et al., 2013). Concerns about breaches of privacy and confidentiality have also been identified where community members are close to or have prior relationships with prospective participants (Molyneux et al., 2013; Shedlin, Decena, Mangadu, & Martinez, 2011; Simon & Mosavel, 2010). g The need for multiple channels of engagement through the respective roles of CCs and CRs was clearly defined within the context of the intervention design as community-based. It was also clear to the research team, driven as they were by the need to reach targets for HST uptake in order to sufficiently power the results but whilst the former was described in the initial recruitment process for both positions, in contrast, the latter as the underlying cause of the changed role of CRs to monitors for CCs was not communicated either to the CCs or the community. It was clear from the impact on subsequent relationships, some of which extended, as we have noted, beyond the duration of the trial itself, that had this been communicated the community-level and longer-term impact might have been mitigated. Balancing research-driven requirements and ethical requirements in the context of such community-based research continues to be ridden by tension and conflict and this paper describes some of these (those of social relationships and trust) clearly. Had greater attention been paid to social relationships in the communities targeted by the intervention, trust might have been maintained. Trust is important for health systems and development because it underpins cooperation throughout the system that is essential for health production and society building (Gilson, 2003). Current literature on trust related to community-based research tends to focus on how community engagement increases trust in research (Molyneux et al., 2005; Quinn, Kass, & Thomas, 2013), trust in research institutions (Marsh et al., 2008), or trust in health delivery systems (Østergaard, 2015; Tibbels, 2015). There is limited litera- ture examining the impacts of research on trust between community volunteers and between these volunteers and the communities they come from. Discussion Our findings show how the use of community volunteers has the potential for both posi- tive and negative impacts during and beyond the end of community-based research, creat- ing both elements of trust and distrust between different project related roles and community members. In particular, we show how different, often conflicting roles and research driven requirements such as recruitment targets can lead to tensions, and that these tensions may impact on social relations during and after the completion of research studies. Specifically, we focus on community representatives recruited as key informants for TB outcomes and mortality and concurrently as representing and reporting commu- nity concerns and community counsellors recruited to provide semi-supervised HIV self- testing within the context of a community-based HIV/TB intervention trial, HitTB, in urban Blantyre, Malawi. y We show that whilst those who design community-based research may have the best of intentions, they are often blind to the implications of recruiting community-based volunteers, especially if these volunteers have differential and hierarchical roles within the study. Whilst we define these roles as community volunteers, their actual work reflects that of fieldworkers, rather than volunteers, since they are in receipt of salaries, and their actual responsibilities are driven by the needs of the study, rather than those of the community. It is perhaps this conflict between their responsibilities and the definition of their roles that creates the tensions that undermine the many positive aspects of employing recruits from the communities targeted in community-based inter- ventions. Similarly to Reynolds et al. (Reynolds, Cousins, Newell, & Imrie, 2013) in work exploring the impact on social relations of the proximity of trial staffto community members affecting HIV surveillance in South Africa, we emphasize that it is imperative to understand these potential implications when designing interventions that rely on and in turn, influence for the future, social relations between communities participating in the research and those charged with delivering it. Communities need to be understood not as homogenous and harmonious, but as diverse groupings with ongoing inequalities and power relationships (Enria et al., 2016) and community-based research should understand these underlying inequalities and power differentials and their potential impact on them. 77 77 77 GLOBAL BIOETHICS Some of our findings reflect research findings reported elsewhere regarding community volunteers and fieldworkers engaged by research. Other studies have found that pressure to manage recruitment creates challenges for the frontline staffresponsible for recruiting. Conclusions Our study shows how multiple roles and the urgency to answer research questions can place pressure on study volunteers and create relations of distrust within communities. Studies need to carefully consider the impact of the roles assigned to community volun- teers and the impact of altering defined roles described during recruitment in the course of study implementation. Changing the pre-defined roles of community volunteers recruited through an engaged, participatory process needs to reflect the engagement aim in itself by involving the community in these redefinitions, ensuring they are informed of these changes and sensitized to their purpose. Assessment of both feasibility and appro- priateness of recruitment targets, and whether these roles and targets might have impli- cations for social relationships and the wellbeing of study volunteers beyond the life of the trial or research is important at the formative phase of any community-based research. Negotiating social relationships within communities can be challenging, especially if com- munity members develop new social roles through informal recruitment within an inter- vention trial. It is important to recognize the long-term impacts of community-based interventions that extend beyond the duration of the research and to take these into account when engaging communities actively in intervention activities, additionally ensur- ing that impact of such engagement activities is monitored and fed back to research teams in a timely feedback system to promote responsive and ethical research engagement. Acknowledgements We would like to thank community members, community counsellors and community represen- tatives for accepting to be part of this study. We also would like to thank Liz Corbett and Augustine Choko for permission to do this study on their study, HIT TB Hard (ISRCTN02004005). Discussion In this respect, our study contributes an additional perspective on the effects of community engagement on trust. We show explicitly how trust is intrinsically located within the social relations sur- rounding participants and delivery agents in community-based interventions. We also demonstrate how the nuances in changing practice in response to the need to achieve research outcomes impact on the fluidity of trust across different groups, particularly how the initial trust in CC roles reinforced through CR roles at the beginning of the inter- vention were lost when the same CRs were required to monitor the integrity of the CCs once trust had been established in the CC role. Thus trust for CCs began with distrust from the community, developed into trust in confidentiality through the support of the CRs and in parallel trust that was originally inherent in CCs due to their representative role towards the community was gradually eroded into distrust both through their role as key informants reporting death and finally through their role as informants or “watch- dogs” on the CCs themselves. This placed the CRs in a challenging social position that M. PHIRI ET AL. 78 eventually extended beyond the duration of the intervention, in some cases changing the very social relationships on which their selection as CRs had been based. y This project was relatively small in scope and whilst the participatory workshop and focus groups generated many interesting ideas we were only able to conduct a small number of interviews. Additional interviews could have provided further perspectives and helped to confirm findings. In addition, some issues were primarily reported by CCs and CRs, and might have benefited from further exploration with community members and the broader research team to understand their views. The study was conducted one year after the trial finished. Ideally, it would have been helpful to conduct this study concurrently with the trial to examine changes in trust as they occurred in situ and to explore changing dynamics that reflected the changing dynamics of intervention design. This would have reduced participants’ reliance on recall and facilitated the ability of the researchers to address issues as they arose in order to reduce both short and long-term impacts on social relations within the study communities. References Anderson, E. E., & Solomon, S. (2013). Community engagement: Critical to continued public trust in research. The American Journal of Bioethics, 13(12), 44–46. doi:10.1080/15265161.2013. 851296 Attree, P., French, B., Milton, B., Povall, S., Whitehead, M., & Popay, J. (2011). The experience of community engagement for individuals: A rapid review of evidence. Health & Social Care in the Community, 19(3), 250–260. Boga, M., Davies, A., Kamuya, D., Kinyanjui, S. M., Kivaya, E., Kombe, F., … Mwalukore, S. (2011). Strengthening the informed consent process in international health research through community engagement: The KEMRI-Wellcome Trust Research Programme Experience. PLoS Medicine, 8(9), e1001089. Brown, B. A., Long, H. L., Weitz, T. A., & Milliken, N. (2001). Challenges of recruitment: Focus groups with research study recruiters. Women & Health, 31(2–3), 153–166. Chantler, T., Otewa, F., Onyango, P., Okoth, B., Odhiambo, F., Parker, M., & Geissler, P. W. (2013). Ethical challenges that arise at the community interface of health research: Village reporters’ experiences in Western Kenya. Developing World Bioethics, 13(1), 30–37. doi:10.1111/dewb. 12023 Choko, A. T., MacPherson, P., Webb, E. L., Willey, B. A., Feasy, H., Sambakunsi, R., … Corbett, E. L. (2015). Uptake, accuracy, safety, and linkage into care over two years of promoting annual self- testing for HIV in Blantyre, Malawi: A community-based prospective study. PLoS Medicine, 12(9), e1001873. Clodagh. (2015). Trust me i am a scientist: Exploring the role of trust within international health research. Retrieved from https://miskellany.me/2015/06/21/trust-me-i-am-a-scientist- exploring-the-role-of-trust-within-international-health-research/ p g Dickert, N., & Sugarman, J. (2005). Ethical goals of community consultation in research. American Journal of Public Health, 95(7), 1123–1127. doi:10.2105/AJPH.2004.058933 f Dunn, A. (2011, June 12). Community engagement – under the microscope. Retrieved from https:// wellcome.ac.uk/sites/default/files/wtvm054326_0.pdf Enria, L., Lees, S., Smout, E., Mooney, T., Tengbeh, A. F., Leigh, B., … Larson, H. (2016). Power, fair- ness and trust: Understanding and engaging with vaccine trial participants and communities in the setting up the EBOVAC-Salone vaccine trial in Sierra Leone. BMC Public Health, 16(1), 203. g p ( ) Gbadegesin, S., & Wendler, D. (2006). Protecting communities in health research from exploitation. Bioethics, 20(5), 248–253. Gilson, L. (2003). Trust and the development of health care as a social institution. Social Science & Medicine, 56(7), 1453–1468. Jahn, A., Crampin, A. C., Glynn, J. R., Mwinuka, V., Mwaiyeghele, E., Mwafilaso, J., … Zaba, B. (2007). Evaluation of a village-informant driven demographic surveillance system in Karonga, Northern Malawi. Demographic Research, 16(8), 219–248. g p Kamuya, D. Disclosure statement No potential conflict of interest was reported by the authors. GLOBAL BIOETHICS 79 GLOBAL BIOETHICS 79 ORCID Mackwellings Phiri http://orcid.org/0000-0001-5765-6340 Kate Gooding http://orcid.org/0000-0003-4926-0287 Deborah Nyirenda http://orcid.org/0000-0002-5867-4687 Moses Kelly Kumwenda http://orcid.org/0000-0003-3091-7330 Mackwellings Phiri http://orcid.org/0000-0001-5765-6340 Kate Gooding http://orcid.org/0000-0003-4926-0287 Deborah Nyirenda http://orcid.org/0000-0002-5867-4687 Moses Kelly Kumwenda http://orcid.org/0000-0003-3091-7330 Funding The project received funding from The Wellcome Trust International Public Engagement Awards under the grant number 104815/Z/14/Z. References M., Marsh, V., Kombe, F. K., Geissler, P. W., & Molyneux, S. C. (2013). Engaging com- munities to strengthen research ethics in Low-income settings: Selection and perceptions of M. PHIRI ET AL. 80 members of a network of representatives in coastal Kenya. Developing World Bioethics, 13(1), 10–20. members of a network of representatives in coastal Kenya. Developing World Bioethics, 13(1), 10–20. Kamuya, D. M., Theobald, S., Munwoki, P. K., Koech, D., Geissler, W. P., & Molyneux, S. C. (2013). Evolving friendships and shifting ethical dilemmas: Fieldworkers’ experiences in a short term community based study in Kenya. Developing World Bioethics, 13(1), 1–9. Kamuya, D. M., Theobald, S., Munwoki, P. K., Koech, D., Geissler, W. P., & Molyneux, S. C. (2013). Evolving friendships and shifting ethical dilemmas: Fieldworkers’ experiences in a short term community based study in Kenya. Developing World Bioethics, 13(1), 1–9. Kingori, P., & Gerrets, R. (2016). Morals, morale and motivations in data fabrication: Medical research fieldworkers views and practices in two Sub-Saharan African contexts. Social Science & Medicine. Retrieved from http://www.sciencedirect.com/science/article/pii/S0277953616 304415 Kumwenda, M. K., Corbett, E. L., Chikovore, J., Phiri, M., Mwale, D., Choko, A. T. … Gutteberg, T. J. (2018). Discordance, disclosure and normative gender roles: Barriers to couple testing within a community-level HIV self-testing intervention in urban Blantyre, Malawi. AIDS and Behavior, 22(8), 2491–2499. Kumwenda, M., Munthali, A., Phiri, M., Mwale, D., Gutteberg, T., MacPherson, E., … Desmond, N. (2014). Factors shaping initial decision-making to self-test amongst cohabiting couples in urban Blantyre, Malawi. AIDS and Behavior, 18(4), 396–404. Marsh, V., Kamuya, D., Rowa, Y., Gikonyo, C., & Molyneux, S. (2008). Beginning community engagement at a busy biomedical research programme: Experiences from the KEMRI CGMRC-Wellcome Trust Research Programme, Kilifi, Kenya. Social Science & Medicine, 67 (5), 721–733. doi:10.1016/j.socscimed.2008.02.007 Molyneux, S., Kamuya, D., Madiega, P. A., Chantler, T., Angwenyi, V., & Geissler, P. W. (2013). Field workers at the interface. Developing World Bioethics, 13(1), ii–iv. doi:10.1111/dewb.12027 Molyneux, C. S., Peshu, N., & Marsh, K. (2005). Trust and informed consent: Insights from com- munity members on the Kenyan coast. Social Science & Medicine, 61(7), 1463–1473. Molyneux, S., Kamuya, D., Madiega, P. A., Chantler, T., Angwenyi, V., & Geissler, P. W. (2013). Field workers at the interface. Developing World Bioethics, 13(1), ii–iv. doi:10.1111/dewb.12027 Molyneux, C. S., Peshu, N., & Marsh, K. (2005). Trust and informed consent: Insights from com- Østergaard, L. R. (2015). References Trust matters: A narrative literature review of the role of trust in health care systems in sub-Saharan Africa. Global Public Health, 10(9), 1046–1059. Pratt, B., Lwin, K. M., Zion, D., Nosten, F., Loff, B., & Cheah, P. Y. (2013). Exploitation and com- munity engagement: Can community advisory boards successfully assume a role minimising exploitation in international research? Developing World Bioethics, 15(1), 18–26. Quinn, S. C., Kass, N. E., & Thomas, S. B. (2013). Building trust for engagement of minorities in human subjects research: Is the glass half full, half empty, or the wrong size? American Public Health Association. Retrieved from http://ajph.aphapublications.org/doi/abs/10.2105/AJPH. 2013.301685 Reynolds, L., Cousins, T., Newell, M.-L., & Imrie, J. (2013). The social dynamics of consent and refusal in HIV surveillance in rural South Africa. Social Science & Medicine, 77(11), 8e125. Sambakunsi, R., Kumwenda, M., Choko, A., Corbett, E. L., & Desmond, N. A. (2015). ‘Whose failure counts?’ A critical reflection on definitions of failure for community health volunteers providing HIV self-testing in a community-based HIV/TB intervention study in urban Malawi. Anthropology & Medicine, 22(3), 234–249. doi:10.1080/13648470.2015.1077202 p gy Shedlin, M. G., Decena, C. U., Mangadu, T., & Martinez, A. (2011). Research participant recruit- ment in hispanic communities: Lessons learned. Journal of Immigrant and Minority Health, 13(2), 352–360. Simon, C., & Mosavel, M. (2010). Community members as recruiters of human subjects: Ethical considerations. The American Journal of Bioethics, 10(3), 3–11. Tibbels, N. (2015). Trust in health system and barriers to healthcare utilization in Sierra Leone. Retrieved from http://www.mamaye.org/sites/default/files/evidence/JHCCP_2015_Lit%20 review%20trust%20and%20barriers%20to%20health%20utilization%20Sierra%20Leone.pdf Tindana, P. O., Singh, J. A., Tracy, C. S., Upshur, R. E. G., Daar, A. S., Singer, P. A., … Lavery, J. V. (2007). Grand challenges in global health: Community engagement in research in developing countries. PLoS Medicine, 4(9), e273. doi:10.1371/journal.pmed.0040273 Wallerstein, N., & Duran, B. (2010). Community-based participatory research contributions to intervention research: The intersection of science and practice to improve health equity. American Journal of Public Health, 100(Suppl 1), S40–S46.
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Rapid quantification of the malaria biomarker hemozoin by improved biocatalytically initiated precipitation atom transfer radical polymerizations
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Analyst PAPER Cite this: DOI: 10.1039/d0an00976h Received 14th May 2020, Accepted 11th September 2020 DOI: 10.1039/d0an00976h rsc.li/analyst Rapid qu hemozo precipita polymer Samuel Racc Michela M. Pe Nico Bruns The fight agains techniques. Diag further transmiss (ATRP), which w cipitation polym investigated usin to greatly reduc ascorbate allows and to the deco dodecyl sulfate ( taining samples amplification tim mL−1. The short of-care diagnos clinical samples. en Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Analyst PAPER Cite this: DOI: 10.1039/d0an00976h Received 14th May 2020, Accepted 11th September 2020 DOI: 10.1039/d0an00976h rsc.li/analyst Rapid qu hemozo precipita polymer Samuel Racc Michela M. Pe Nico Bruns The fight agains techniques. Diag further transmiss (ATRP), which w cipitation polym investigated usin to greatly reduc ascorbate allows and to the deco dodecyl sulfate ( taining samples amplification tim mL−1. The short of-care diagnos clinical samples. en Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Analyst PAPER Cite this: DOI: 10.1039/d0an00976h Received 14th May 2020, Accepted 11th September 2020 DOI: 10.1039/d0an00976h rsc.li/analyst n Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. †Electronic supplementary information (ESI) available: Influence of crosslinker on the precipitation polymerization. Influence of the measuring wavelength. Influence of the concentration of the cosolvent ethanol. Hemozoin absorption spectra at SDS concentration 0 mM, 0.170 μM and 34.7 mM. Short review on the sodium ascorbate degradation pathway. Additional measurement underlying the function of sodium pyruvate. MATLAB script used to calculate the rate of turbid- ity formation. See DOI: 10.1039/d0an00976h ‡Current address: Department of Materials and Department of Bioengineering, Institute of Biomedical Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, UK. aAdolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland bDepartment of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, UK. E-mail: nico.bruns@strath.ac.uk Rapid quantification of the malaria biomarker hemozoin by improved biocatalytically initiated precipitation atom transfer radical polymerizations† Samuel Raccio, a Jonas Pollard,a Ashley Djuhadi, a Sandor Balog, a Michela M. Pellizzoni, a Kyle J. Rodriguez, a Omar Rifaie-Graham ‡a and Nico Bruns *a,b The fight against tropical diseases such as malaria requires the development of innovative biosensing techniques. Diagnostics must be rapid and robust to ensure prompt case management and to avoid further transmission. The malaria biomarker hemozoin can catalyze atom transfer radical polymerizations (ATRP), which we exploit in a polymerization-amplified biosensing assay for hemozoin based on the pre- cipitation polymerization of N-isopropyl acrylamide (NIPAAm). The reaction conditions are systematically investigated using synthetic hemozoin to gain fundamental understanding of the involved reactions and to greatly reduce the amplification time, while maintaining the sensitivity of the assay. The use of excess ascorbate allows oxygen to be consumed in situ but leads to the formation of reactive oxygen species and to the decomposition of the initiator 2-hydroxyethyl 2-bromoisobutyrate (HEBIB). Addition of sodium dodecyl sulfate (SDS) and pyruvate results in better differentiation between the blank and hemozoin-con- taining samples. Optimized reaction conditions (including reagents, pH, and temperature) reduce the amplification time from 37 ± 5 min to 3 ± 0.5 min while maintaining a low limit of detection of 1.06 ng mL−1. The short amplification time brings the precipitation polymerization assay a step closer to a point- of-care diagnostic device for malaria. Future efforts will be dedicated to the isolation of hemozoin from clinical samples. This journal is © The Royal Society of Chemistry 2020 Rapid quantification of the malaria biomarker hemozoin by improved biocatalytically initiated precipitation atom transfer radical polymerizations† Samuel Raccio, a Jonas Pollard,a Ashley Djuhadi, a Sandor Balog, a Michela M. Pellizzoni, a Kyle J. Rodriguez, a Omar Rifaie-Graham ‡a and Nico Bruns *a,b Rapid quantification of the malaria biomarker hemozoin by improved biocatalytically initiated precipitation atom transfer radical polymerizations† Samuel Raccio, a Jonas Pollard,a Ashley Djuhadi, a Sandor Balog, a Michela M. Pellizzoni, a Kyle J. Rodriguez, a Omar Rifaie-Graham ‡a and Nico Bruns *a,b Analyst View Article Online View Journal aAdolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland bDepartment of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, UK. Introduction Hemozoin can be qualified as a pan-malaria bio- marker since all Plasmodium sp. generate it during their intraerythrocytic life stages. However, its concentration varies greatly depending on the species as well as the life stage.33,34 Nevertheless, hemozoin has been investigated as a malaria biomarker for diagnostic purposes via several physical detec- tion techniques such as laser desorption mass spectrometry,35 multiple-angle polarization scatter separation,36 magnetically induced dichroism,37–39 laser-induced nanobubble for- mation,40 and Raman spectroscopy.41 Our polymerization- amplified hemozoin detection technique is a very sensitive method for detecting hemozoin concentrations down to 0.85 ng mL−1 at a confidence level of 95%.28 To evaluate the per- formance of the technique, hemozoin was extracted from blood samples spiked with different amounts of cultured para- sites. A limit of detection (LOD) of 10 infected red blood cells per μL was achieved, demonstrating the possible application of this technique as a sensitive diagnostic test for malaria. However, the amplification time required for the assay was 37 ± 5 min excluding sample collection and processing, which is too long for practical application. One paramount feature of point of care diagnostics is that the analysis must be done rapidly, ideally within the time of the medical consultation of approximately 20 min.1,7,10,11 techniques are generally used. The most famous of these is the polymerase chain reaction (PCR) that has revolutionized bio- medical sciences. Radical polymerizations have also been used for diagnostics because the successive addition of many mono- mers to a growing polymer chain in the radical chain reaction is an effective way to concentrate molecules at a site of biode- tection, thus amplifying a sensing signal.14 Polymerization- based amplification techniques (PBA) refer to the use of a radical chain reaction to molecularly amplify the recognition of a target analyte.15,16 In the field of malaria, Sikes et al. have proposed the combination of PBA in association with a paper based immunoassay.17 Free radical polymerization was initiated by an antibody-bound photoinitiator in the presence of a dye at the site where Plasmodium falciparum histidine-rich protein 2 (Pf HRP2) was identified by immunorecognition. After washing steps, trapped dye in the polymer revealed the presence of Pf HRP2 at the recognition site. Introduction acronym ASSURED (affordability, sensitivity, specificity, user friendliness, rapid and robust, equipment free, and deliverable to end-user). Since then, many authors have emphasized the need for rapid tests to meet the needs of the developing world.2–7 To ensure that the patient receives treatment before leaving the medical facility, it is important that the results are established during the visit. Rapid tests allow for an immedi- ate case-management and prevent the patient from having to return to the clinic several times. This is a very important factor in rural areas where remote contact is impossible and where the patient is unable to visit the test site several times due to lack of transportation or financial means. At the popu- lation level, immediate case management helps to decrease transmission.8,9 In some cases, assay time can even outweigh sensitivity as described for Chlamydia trachomatis10 and for syphilis.11 In 2003, the World Health Organization Special Program for Research and Training in Tropical Diseases (WHO/TDR) pub- lished a list of criteria that an ideal test for infectious tropical diseases must fulfill.1,2 This criteria list is known as the †Electronic supplementary information (ESI) available: Influence of crosslinker on the precipitation polymerization. Influence of the measuring wavelength. Influence of the concentration of the cosolvent ethanol. Hemozoin absorption spectra at SDS concentration 0 mM, 0.170 μM and 34.7 mM. Short review on the sodium ascorbate degradation pathway. Additional measurement underlying the function of sodium pyruvate. MATLAB script used to calculate the rate of turbid- ity formation. See DOI: 10.1039/d0an00976h The specific detection of DNA sequences, proteins or other biomolecules in very small quantities is of primary diagnostic interest.12 Early and rapid detection even before symptoms appear maximizes the chances of recovery while reducing health costs.13 To achieve this objective, analyte amplification ‡Current address: Department of Materials and Department of Bioengineering, Institute of Biomedical Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, UK. This journal is © The Royal Society of Chemistry 2020 Analyst Analyst View Article Online View Article Online Paper Analyst Plasmodium sp. parasites.30 The digestion of hemoglobin releases a significant amount of free heme that is toxic to the parasite. In order to disable the free heme, the parasite crystal- lizes it into centrosymmetric μ-propionate dimers of heme (hemozoin).31,32 During the life of the parasite, more and more hemozoin crystals are formed in the parasite’s food vacuole. Introduction PBA systems show great sensitivity, although their specificity is achieved, in most cases, by immunorecognition.16 This allows to detect a wide variation of analytes, but manufacturing of sensor surfaces and reagents is complex, and the need for several washing steps is laborious. Label-free detection can overcome these dis- advantages, e.g., by using the intrinsic catalytic activity of the target analyte. For example, heme-containing enzymes and hemin are known for their ability to initiate polymerizations,18–20 and for their use as catalysts for revers- ible-deactivation radical polymerizations (also termed con- trolled radical polymerizations) such as atom transfer radical polymerizations (ATRP).21–27 Recently, our group showed that a biomarker of malaria, hemozoin (Fig. 1a), can be used to cata- lyze radical polymerizations.28 We exploited this phenomena to design a polymerization-amplified assay for the detection of hemozoin and, therefore, of malaria parasites. Hemoglobin can also be detected by the assay in a sensitive way.29 Hemozoin is a product of hemoglobin digestion by Experimental section 2-Hydroxyethyl 2-bromoisobutyrate (≥95%, HEBIB), N,N′- methylenebisacrylamide (BA), mineral oil (BioUltra grade), sodium phosphate monobasic (≥99.0%), sodium phosphate dibasic (≥99.0%), (+)-sodium L-ascorbate (≥99.0%, Asc), sodium pyruvate (≥99.0%, Pyr), ethanol (≥99.8%, EtOH), sodium hydroxide (≥98.0%), and sodium dodecyl sulfate (≥99.0%, SDS) were purchased from Sigma Aldrich and used as received. N-Isopropyl acrylamide (97%, NIPAAm) was pur- chased from Sigma Aldrich and was recrystallized twice from hexane. Synthetic hemozoin (sHz) was purchased from InvivoGen (San Diego, CA) and was used as received. Ultrapure water was made with Purelab Flex II (ELGA-Veolia Water System) at 18.2 mΩ using the purification pack LC208. UV-vis measurements were performed on an Analytik Jena Specord 50 Plus spectrophotometer equipped with a 6-cell changer that was thermostatted with a Julabo heating circulator TD-6. Semi- micro UV-vis cuvettes (path length 10 mm, optical glass) were obtained from Hellma Analytics. UV-vis extinction measurements were recorded at 410 nm every 22.2 s with an integration time of 0.1 s. (Please see ESI Fig. 1† that provides details for the choice of wavelength). Extinction is the sum of the contributions of absorbance and light scattering on the transmission of light through a cuvette, i.e., −log[T], where T is the transmission at a defined wave- length. The rates of turbidity formation ΔE Δt were determined using a MATLAB script (see ESI: MATLAB Script†). The script calculates a linear regression, fitted at every two points over the entire reaction time. Since turbidity formation is sigmoidal over time, the inflection point corresponds to the location where the slope is the highest. The highest linear regression slope was, therefore, used as a measure of the rate of turbidity formation ΔE Δt . The decision limit and the detection limit were calculated according to Hubaux and Vos.42 This method con- nects a linear calibration curve with the confidence limit. The decision limit corresponds, a priori, to the lowest signal differ- entiable from the non-catalyzed reaction. The detection limit is, a priori, the lowest signal that cannot be confused with the blank. The maximum amplification time was defined as the time needed for the blank reaction to reach maximum ΔE Δt . In a typical experiment, NIPAAm (639 mg, 5.65 mmol) and HEBIB (22.42 µL, 32.6 mg, 296 µmol) were added to a volu- metric flask. Sodium phosphate buffer (pH 7.0, 0.1 mM, 10% v/v EtOH) was used to dissolve the reagents to a final volume of 6 mL. ccess Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. For experiments using pyruvate, sodium pyruvate (604 mg, 5.49 mmol) was dissolved in sodium phosphate buffer (8 mL, pH 7.0, 0.1 M, 10% EtOH). The resulting solution was further used to prepare the solutions of NIPAAm, HEBIB and sodium ascorbate. Experimental section 850 µL of this solution was introduced into a cuvette. 50 µL of 0.4 M sodium hydroxide solution in ultrapure water containing various hemozoin concentrations was added. The solution was then sealed from ambient air by overlaying it with mineral oil (400 µL) and incubated in the cell changer for 3 min. During this time, sodium ascorbate (158 mg, 0.80 mmol) was dissolved in a volumetric flask (1 mL) with sodium phosphate buffer (pH 7.0, 0.1 M, 10% EtOH). Reactions were started by the addition of 100 µL sodium ascor- bate solution to the reaction mixture, using a pipette tip to inject the solution through the oil layer. Final reagent concen- trations were 36.3 mM HEBIB, 800 mM NIPAAm, and 80 mM Asc. The pH indicated in this paper refers to the pH of the buffer used for the preparation of solutions. For sodium phos- phate buffer pH 7.0 (0.1 M, 10% EtOH), the pH after mixing of all reagents was 7.07. UV-vis spectra were measured from 350 nm to 800 nm with a Δλ of 1 nm at a scan speed of 10 nm s−1, unless otherwise noted. Kinetic UV-vis spectra were followed by spectral scans between 320 to 700 nm, at a speed of 50 nm s−1 and with Δλ = 5 nm. This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The motivation of the work described herein is to gain a greater understanding of the chemistry behind the hemozoin- catalyzed precipitation polymerization assay, and to optimize the reaction conditions to perform rapid and sensitive detec- tion of hemozoin. Thus, we probed how the reagents used in Fig. 1 Hemozoin-catalyzed precipitation polymerization of NIPAAm. (a) Scheme of crystal structure of hemozoin. (b) Turbidity formation during an assay catalyzed by 100 ng mL−1 of synthetic hemozoin (800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 650 mM Pyr, 170 μM SDS, 45 °C and pH 7.5) over time. (c) Reaction scheme of the polymerization. Fig. 1 Hemozoin-catalyzed precipitation polymerization of NIPAAm. (a) Scheme of crystal structure of hemozoin. (b) Turbidity formation during an assay catalyzed by 100 ng mL−1 of synthetic hemozoin (800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 650 mM Pyr, 170 μM SDS, 45 °C and pH 7.5) over time. (c) Reaction scheme of the polymerization. This journal is © The Royal Society of Chemistry 2020 This journal is © The Royal Society of Chemistry 2020 View Article Online Analyst Paper the assay influence the outcome of the reaction. These reagents include the crosslinker N,N′-methylenebis(acryl- amide) (BA), ethanol as cosolvent, exogenous oxygen, the redu- cing agent ascorbate, the ATRP initiator 2-hydroxyethyl 2-bro- moisobutyrate (HEBIB), the monomer N-isopropyl acrylamide (NIPAAm), the surfactant sodium dodecyl sulfate (SDS), and the reactive oxygen scavenger sodium pyruvate. Moreover, the effects of temperature and pH on the reactions were investi- gated in order to reduce the amplification time. Using opti- mized reaction parameters, the hemozoin assay was able to be performed in less than 4 minutes with a sensitivity of 1.06 ng mL−1 for synthetic hemozoin. were added to sodium phosphate buffer (7 mL, pH 6.0, 0.1 M). Buffers with EtOH concentrations 0, 1, 5, 10, 20 and 30% v/v were made. NIPAAm (639 mg, 5.75 mmol) and HEBIB (60 µL, 87.2 mg, 719 µmol) were dissolved in phosphate buffer con- taining EtOH (6 mL). 850 µL of this solution was introduced into a cuvette. The rest of the procedure was performed as described above. Degassed solutions were obtained by bubbling argon (ALPHAGAZ 1 AR, 99.999%) for 20 min through the solutions under stirring. Solutions were then introduced with a syringe to the cuvette below the oil layer. General description of the hemozoin assay The assay discussed herein is based on the thermoresponsive properties of poly(N-isopropylacrylamide) (PNIPAAm). When heated higher than 34 °C, PNIPAAm precipitates in aqueous solution while the monomer remains soluble (Fig. 1b).43–45 The radical polymerization of NIPAAm at elevated tempera- tures, therefore, results in the precipitation of the formed chains. This changes the macroscopic appearance of the solu- tion from transparent to milky due to the scattering of light by the suspension of PNIPAAm particles. The addition of a cross- For experiments with various ethanol concentrations, solu- tions containing water and EtOH in different ratios (3 mL) This journal is © The Royal Society of Chemistry 2020 Analyst View Article Online Paper Analyst begins and the extinction of the solution increases. The turbid- ity formation rate follows an almost linear increase, which allows the turbidity formation rate (ΔE/Δt) to be determined by a linear regression calculated at the steepest part of the extinction curve (Fig. 2, black dashed line). We have previously shown that the rate of turbidity formation depends on the hemozoin concentration, making the test quantitative.28 The higher the concentration of dissolved hemozoin is, the faster the rate of turbidity formation is. However, turbidity forms even in the absence of a catalyst (Fig. 2, blue curve), albeit at a very slow rate and after a longer lag-phase than for hemozoin- catalyzed reactions (Fig. 2, red curve). Therefore, the presence of the catalyst and its concentration is assessed by the differ- ence between the rate of turbidity formation of an analytical sample and a non-catalyzed background reaction. linker to the reagent mix would create crosslinked PNIPAAm particles, but we found that the crosslinker bisacrylamide lowered the performance of the assay (ESI Fig. 1†). Therefore, crosslinkers are not used in the precipitation polymerization. The reaction conditions of the assay are similar to those of activators regenerated by electron transfer (ARGET) ATRP (Fig. 1c). HEBIB is used as an initiator. An excess of sodium ascorbate is employed to regenerate the catalyst and to allow the system to tolerate the presence of oxygen. Hemozoin, which is insoluble in physiological conditions, is dissolved in aqueous sodium hydroxide. Once dissolved, hemozoin is acti- vated by sodium ascorbate which reduces the iron of hemozoin from the Fe(III) oxidation state to Fe(II). The reduced hemozoin can homolytically cleave the bromine–carbon bond of the ATRP initiator HEBIB. Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Another key parameter of our method is the amplification time, i.e. the time that the assay takes until it gives a quantifi- able result. This is the time needed for a polymerization to reach maximum ΔE/Δt. Because a precipitation assay with an analytical sample of unknown hemozoin concentration would have to be run until the non-catalyzed reaction reached maximum ΔE/Δt, we define the maximum amplification time of the assay to be the time taken by the non-catalyzed reaction to achieve maximum ΔE/Δt (Fig. 2, green dotted line). This is the measure with which we compare the performance of the assay under various reaction conditions. It should be noted that catalyzed reactions react faster than non-catalyzed reac- tions, so that they reach the highest ΔE/Δt faster (Fig. 2, orange dotted line vs. green dotted line). The precipitation of PNIPAAm can be monitored by measur- ing the extinction of the reaction mixture with a UV-vis spectro- photometer at a specific wavelength (Fig. 2). As detailed in the ESI,† we chose to use 410 nm as the measuring wavelength (ESI Fig. 2†). During the initial lag-phase, no turbidity for- mation is observed because oxygen inhibits the polymeriz- ation. The generated radicals as well as the excess of reducing agent overcome the inhibition of the polymerization by oxygen. Indeed, ascorbate oxidizes in the presence of oxygen and thus, leads to a rapid decrease of the dissolved oxygen concentration.46–49 After the lag phase, the polymerization At the end of the hemozoin-catalyzed reactions, the rate of turbidity formation decreases in the catalyzed reactions because the catalyst copolymerizes into the polymer.28 Heme has two vinyl groups that make it susceptible to radical polymerization. Precipitation of the catalyst with the polymer leads to the loss of catalytic activity and, therefore, the rate of turbidity formation decreases. Fig. 2 Schematic representation of time-dependent extinction measurements of precipitation polymerizations to illustrate assay results for a catalyzed (red curve) and a non-catalyzed reaction (blue curve). The rate of turbidity formation (ΔE/Δt) is calculated by linear regression (black dashed lines) over the quasi-linear increase of extinction. The amplification time (orange and green dotted lines) correspond to the minimal time needed to reach maximum ΔE/Δt. The maximum amplifi- cation time (green dotted line) is the time needed for the non-catalyzed reaction to reach maximum ΔE/Δt. Cosolvent Dimethylformamide was used as a co-solvent in our previous publication to solubilize HEBIB in the assay.28 However, DMF is harmful by skin contact, causes severe eye irritation and may harm the fetus. In order to overcome this toxicity problem and to make the assay safer, the experiments presented here were performed using ethanol, which poses a smaller risk and is more accepted in the medical community. The optimum ethanol concentration for the assay was found to be 10 vol% because it resulted in the highest resolution between catalyzed and non-catalyzed reactions (ESI Fig. 3†). Fig. 2 Schematic representation of time-dependent extinction measurements of precipitation polymerizations to illustrate assay results for a catalyzed (red curve) and a non-catalyzed reaction (blue curve). The rate of turbidity formation (ΔE/Δt) is calculated by linear regression (black dashed lines) over the quasi-linear increase of extinction. The amplification time (orange and green dotted lines) correspond to the minimal time needed to reach maximum ΔE/Δt. The maximum amplifi- cation time (green dotted line) is the time needed for the non-catalyzed reaction to reach maximum ΔE/Δt. General description of the hemozoin assay The transfer of bromine from the initiator to the catalyst forms a tertiary carbon radical which initiates radical polymerization. The catalyst can transfer bromine back to the growing polymer chain. However, the reaction does not proceed as typical reversible-deactivation radical polymerization due to the heterogeneous nature of the reaction mixture.28 Even though oxygen interferes with radical polymerizations, the solution is not deoxygenated to allow for point-of-care handling of the assay. Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. As reported in our first communication, degassing of the solutions resulted in stark improvements when compared to the non-degassed catalyzed reactions.28 The lag phase is elimi- nated, i.e. reactions started immediately after the injection of the reducing agent. Moreover, the rate of turbidity formation is faster. The absence of the lag phase indicates that oxygen must be first consumed before the polymerization can start. Moreover, excess ascorbate and initiator initially used to consume oxygen are now available for the catalyzed reaction resulting in faster polymerization kinetics. The impact of dis- solved oxygen on the non-catalyzed blank reaction was not studied previously. Oxygen has a less pronounced effect on the non-catalyzed reaction than on the catalyzed reaction (Fig. 3), indicating that the formation of turbidity in the absence of catalyst is not directly dependent of oxygen. As a conclusion, The removal of HEBIB from the catalyzed reaction reduced the rate of turbidity formation by an order of magnitude (Fig. 4 pale green vs. purple lines). Without an initiator, the presence or absence of hemozoin in the reactions cannot be differentiated (Fig. 4 purple vs. pink lines). In the latter case, only monomer and ascorbate were left in the reagent mix. However, slight precipitation was still observed. On the other hand, turbidity formation was completely suppressed when the reaction mixture consisted of initiator and monomer but no reducing agent, indicating that the monomer did not self- polymerize and that the initiator was stable over the reaction time (Fig. 4 black line). It would therefore appear that sodium Fig. 3 Influence of the presence of oxygen on the hemozoin-catalyzed precipitation polymerization. Reaction catalyzed by 100 ng mL−1 sHz purged with argon (black line) and non-degassed (red curve) are com- pared to the non-catalyzed reaction purged with argon (pale green) and non-degassed (blue curve). Reaction conditions: 800 mM NIPAAm, 80 mM Asc, 36 mM HEBIB, 170 μM SDS, 45 °C, pH 7. Fig. 4 Contribution of individual reagents on the precipitation polymerization. Normal catalyzed reaction condition (pale green, 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS, 100 ng mL−1 sHz, 45 °C, pH 7), non-catalyzed reaction (dark green), catalyzed reac- tion without HEBIB (purple), non-catalyzed reaction without HEBIB (pink), and non-catalyzed reaction without sodium ascorbate (black). Fig. 4 Contribution of individual reagents on the precipitation polymerization. Presence of air Oxygen quenches radical polymerizations. If a polymerization is carried out in the presence of air, enough radicals have to be created to consume the present oxygen before the polymer- ization can start. Oxygen can therefore inhibit the polymeriz- ation and result in a lag phase at the beginning of the reaction and, thereby, increase the time needed for the hemozoin assay This journal is © The Royal Society of Chemistry 2020 Analyst Analyst View Article Online Analyst Analyst Paper Paper to give a meaningful result. One way to shorten the amplifica- tion time is to conduct the polymerization in the absence of oxygen, which is usually achieved by physically degassing the reaction solution, e.g. by purging the solution with an inert gas. However, such procedures are not feasible for an assay that is intended for use outside of laboratories. To reduce the effect of oxygen on the hemozoin-catalyzed polymerization, an excess of sodium ascorbate, a reducing agent for the hemozoin catalyst, was used to consume the oxygen that is present in the system. Moreover, an oil layer was added on top of the reaction mixture to prevent the diffusion of oxygen from the headspace into the solution. This technique has the advantage of being easy to use because no inert gas handling is required. The oxygen present at the start of the reaction leads to an inhi- bition of the polymerization causing the consumption of initiator, monomer and ascorbate. In addition, ascorbate oxi- dizes in the presence of oxygen. To assess the impact of oxygen consumption on reaction parameters, reagents and reaction mixtures were degassed by argon bubbling. Then, degassed reactions were compared to non-degassed reactions (Fig. 3). the presence of oxygen diminishes the performance of the assay by slowing down the catalyzed reaction while having little effect on the blank reaction. Although degassing would be advantageous to improve the performance of the assay, it is not feasible for point-of-care applications. Deciphering the role of individual reagents The individual role of each reagent must be understood to identify the key parameters that can influence the turbidity for- mation rate, the amplification time and the differentiation between catalyzed and non-catalyzed reactions. Theoretically, in the absence of catalyst, no turbidity formation should occur because only the catalyst should be able to cleave the C–Br bond of the initiator and, thus, initiate a radical chain reac- tion. However, experimentally, even in the absence of a cata- lyst, turbidity is observed after some time indicating that some reagents are not stable under the reaction conditions and that compounds capable of initiating free radical polymerization are generated in situ. It is therefore crucial to understand how the reagents and the parameters interact to affect the catalyzed and the non-catalyzed reactions. The initiator (HEBIB), the catalyst (sHz), and ascorbate were independently removed from the reaction mixture to assess their contributions to the overall reaction (Fig. 4). This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Surfactants are widely used to stabilize colloidal dispersions.50 The use of surfactants, such as SDS, shifts the LCST of PNIPAAm to a higher temperature.51 Moreover, when NIPAAm is polymerized at temperatures higher than its LCST in the presence of SDS, more particles of a more uniform size can be obtained due to the stabilization of the microgel nuclei over the nucleation and growing phases.44 In order to observe the effect of the surfactant on the precipitation polymerization under the conditions of our system, SDS was added to non- catalyzed and catalyzed reactions up to a concentration of 34.7 mM (Fig. 6) The critical micellar concentration of SDS is approx. 8 mM at 25 °C in water and falls to approx. 2 mM in the presence of salts.52 Thus, it can be expected that SDS con- centrations of 0.9 mM and lower, as used for most experiments herein, did not result in the formation of micelles. In order to understand the role of the reducing agent better, the ascorbate concentration was varied and precipi- tation kinetics were recorded in the presence of monomer (Fig. 5). Catalyst and initiator were omitted from the reaction mixtures. For all the studied concentrations, ranging from 4 mM to 80 mM of ascorbate, polymerization was initiated. Increasing the ascorbate concentration from 4 mM to 80 mM reduced the lag phase from 26 min to 2 min. This indicates that a higher concentration of ascorbate makes it possible to overcome the presence of oxygen inhibition more quickly. Moreover, the lowest concentrations of ascorbate resulted in the fastest rates of turbidity formation in these uncatalyzed reactions. Because longer amplification times and fast blank reactions are unfavorable to the purpose of the assay, low ascorbate concentrations should be avoided. Ascorbate con- centrations of more than 80 mM lead to the saturation of the solution, making it difficult to solubilize the reagents. In order to maximize the speed of the reaction, an ascorbate concen- tration of 80 mM was chosen for further experiments. Concerning the catalyzed reaction, addition of SDS increased the rate of turbidity formation strongly until it reached a maximum of 5.23 ms−1 at 0.170 mM SDS, most likely by increasing the number of formed PNIPAAm particles.44,51 At higher concentrations, the assay read-out decreased again. Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This decrease can be attributed to a partial solubilization of precipitating PNIPAAm51 and a decrease in apparent activity of the catalyst either by preventing the initiator to react with the catalyst, by heme degradation,53,54 or by transition of heme dimers to heme monomers.55 In order to gain insight into the interaction of SDS with hemozoin, UV- vis spectra of the catalyst were recorded in the presence and To summarize, the non-catalyzed reaction is caused by the initiator interacting with the reducing agent and by the redu- cing agent itself. The reducing agent concentration influences the lag phase and the rate of turbidity formation of the blank reaction. However, sodium ascorbate is needed in the assay to reduce hemozoin (which is an Fe(III) complex) and thereby create the activating catalyst species in the ATRP reaction. For Fig. 5 Contribution of ascorbate and oxygen to the non-catalyzed pre- cipitation polymerization. Sodium ascorbate concentrations were inves- tigated at 0 mM (black), 4 mM (orange), 8 mM (pale green), 40 mM (green) and 80 mM (pink). Reaction conditions: 800 mM NIPAAm, 0 mM HEBIB, 170 μM SDS, no sHz, 45 °C, pH 7. Please note: This noisy signal arises when PNIPAAm particles sediment, i.e. when larger polymer par- ticles form. Fig. 6 Influence of concentration of sodium dodecyl sulfate on the rate of turbidity formation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-catalyzed reactions (blue triangle) (average of n = 3 and SD). The amplification time of the non-catalyzed reaction (grey square, average of n = 3 and SD) is displayed on the second y axis (grey square). Reaction conditions were 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 45 °C and pH 7. Analyst This journal is © The Royal Society of Chemistry 2020 Fig. 6 Influence of concentration of sodium dodecyl sulfate on the rate of turbidity formation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-catalyzed reactions (blue triangle) (average of n = 3 and SD). The amplification time of the non-catalyzed reaction (grey square, average of n = 3 and SD) is displayed on the second y axis (grey square). Reaction conditions were 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 45 °C and pH 7. Fig. Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Normal catalyzed reaction condition (pale green, 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS, 100 ng mL−1 sHz, 45 °C, pH 7), non-catalyzed reaction (dark green), catalyzed reac- tion without HEBIB (purple), non-catalyzed reaction without HEBIB (pink), and non-catalyzed reaction without sodium ascorbate (black). Fig. 3 Influence of the presence of oxygen on the hemozoin-catalyzed precipitation polymerization. Reaction catalyzed by 100 ng mL−1 sHz purged with argon (black line) and non-degassed (red curve) are com- pared to the non-catalyzed reaction purged with argon (pale green) and non-degassed (blue curve). Reaction conditions: 800 mM NIPAAm, 80 mM Asc, 36 mM HEBIB, 170 μM SDS, 45 °C, pH 7. Fig. 4 Contribution of individual reagents on the precipitation polymerization. Normal catalyzed reaction condition (pale green, 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS, 100 ng mL−1 sHz, 45 °C, pH 7), non-catalyzed reaction (dark green), catalyzed reac- tion without HEBIB (purple), non-catalyzed reaction without HEBIB (pink), and non-catalyzed reaction without sodium ascorbate (black). Fig. 3 Influence of the presence of oxygen on the hemozoin-catalyzed precipitation polymerization. Reaction catalyzed by 100 ng mL−1 sHz purged with argon (black line) and non-degassed (red curve) are com- pared to the non-catalyzed reaction purged with argon (pale green) and non-degassed (blue curve). Reaction conditions: 800 mM NIPAAm, 80 mM Asc, 36 mM HEBIB, 170 μM SDS, 45 °C, pH 7. This journal is © The Royal Society of Chemistry 2020 Analyst View Article Online Paper Analyst ascorbate initiated the polymerization and generated turbidity in solution. If monomer, initiator, and reducing agent were present in the reaction mixture (i.e. in a non-catalyzed blank reaction), the turbidity formation was twice faster than in the absence of the initiator (Fig. 4 dark green vs. pink lines). Therefore, the turbidity observed for these non-catalyzed reac- tions resulted from an interaction between the initiator and the reducing agent that led to polymerization, as well as a background polymerization caused by the ascorbate itself. practical reasons in this study, high doses of sodium ascorbate and initiator were used to help to overcome the oxygen inhi- bition, which in return resulted in turbidity formation even in the absence of catalyst. Despite the complexity of the non-cata- lyzed reaction, it remains reproducible and, therefore, allows hemozoin to be detected and quantified. ccess Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. For the non-catalyzed reactions, increasing the SDS concen- tration up to 34.7 mM raised the rate of turbidity formation from 0.45 to 1.57 ms−1. Moreover, the addition of SDS shor- tened the maximum amplification time from 20 ± 0.5 min (no SDS) to 7.0 ± 0.5 min (at 34.7 mM SDS). Fig. 7 Impact of the pyruvate concentration on the rate of turbidity for- mation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-catalyzed reactions (blue triangle) (average of n = 3 and SD). The amplification time of the non-catalyzed reaction (grey square, average of n = 3 and SD) is displayed on the second y axis. Reaction conditions: 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS, 45 °C, pH 7. In conclusion, addition of SDS up to a concentration of 0.170 mM increased the differentiation between the catalyzed and non-catalyzed reaction by a factor of 2.5 and reduced the maximum amplification time to 15 min. Higher SDS concen- trations would decrease the amplification time further, but at the cost of a loss in sensitivity. generated by ascorbate oxidation. To further investigate the role of pyruvate under the assay conditions, we measured the influence of pyruvate on the non-catalyzed reaction caused by ascorbate in the absence of initiator (ESI Fig. 7†). The addition of pyruvate (650 mM) reduced ΔE/Δt from 0.23 ms−1 to 0.01 ms−1 while maintaining the same lag phase. The use of pyruvate therefore considerably reduced the turbidity resulting from the decomposition of ascorbate. When HEBIB was added to the system, ΔE/Δt increased. Although pyruvate significantly decreases the contribution of ascorbate to the non-catalyzed reaction, it does not decrease the contribution of ascorbate together with the initiator. Bruce et al. have shown that the type and concentration of ions can have significant effects on the lower critical solution temperature (LCST) of PNIPAAm.62 The addition of sodium pyruvate at a high concentration of 650 mM might have decreased the LCST of PNIPAAm allowing the polymer chains to precipitate faster. Most likely, the ROS scavenging properties of pyruvate were offset by the change of ionic strength and ionic environment in the solution upon addition of pyruvate. Use of pyruvate as a ROS scavenger The presence of oxygen in the assay seems to play many roles. Apart from quenching radicals, it might also form reactive oxygen species (ROS) with sodium ascorbate, which in turn can contribute to the blank reaction (ESI Fig. 5†). Hydrogen peroxide can form by oxidation of sodium ascorbate.57–59 In the presence of metal traces, hydrogen peroxide degrades into reactive hydroxyl radicals through the Fenton reaction and can initiate polymerizations. It is also important to note that hydrogen peroxide is known for its capacity to degrade heme.53,60 In the case of hemoglobin, Nagababu et al. showed that superoxide forms when ferrylhemoglobin (Fe(II)) is in contact with hydrogen peroxide, which leads to the degra- dation of the heme moiety. Pyruvate has been used to scavenge ROS because it reacts with hydrogen peroxide to form inert CO2, water, and acetate.61 In order to assess whether pyruvate can be used to improve the performance of the assay, the effect of pyruvate was investigated on catalyzed and non-cata- lyzed reactions at pyruvate concentrations varying from 0 mM to the solubility limit of 650 mM (Fig. 7). In conclusion, in the presence of pyruvate, the non-cata- lyzed reaction is slightly faster but this effect is outweighed by the acceleration of the catalyzed reaction. As an outcome, pyru- vate improved the performance of the assay by increasing the differentiation between the catalyzed and non-catalyzed reac- tion by 800%, while not affecting the maximum amplification time. Addition of pyruvate considerably increased the rate of tur- bidity formation linearly from 5.32 ms−1 to 37.0 ms−1 for the sHz-catalyzed reactions. Concerning the non-catalyzed reac- tions, the rate of turbidity formation increased slightly with the addition of pyruvate from 0.86 ms−1 to 1.63 ms−1 while maintaining similar amplification times. Thus, pyruvate makes the test more efficient by increasing the differentiation between the catalyzed and non-catalyzed reactions. UV-vis spectra of the dissolved hemozoin show significant differences in the visible Q-band region when pyruvate is added to the assay (ESI Fig. 6†). Possibly, pyruvate coordinates to solubil- ized hemozoin and increases its catalytic activity. For the non- catalyzed reaction, the observed effect is not expected because the use of a ROS scavenger should have reduced the rate of tur- bidity formation by preventing the in situ formation of radicals This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 6 Influence of concentration of sodium dodecyl sulfate on the rate of turbidity formation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-catalyzed reactions (blue triangle) (average of n = 3 and SD). The amplification time of the non-catalyzed reaction (grey square, average of n = 3 and SD) is displayed on the second y axis (grey square). Reaction conditions were 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 45 °C and pH 7. Fig. 5 Contribution of ascorbate and oxygen to the non-catalyzed pre- cipitation polymerization. Sodium ascorbate concentrations were inves- tigated at 0 mM (black), 4 mM (orange), 8 mM (pale green), 40 mM (green) and 80 mM (pink). Reaction conditions: 800 mM NIPAAm, 0 mM HEBIB, 170 μM SDS, no sHz, 45 °C, pH 7. Please note: This noisy signal arises when PNIPAAm particles sediment, i.e. when larger polymer par- ticles form. This journal is © The Royal Society of Chemistry 2020 Fig. 7 Impact of the pyruvate concentration on the rate of turbidity for- mation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-catalyzed reactions (blue triangle) (average of n = 3 and SD). The amplification time of the non-catalyzed reaction (grey square, average of n = 3 and SD) is displayed on the second y axis. Reaction conditions: 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS, 45 °C, pH 7. Paper View Article Online View Article Online Analyst Pape absence of SDS (ESI Fig. 4†). SDS causes changes in the spec- trum of solubilized hemozoin, indicating a direct interaction of the catalyst with SDS, which is in agreement with literature.53,54,56 Even though the unambiguous speciation of the hematin species that are present under certain conditions is beyond the context of this report, the UV-vis spectra indicate that without SDS and at 0.170 mM SDS, dimers (most likely π–π dimers) of ferriprotoporphyrin IX might dominate while at high SDS concentrations monomers might be present.55 Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 8 Influence of temperature on the rate of turbidity formation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-catalyzed reactions (blue triangle) (average of n = 3 and SD). The amplification time of the non-catalyzed reaction is displayed on the second y axis (grey square, average of n = 3 and SD). Reaction con- ditions: 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS and pH 7. Fig. 9 Effect of pH on the rate of turbidity formation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-cata- lyzed reactions (blue triangle) (average of n = 3 and SD). The amplifica- tion time of the non-catalyzed reaction is displayed on the second y axis (grey square, average of n = 3 and SD). Reaction conditions: 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS and 45 °C. time greatly diminished from 59 ± 13 min down to 3.1 ± 0.5 min by raising the pH from 6 to 7.5. Importantly, a higher pH benefitted the catalyzed reaction more than the non-cata- lyzed reaction. This could be due to the increasing deprotona- tion of ferriprotoporphyrin IX with increasing pH. At pH 6, no difference between the catalyzed and non-catalyzed reaction was observed. This could be explained by catalyst precipitation, as its solubility decreases when reducing the pH.32,67 The effect of pH on the non-catalyzed reaction might be explained by the influence of pH on the ascorbate species in solution. Fully protonated ascorbic acid (AscH2) is mainly found below pH 4.1, AscH−is found in majority up to pH 11.8, and at higher pH values Asc2−is dominant.47,68 Asc2−is supposed to be the only compound capable of oxidizing in the absence of metal traces such as copper or iron.47 At lower pH, the concen- tration of Asc2−is lower. The reactivity of Asc with dissolved oxygen, which leads to the blank reaction, is therefore much slower at lower pH values. and catalyzed reactions, ΔE/Δt increased by a factor of 2.5 ± 0.6 for every 10 °C. Concerning the amplification time, increas- ing the temperature from 45 °C to 60 °C reduced the maximum amplification time from 20 ± 0.5 min to 6.3 ± 0.5 min. Temperature, pH, and wavelength Temperature, pH and measuring wavelength were investigated as key reaction parameters and data collection parameter to reduce the amplification time while keeping high sensitivity. The temperature was varied between 35 °C and 60 °C (Fig. 8). As expected, raising the temperature of the reaction increased the rate of turbidity formation. For the non-catalyzed Temperature, pH and measuring wavelength were investigated as key reaction parameters and data collection parameter to reduce the amplification time while keeping high sensitivity. The temperature was varied between 35 °C and 60 °C (Fig. 8). As expected, raising the temperature of the reaction increased the rate of turbidity formation. For the non-catalyzed This journal is © The Royal Society of Chemistry 2020 Analyst Analyst Fig. 9 Effect of pH on the rate of turbidity formation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-cata- lyzed reactions (blue triangle) (average of n = 3 and SD). The amplifica- tion time of the non-catalyzed reaction is displayed on the second y axis (grey square, average of n = 3 and SD). Reaction conditions: 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS and 45 °C. Analyst View Article Online Fig. 9 Effect of pH on the rate of turbidity formation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-cata- lyzed reactions (blue triangle) (average of n = 3 and SD). The amplifica- tion time of the non-catalyzed reaction is displayed on the second y axis (grey square, average of n = 3 and SD). Reaction conditions: 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS and 45 °C. Analyst View Article Online Fig. 8 Influence of temperature on the rate of turbidity formation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-catalyzed reactions (blue triangle) (average of n = 3 and SD). The amplification time of the non-catalyzed reaction is displayed on the second y axis (grey square, average of n = 3 and SD). Reaction con- ditions: 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS and pH 7. Fig. 9 Effect of pH on the rate of turbidity formation for precipitation polymerizations catalyzed by 100 ng mL−1 sHz (red dot) and non-cata- lyzed reactions (blue triangle) (average of n = 3 and SD). Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Measurements at 35 °C and 40 °C are close to the LCST of poly(NIPAAm), which is reported to be around 31–34 °C.44,45,63 To ensure an efficient precipitation, tempera- tures close to the LCST should be avoided. Using a tempera- ture below 60 °C prevents possible hazards for the operator and reduces the energy consumption of the heating system. With these considerations, the operating temperature range should ideally be between 45 °C and 60 °C. Unless otherwise stated, we used 45 °C. pH is an important factor affecting the decomposition of ascorbate.47,64,65 Higher pH increases its decomposition into dehydro-L-ascorbic acid. The disproportionation of the mono- dehydroascorbate radical anion (Asc•−) is pH-dependent as well. Additionally, the oxidation of ascorbate by oxygen can be highly promoted by the presence of metal traces such as iron or copper at neutral and basic pH.66 Moreover, the pKa of hemozoin is around 7.55 Its exact value depends on the specia- tion of ferriprotoporphyrin IX which is influenced by the solvent composition. pH values lower than 6 were not investi- gated because below this pH the solubility of hemozoin decreases leading to its precipitation and therefore a loss of catalytic activity.32,67 In order to assess the pH-dependency of the precipitation polymerization assay, reactions were followed at pHs ranging from 6 to 8 at hemozoin concentrations of 0 and 100 ng mL−1 (Fig. 9). Reactions that were carried out at pH 8 were too fast to be followed with our experimental set-up, which requires a minute before measuring the extinction. In conclusion, pH is an essential parameter that influences the catalyzed and the non-catalyzed precipitation polymeriz- ations and the overall amplification time. Because of the reac- tion speed, pH 7.5 was used in further experiments. Having a maximum amplification time of 3.1 ± 0.5 min is a great advan- tage towards a rapid diagnostic test. Moreover, these experi- ments clearly demonstrate that the pH must be precisely con- trolled by appropriate buffers to ensure the robustness of the test. Otherwise, a small increase in pH will result in a signifi- cant change of ΔE/Δt. Temperature, pH, and wavelength The amplifica- tion time of the non-catalyzed reaction is displayed on the second y axis (grey square, average of n = 3 and SD). Reaction conditions: 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 170 μM SDS and 45 °C. Paper Analyst View Article Online View Article Online Paper Paper Ana Analyst Conclusion The use of 170 μM SDS, 650 mM pyruvate, and pH set to 7.5 have made it possible to reduce the amplification time to 3 ± 0.5 min while maintaining a low detection limit for synthetic hemozoin of 1.06 ng mL−1. The short amplification time is very important for the practical implementation of the assay in malaria diag- nostics. Ultra-sensitive malaria diagnostic methods, such as PCR, require hours.71 Fast diagnostics such as malaria rapid diagnostic immunotests are not sensitive enough to detect low levels of parasitemia in non-symptomatic humans.71 In con- trast, the optimized precipitation polymerization assay for hemozoin is both rapid and highly sensitive. A malaria test based on hemozoin-catalyzed polymerizations will consist of several steps that include blood sample collection, the extrac- tion of hemozoin from blood, and the precipitation polymeriz- ation assay for hemozoin. As demonstrated earlier, natural hemozoin can be extracted from parasite-containing blood samples.28 However, also this step needs to be simplified and accelerated. Ongoing research in our laboratories aims to develop a simple and rapid extraction method of hemozoin from blood samples that provides good isolation efficiency and specificity for hemozoin, e.g. by the use of paper-based micro- fluidics. Finally, the chemical assay and a suitable extraction method need to be implemented into a diagnostic device, which will then have to be validated in field studies to assess maximum amplification time. In order to determine the col- lective effect of these improvements, the dose response curve of the assay was measured between 0 and 4 ng mL−1 sHz using 650 mM pyruvate, 170 μM SDS and 0.1 M sodium phosphate buffer (pH 7.5, 10% EtOH) (Fig. 10). The limit of detection (LOD) of the assay in the presence of SDS, pyruvate, and at pH 7.5 was 1.06 ng mL−1 with a confi- dence level of 95%. The limit of decision was 0.62 ng mL−1. The previous generation of the assay had a LOD of 0.85 ng mL−1 and a decision limit of 0.49 ng mL−1 natural hemozoin without the use of SDS and pyruvate.28 Thus, the sensitivity of the assay did not change substantially with the change in reac- tion parameters. The most important improvement of assay performance is, however, the amplification time. The assay was completed within 3 ± 0.5 min. Conclusion Fig. 10 Dose–response curve of the optimized hemozoin-catalyzed precipitation polymerization assay at low concentrations of synthetic hemozoin (average of n = 5 and SD, linear data fit (black line), 95% confi- dence interval (orange dotted line), decision limit (green vertical line) and limit of detection (red vertical line)). Maximum amplification time, i.e. the time of the non-catalyzed reaction is displayed on the second y axis (grey square, average of n = 5 and SD). Reaction conditions were 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 650 mM Pyr, 170 μM SDS, 45 °C and pH 7.5. Fig. 10 Dose–response curve of the optimized hemozoin-catalyzed precipitation polymerization assay at low concentrations of synthetic hemozoin (average of n = 5 and SD, linear data fit (black line), 95% confi- dence interval (orange dotted line), decision limit (green vertical line) and limit of detection (red vertical line)). Maximum amplification time, i.e. the time of the non-catalyzed reaction is displayed on the second y axis (grey square, average of n = 5 and SD). Reaction conditions were 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 650 mM Pyr, 170 μM SDS, 45 °C and pH 7.5. In conclusion, the chemistry of the hemozoin-catalyzed pre- cipitation polymerization assay is complex. The reagents ascor- bate and HEBIB, as well as dissolved oxygen cause secondary reactions that lead to NIPAAm polymerization and, therefore, turbidity formation even in the absence of the catalyst. However, each of the reagents is essential for the proper func- tioning of the assay, and oxygen cannot be excluded because of practical reasons. The catalyzed and the non-catalyzed reac- tions, although complex, are reproducible and allow for a robust and highly sensitive assay. The addition of pyruvate considerably increased the catalytic activity of dissolved hemo- zoin, and the use of the surfactant SDS significantly improved the differentiation between catalyzed and non-catalyzed reac- tion. pH strongly influences the assay, with more basic con- ditions accelerating the reactions. Thus, control of pH is essen- tial for the proper functioning of the test, especially at neutral to basic pHs where a small change in pH can lead to a signifi- cant change in the rate of turbidity formation. Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This journal is © The Royal Society of Chemistry 2020 Assessment of the assay performance As elaborated above, the addition of 650 mM pyruvate and 170 μM SDS improved the difference in the rate of turbidity formation between catalyzed and non-catalyzed reactions sig- nificantly, which facilitates the assignment of a sample as a hemozoin-containing sample or as a control or negative. In addition, increasing the pH to 7.5 greatly reduced the The increase in pH had a considerable effect on precipi- tation kinetics, with an exponential increase in the rate of tur- bidity formation as a function of pH for both the catalyzed and the non-catalyzed reaction. The maximum amplification This journal is © The Royal Society of Chemistry 2020 Analyst Analyst View Article Online Fig. 10 Dose–response curve of the optimized hemozoin-catalyzed precipitation polymerization assay at low concentrations of synthetic hemozoin (average of n = 5 and SD, linear data fit (black line), 95% confi- dence interval (orange dotted line), decision limit (green vertical line) and limit of detection (red vertical line)). Maximum amplification time, i.e. the time of the non-catalyzed reaction is displayed on the second y axis (grey square, average of n = 5 and SD). Reaction conditions were 800 mM NIPAAm, 36 mM HEBIB, 80 mM Asc, 650 mM Pyr, 170 μM SDS, 45 °C and pH 7.5. Analyst Analyst Paper Analyst life cycle of the parasite and its species. From the literature, it is possible to obtain a raw estimation of parasitemia from hemozoin concentration.28,69,70 As discussed in our previous report, a concentration of 0.85 ng mL−1 would correspond to a parasitemia of 1.4 iRBCs µL−1.28 Thus, an LOD of 1.06 ng mL−1 would correspond to a parasitemia in the range of 1 to 10 iRBCs µL−1, which is around 20 times better than the current malaria rapid diagnostic tests (MRDTs) and more than 5 times more sensitive than microscopy.71 References 27 K. J. Rodriguez, B. Gajewska, J. Pollard, M. M. Pellizzoni, C. Fodor and N. Bruns, ACS Macro Lett., 2018, 7, 1111– 1119. 1 D. Mabey, R. W. Peeling, A. Ustianowski and M. D. Perkins, Nat. Rev. Microbiol., 2004, 2, 231–240. 28 O. Rifaie-Graham, J. Pollard, S. Raccio, S. Balog, S. Rusch, M. A. Hernandez-Castaneda, P.-Y. Mantel, H.-P. Beck and N. Bruns, Nat. Commun., 2019, 10, 1–8. 2 H. Kettler, K. White and S. J. 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Ross, I. Felger, N. E. Hofmann, L. Robinson, J. Cook, B. P. Gonçalves, A. Björkman, A. L. Ouedraogo, U. Morris, M. Msellem, C. Koepfli, I. Mueller, F. Tadesse, E. Gadisa, S. Das, G. Domingo, M. Kapulu, J. Midega, S. Owusu-Agyei, C. Nabet, R. Piarroux, O. Doumbo, S. N. Doumbo, K. Koram, N. Lucchi, V. Udhayakumar, J. Mosha, A. Tiono, D. Chandramohan, R. Gosling, F. Mwingira, R. Sauerwein, R. Paul, E. M. Riley, N. J. White, F. Nosten, M. Imwong, T. Bousema, C. Drakeley and L. C. Okell, Nat. Commun., 2019, 10, 1433. 34 H. C. Slater, A. Ross, I. Felger, N. E. Hofmann, L. Robinson, 7 K. J. Land, D. I. Boeras, X.-S. Chen, A. R. Ramsay and R. W. Peeling, Nat. Microbiol., 2019, 4, 46–54. J. Cook, B. P. Gonçalves, A. Björkman, A. L. Ouedraogo, U. Morris, M. Msellem, C. Koepfli, I. Mueller, F. Tadesse, 8 S. O. Aral, K. K. Holmes, N. S. Acknowledgements 20 S.-I. Shoda, H. Uyama, J.-I. Kadokawa, S. Kimura and S. Kobayashi, Chem. Rev., 2016, 116, 2307–2413. We thank Jan Eisenmann and Patrick Weber for their contri- bution to the optimization of reaction conditions. The Matlab script was written by Jerôme Brouchoud. This work was finan- cially supported by the Swiss National Science Foundation through projects PP00P2_144697, PP00P2_172927 and PZ00P2_179865, the National Centre of Competence in Research (NCCR) Bio-Inspired Materials, and a BRIDGE Proof- of-Concept grant (20B1-1_173771). Further financial support of the project came from the Novartis Foundation for Biological-Medical Research (16A025) and the Gebert Rüf Foundation (GRS086/17). 21 S. J. Sigg, F. Seidi, K. Renggli, T. B. Silva, G. Kali and N. Bruns, Macromol. Rapid Commun., 2011, 32, 1710–1715. 22 Y.-H. Ng, F. di Lena and C. L. L. Chai, Polym. Chem., 2011, 2, 589–594. 23 Y.-H. Ng, F. di Lena and C. L. L. Chai, Chem. Commun., 2011, 47, 6464–6466. 24 T. B. Silva, M. Spulber, M. K. Kocik, F. Seidi, H. Charan, M. Rother, S. J. Sigg, K. Renggli, G. Kali and N. Bruns, Biomacromolecules, 2013, 14, 2703–2712. 25 A. Simakova, M. Mackenzie, S. E. Averick, S. Park and K. Matyjaszewski, Angew. Chem., Int. Ed., 2013, 52, 12148–12151. 26 C. Fodor, B. Gajewska, O. Rifaie-Graham, E. A. Apebende, J. Pollard and N. Bruns, Polym. Chem., 2016, 7, 6617–6625. Open Access Article. Published on 17 September 2020. Downloaded on 10/7/2020 11:19:20 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 16 S. Kim and H. D. Sikes, Polym. Chem., 2020, 11, 1424–1444. 17 S. Lathwal and H. D. Sikes, Lab Chip, 2016, 16, 1374–1382. 18 A. Singh, S. Roy, L. Samuelson, F. Bruno, R. Nagarajan, J. Kumar, V. John and D. Kaplan, J. Macromol. Sci., Part A: Pure Appl.Chem., 2001, 38, 1219–1230. 19 F. Hollmann and I. W. C. E. Arends, Polymers, 2012, 4, 759– 793. Conclusion This is significantly shorter than the time needed for the assay under the originally pub- lished assay conditions where the amplification time was 37 ± 5 min.28 A practically viable diagnostic assay should take 20 min or less.7 Thus, the amplification time is now within that range and leaves time that can be dedicated to the sample preparation, i.e. the extraction of hemozoin from an infected blood sample. It is interesting to note that despite the improvement made to the speed of the test, the sensitivity did not increase. The major factor is that the change in reaction parameters did not only increase the rate of the catalyzed precipitation polymeriz- ation, but also the rate of the blank reaction. Nevertheless, the detection limit of the assay is very high and within a range that is relevant for the detection of low parasitemia patients.28 Classically, LOD is described in terms of infected red blood cells per μL (iRBCs µL−1), but the translation from a hemozoin concentration to a parasitemia in iRBCs µL−1 is not so straight- forward. The hemozoin concentration varies according to the This journal is © The Royal Society of Chemistry 2020 Analyst Analyst View Article Online Paper Analyst its sensitivity and specificity as a point-of-care test in low resource settings. 11 P. W. Smit, D. Mabey, J. Changalucha, J. Mngara, B. Clark, A. Andreasen, J. Todd, M. Urassa, B. Zaba and R. W. Peeling, PLoS One, 2013, 8, e75327. 12 S. Goggins and C. G. Frost, Analyst, 2016, 141, 3157–3218. 13 O. Golubnitschaja and V. Costigliola, EPMA J., 2012, 3, 14. Conflicts of interest 14 X. Lou, M. S. Lewis, C. B. Gorman and L. He, Anal. Chem., 2005, 77, 4698–4705. The authors declare that they have submitted a patent appli- cation that covers the technology. N. B., O. R.-G., and J. P. are the inventors on this patent. Moreover, the authors are in the process of establishing a spin-out company with the aim to commercialize malaria diagnostic assays based on these polymerizations. 15 K. H. Malinowska and M. A. Nash, Curr. Opin. Biotechnol., 2016, 39, 68–75. Paper N. Sheibani and M. Habibi-Rezaei, J. Photochem. Photobiol., B, 2014, 133, 11–17. 35 P. F. Scholl, D. Kongkasuriyachai, P. A. Demirev, A. B. Feldman, J. S. Lin, D. J. Sullivan Jr. and N. Kumar, Am. J. Trop. Med. Hyg., 2004, 71, 546–551. 54 W. Liu, X. Guo and R. Guo, Int. J. Biol. Macromol., 2007, 41, 548–557. 36 G. Campuzano-Zuluaga, T. Hänscheid and M. P. Grobusch, Malar. J., 2010, 9, 346. 55 C. Asher, K. A. de Villiers and T. J. Egan, Inorg. Chem., 2009, 48, 7994–8003. 37 D. M. Newman, R. J. Matelon, M. L. Wears and L. B. Savage, IEEE J. Sel. Top. Quantum Electron., 2009, 16, 573–580. 56 A. A. Moosavi-Movahedi, M. R. Dayer, P. Norouzi, M. Shamsipur, A. Yeganeh-faal, M. J. Chaichi and H. O. Ghourchian, Colloids Surf., B, 2003, 30, 139–146. 38 Á. Orbán, M. Rebelo, P. Molnár, I. S. Albuquerque, A. Butykai and I. Kézsmárki, Sci. Rep., 2016, 6, 23218. 57 J. Du, J. J. Cullen and G. R. Buettner, Biochim. Biophys. Acta, 2012, 1826, 443–457. 39 A. Butykai, A. Orbán, V. Kocsis, D. Szaller, S. Bordács, E. Tátrai-Szekeres, L. F. Kiss, A. Bóta, B. G. Vértessy, T. Zelles and I. Kézsmárki, Sci. Rep., 2013, 3, 1431. 58 A. R. Giandomenico, G. E. Cerniglia, J. E. Biaglow, C. W. Stevens and C. J. Koch, Free Radicals Biol. Med., 1997, 23, 426–434. 40 E. Y. Lukianova-Hleb, K. M. Campbell, P. E. Constantinou, J. Braam, J. S. Olson, R. E. Ware, D. J. Sullivan and D. O. Lapotko, Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 900– 905. 59 M. Gutowski and S. Kowalczyk, Acta Biochim. Pol., 2013, 60, 1–16. 60 E. Nagababu and J. M. 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Hosseinzadeh, Analyst Analyst
https://openalex.org/W2750987583
http://deepblue.lib.umich.edu/bitstream/2027.42/113850/1/39015003269373.pdf
English
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Ideals and purposes in American forestry
Deep Blue (University of Michigan)
1,942
public-domain
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.,u_ a , . p 'r; * h 9 j$X rI IDEALS AND PURPOSES IN ALiRICAN FORESTRY by riarshall NevJin Palley Thesis submitted in partial fulfillment of the requirements for the degree of Laster of Forestry. 1942 To k1, For this and lots more to come. To k1, For this and lots more to come. To k1, To k1, For this and lots more to come. TAi BLE OFCOLiT CHAPTER 'I. Ill. ITTV. Tihe Spiri.t of P ublic Service Two Ki*nds of Foresters Niew Directions The Forest and the Trees s. 8. 34. THE SPIRIT OF PUBLIC SERVICE From the early days of the settlement of this continent the predominating philosophy of our civilization has been the philosophy of business enterprise and mate- rial progress. As older civilizations have exalted the lover of wisdom, the creator of artistic beauty or the saintly man, so have we honored and envied the man of busi- ness and the man of science. While in other ages men have seen the chief interest and significance of life in a man's relation to other men or to God, in our own times the most compelling interest of men has been the extent of their control over the world's goods or the forces of na- ture. So thoroughly does this ideal pervade every area of life it is sometimes difficult for a person breathing the air of our culture to imagine that in other times the ba- sic outlook of people on life could have been fundamen- tally opposed to our own. For all the dominance of this business ideal in the western world generally, no less than in America, there has nevertheless been a stubborn tradition of op- position to it that has steadfastly denied the validity of this de-humanized, life denying ideal. If Franklin and the Puritans are representative Americans, so also is Henry Thoreau. Franklin might degrade time into money, but Tho- reau at Walden Pond, hoeing his beans for an hour or two in the morning and spending the rest of the day in tramps -2- through the woods and contemplation, knew better. The romantic movement in the arts of the early nineteenth century, is also a part of this current which denied the pretensions of the economic philosophy of life. If the romantics in their enthusiasm sometimes were led to ex- tremes, they were on the side of the angels in proclaim- ing that striving for artistic beauty and seeking a direct and personal experience of nature were more worthy of men's energies and devotion than struggling for money and indus- trial power. A central article in the businessman's creed was the doctrine of enlightened self-interest. The indi- vidual enterpriser was obliged only to make his business decisions in accord with his own best self-interest; from the automatic workings out of the laws of economics would follow, as surely as the night the day, the greatest good to all society. THE SPIRIT OF PUBLIC SERVICE This happy state of affairs relieved a man from troublesome moral questions that might otherwise have vexed him in the day-to-day conduct of his business. If market conditions dictated the enmloyment of children in ill-lighted factories or the discharge of a number of faithful employees when a machine had been devised to do their work, it was all for the best. A farmer might, by a system of one-crop farming, destroy the fertility of his land and leave it gullied and worthless. A lumberman, in pursuit of his legitimate interest might lay waste a a forest and leave fire in his wake, his only concern be- -3- -3- ing to realize the maximum in immediate gain. As time went on, the gap between the claims made for the principle of self-interest and its actual results became more and more evident. If religion and ethical standards of conduct had been on the decline steadily for several centuries and had been largely di- vorced from men's daily activities, there were still e- nough people, as we have suggested, who took them seri- ously to raise their voices in protest against the prac- tices prevalent generally in society. The American.forestry movement is best under- stood as a part of this protest against the empire of business and the philosophy of life it generated. It is no accident that forestry came into its own in this coun- try at a time when some of the evils of big business were being brought to light and effectively challenged by gov- ernment for the first time. Of all the things that Gif- ford Pinchot and Theodore Roosevelt had in cormon, their frequently expressed desire to make private business serve the common good was not the least. As it first developed in this country, forestry offered the young men who chose it as their work a chance to realize their personalities in another world from that of the business man, a world in which human values and social needs were of primary im- portance, rather than the indirect consequences of profit motivated actions. In another, and related respect, for- estry, and conservation generally, marked a departure from N4- existing ways of doing things. If in business affairs considerations of the immediate future were of necessity paramount, in the conservation philosophy the long-term consequences of methods of handling resources were given their rightful share of attention. THE SPIRIT OF PUBLIC SERVICE In The Fight for Conservation (8), a collection of articles and speeches made by Pinchot during the years when the .new philosophy was first being put into action, we have a good picture of the prevailing spirit among for- esters in those early days. Conservation, Pinchot forcibly asserts, is a great moral issue, and matters of economics, rather than being morally neutral as the business theorists have claimed, are on the contrary heavily charged with. considerations of right and wrong: The present economic order, with its face turned away from equality of opportunity, involves a bitter moral wrong, which must be corrected for moral rea- sons and along moral lines. (p. 69) The present economic order, with its face turned away from equality of opportunity, involves a bitter moral wrong, which must be corrected for moral rea- sons and along moral lines. (p. 69) Conservation, as Pinchot sees it, adds another dimension to utilitarianism: the rightful aim of man and society be- comes realizing the greatest good for the greatest number for the longest possible time. Over against the business man, seeking his own interest and gain Pinchot sets up the ideal of the devoted public servant: Conservation, as Pinchot sees it, adds another dimension to utilitarianism: the rightful aim of man and society be- comes realizing the greatest good for the greatest number for the longest possible time. Over against the business man, seeking his own interest and gain Pinchot sets up the ideal of the devoted public servant: Public spirit is patriotism in action; it is the application of Christianity to the commonwealth; it is effective loyalty to our country, to the brother- hood of man, and to the future. It is the use of a man by himself for the general good. (pp. 95,96) Public spirit is patriotism in action; it is the application of Christianity to the commonwealth; it is effective loyalty to our country, to the brother- hood of man, and to the future. It is the use of a man by himself for the general good. (pp. 95,96) ndeed, in many ways, Pinchot regarded bid businessmen -5- -5- and the "interests" as morally corrupt. There are many men who believe, and always will believe in the divine right of money to rule. With such men argument, compromise, or conciliation is useless or worse. THE SPIRIT OF PUBLIC SERVICE The only thing to do with them is to fight them and beat them. It has been done and it can be done again. It was in the spirit of public service described by Pinchot that American forestry deserted the libraries and classrooms and at last made for the woods in 1905. The challenge of those first years was great and the ob- stacles many, but the foundations for the protection and administration of the National Forests were soundly and surely laid. It was a pioneer undertaking, and working together at a com ion task though often separated widely in space, that spirit of the first ;American foresters, so fondly recalled in recent years by some of them, developed and flourished. Walter Mulford, writing in 1925 spoke of it in this way: In his early years, service--public service--was written on his heart in what it was hoped would be indelible characters. And the spirit of the pioneer was his. Without these two qualities, in those days, he would not have been. (47) The devotion to human needs and the generous spirit of the first years of American forestry are emhasized by Raphael Zon: The first period in the forestry ?ovement nay be characterized by greit spiritual richness, the spirit of the proselyte and the missionary . . . The qualities that v ere most prominent were those of human understanding applied to the solution of a thousand human problems which arose as a result of a change in the national policy toward the public domain and above all the spirit of public service. (22j In addition to these retrospective judgments In addition to these retrospective judgments -6- there is contemporary evidence that bears witness to the life-directed emphasis of pre-war forestry. Thus, at the meeting of the Society of American Foresters in San Fran- cisco in 1915, we find a young forester named Coert Du]Bois giving an interesting talk entitled "The American Forester: His Opportunities", which is worthy of some discussion. (23) The chief concern of a forester, DuBois holds, must be ministering to human needs: There are foresters whose vision sticks in the woods and does not pierce through to the fact that the wel- fare of the people, not the welfare of a comiunity of trees, must be the aim of the forester's endeavor. Forests are only the medium through which he works. THE SPIRIT OF PUBLIC SERVICE The most successful forester is the ono whose life and work contributes most fully to the necessity, conven- ience, and pleasure of the greatest number of people,- not necessarily the one who grows the most wood per acre in the shortest time. DuBois does not stop at generalities. He speaks of the re- sponsibilities of the forester to the backwoods dweller on his ranger district, of the opportunities he has to open the world of books to the children of these wilderness people, of his chance to help in settling feuds, in suggesting better farming methods: better farming methods: Part of the training of every forester should be a course in chewing tobacco and whittling and sitting on corral fences in order to fit him for the responsible task of the after-supper tPlk with the great American nestor. Another evidence of this spirit of social re- sponsibility so strong in all this early period is to be seen in the .response of foresters to unrest among woods workers widespread at the time of the war and for a while thereafter. They maintained that it was partly the result -7- of the organization of the destructive logging industry with its cut-out and get-out policy and that the remedy lay in permanent forests. As F. A. Silcox, later Chief the Forest Service and at the time a skilled and suc- cessful conciliator of these labor difficulties put it: The "womanless, voteless, and part of the year workless," "blanket stiff" lumberjack is just as much a product of forest exploitation by devasta- tion as the community, home-owning citizen is of forest conservation. (60) Benton MacKaye (45), since a leader in the Regional Planning movement, urged that the Forest Service take stable employment and community life into account in planning working circles, with self-governmnt, adeguste educational opportunities for children, and provision made for moving communities as a whole, if the require- ments of management plans could be met only in that way. CHAPTER II TWO KINDS OF FORESTERS The idealism, the spirit of public service and the unity of purpose that characterized American forestry in its period of development were deeply felt and shared by all the nien who were associated with the early workings of the Forest Service. But even in the time of Pinchot's years as Forester in the golden day of American forestry a few voices of dissent were raised within the profession. This dissent took the form of an advocacy of the cause of the lumberman, a defense of his motives, morals and con- tribution to society. One of the first to undertake this justification of the private owner in the councils of foresters was Aus- tin Cary, who remained to the end of his life one of the most reasonable and effective spokesmen for this point of view. Cary could justly lay claim to being the oldest practicing forester in the country: in 1898 when Fernow was setting up the first school for technical training in forestry and when Pinchot was just taking over the reins of the landless Division of Forestry in the Department of Agriculture, Austin Cary was forester for a large lumber and pulpwood operation in his native state of iaine. He was an older man than Pinchot and the other leaders of the forestry movement and could look back to boyhood days spent in Maine lumber camps belonging to his uncles. As early as 1908 we find Cary speaking his mind -9- at a gathering of the Society of American Foresters in Washington, perhaps on one of those celebrated Baked Apple evenings, when this favorite dish of Pinchot's topped off the evening's entertainment. Whether Cary's paper entitled "Influence of Lumbering upon Forestry" (12) was followed by that appetizing fruit dish or not, it must have made all of the young foresters assembled on that occasion pause and reflect. After acknowledging the great advance that had been made along some lines in the past 15 years, he warmed to his message of the even- ing: In some other lines, foresters seem to me to have been surprisingly blind, and the record of their a- chievements extremely small. I refer now to the ap- plication of forestry ideas to the actual management of timber lands. TWO KINDS OF FORESTERS It seemed to Cary that the private owner would be brought to practice forestry only if he could be shown in concrete terms that it was a promising business undertaking: Foresters up-to-date have been mainly interested in demonstrating forestry principles abstractly, not in using their knowledge for the benefit of forest- owners. Foresters up-to-date have been mainly interested in demonstrating forestry principles abstractly, not in using their knowledge for the benefit of forest- owners. He chided his colleagues for their unrealistic approach He chided his colleagues for their unrealistic approach to the problems of the lumberman: They have not as a rule understood lumbering ope- rations on the mechanical and financial side. A thing may be desirable in itself and yet cost so much that a man is better off without it. This whole conception of a forester who is not also a lumberman as far as I know, is temporary and local. The German forest officer is not such a man. There were a number of reasons, however, for foresters to There were a number of reasons, however, for foresters to el hopeful about the future. The old-time lumberman, -10- he freely condeded, was hopeless. Economic forces and industrial develooments seemed to be on the side of for- estry. In the pulp and paper industry, the heavy capital investment in processing eiuipment that was required would give the owrners an interest in insuring permanent supplies of raw material to keep their mills operating. The ge- neral interest rate seemed to be falling, making timber growing economically more attractive. And, generalizing on the basis of his New England exPerience, residence, fam- ily considerations and other property interests near-by tended to dispose ovners to keep their lands in permanent productivity. Austin Cary was by no -mieans alone in his advo- cacy of lumbering and the private operator as the starting point for forestry. Early in the history of the profession men found their way into the employ of the private tiiber- men and under these circumstances they were chiefly en- gaged in harvesting virgin timber. As foresters they re- tained an interest in regenerat ing lands cut over under their direction, but in their daily work considerations of profit and loss had to be uppermost. A mutation had taken place: along side the forester there now stood the logging engineer. TWO KINDS OF FORESTERS Judson Clark, author of the International Log Rule, justified this new calling at the Society meetings in San Francisco in 1915. The most promising approach to forestry in private industry lay in more efficient and bet- ter organized logging. As Clark put the whole matter in -11- concise terms: Logging engineering is at present our best hope for the larger stumpage returns so necessary for the extension of forestry methods. (19) Donald Bruce spoke to much the same effect with an added emphasis on improved marketing: emphasis on improved marketing: But if (the forester) lives in a state which at the present rate of cutting has a three hundred years'supply of mature timber in sight, surely he can be forgiven if for the moment he concentrates his greatest efforts on its economical logging and marketing. (11) This point of view was not allowed to pass unchallenged. This point of view was not allowed to pass unchallenged. Olmsted insisted on the fundamental difference in aim of f d l b i Olmsted insisted on the fundamental difference in aim of forestry and lumbering: forestry and lumbering: It must be granted without argument, I think, that forestry, as distinguished from lumbering, aims chief- ly at providing trees for the future, for otherwise the forester would be simply a lumberman. These first debates regarding the proper scope of forestry and the relation of lumbering to forestry brieg out the reaction that was setting in against the high i- dealism of the first years. In one section of the profes- sion the lumberman was fast regaining status and becoming the object of respectful consideration. In 1916 we find Austin Cary speaking up again, his subject significantly enough being "How Lumbermen in Following their Own Inter- ests Have Served the Public." (13) Cary began by describ- ing the democracy of the industry, drawing on his memo- ries of the old New England logging camp and its traditions of hardihood and fellowship. He pointed to the moral val- ues of the business struggle, to the "discipline of indus- try:" We know that business to-day attracts many of the ablest and strongest in this country. As I see it, those men congregate to it in part because of the vi- gor and test of it -- in other words, they crave as well to make good as to make money. Industry, moreover, was good and necessary because it pro- vided employment for workers and needed articles and mate- rials for society. It had done its job pre-eminently well in the past and there was every reason for thinking that it would do so in the years to come: This continent was conquered and civilized not un- der the direction of a despot or by a carefully planned system, but through individual initiative, by the push and energy of a free people. Burt P. Kirkland, in a rejoinder (34), pointed to the shortcomings of unregulated private industry. The timber supply was being fast depleted and at the present rates of cutting only a 50 or 70 year supply was on hand. The American people are turning quite definitely from the laissez-faire system of handling industry to control in the interest of the whole community . .0 . The apparent freedom obtained by absence of any control means only freedom for the strong to over-run the rights of the weak. A division, then, was taking form in the ranks of American foresty, a division that is in existence at the present day. The business point of view was too firmly ingrained in people's minds, the need for young foresters to find jobs and support themselves and their families too compelling for matters to be otherwise. For men with se- cure places in the Forest Service, for those with inherited wealth like Pinchot and Robert Marshall, idealism has been -13- an easy matter. The defection of many younger men to private industry and the point of view of private indus- try has been a partial result of the inability of govern- ment agencies to employ more than a small proportion of the graduates of the numerous forestry schools of the country. Franklin W. try:" Reed (54) has well described this central cleavage in the ranks of American forestry: We have, it would seem, in our profession two kinds of foresters: first, the forest idealist who sees things as they ought to be, and looks upon forestry as a cause to be worked for; and second, the forest pragmatist who takes things as they are, and looks upon forestry as a business to be worked at. If a division had been building up in the ranks of American foresters in the years preceding entry into the war; it was widened to an open breach shortly after the peace was concluded. Early in 1919 Henry S. Graves, nearing the end of his term as forester initiated what was to be a long drawnout and bitterly contested fight to bring about the practice of forestry on privately owned timberlands. In a series of speeches Graves put forward a program that called for an attack on the mroblem from to directions. Destructive methods of cutting were to be pro- hibited by law; and at the same time energetic steps were to be taken to remove some of the major obstacles in the way of private forestry such as the serious fire danger and taxation methods that favored liquidation logging. In March of that same year, Frederic Olmsted, then president of the Society of American Foresters, sooke out in the Joix - nal in more emphatic terms. (49) He reminded his colleagues -14- that "the wiping out of wealth, production, and employ- ment over extensive areas good for nothing but the growing of trees," that had called their profession into existence went on almost unchecked. He held out no hope for efforts directed towards persuading the private owner to practice forestry, pointed to years of unsuccessful efforts in that. direction and enlarged on the backwardiness and inefficiency of the lumber industry. There was only one course of ac- tion that showed promise of results: "The Dublic must com- pel the lumberman to treat his forests decently." A Comittee for the Application of Forestry was formed under Pinchot's chairmanship and its report appeared in the Journal of Forestry for December, 1919 preceded by a militant article by Pinchot entitled "The Lines are Drawn." (52) Compulsion was needed to get forest omners to manage their lands in harmony with the public good. try:" It was the job of foresters to see that this compulsion was applied: The field is cleared for action and the lines are plainly drawn. He who is not for forestry is against it. The choice lies between the convenience of the lumberman and the public good. The report of the Committee which followed Pin- chot's call to arms was entitled "Forest Devastation: A National Danger and a Plan to Meet it." (72) With three times as much timber being used as was growing a shortage was clearly building up with exhaustion in sight within 50 years. Of the unfortunate social consequences of forest devastation not the least was its effect on the men who -15- worked in the woods. Housing, sanitary arrangements, and hours of labor too often have been outrageous and living conditions intolerable, and this because the lumber camp and the lumber town exist only long enough to skin the timber from the land. Many benefits were likely to flow from the establishment of permanent forests. A greater degree of control over floods and drought could be expected and soil erosion and the silting up of streams and rivers would be lessened. Hunting and fishing and other forms of forest recreation would be favored and Dublic health values would be im- proved in well-forested localities. To the committee's report was appended a draft of proposed legislation on the part of the federal government to make this plan a reality. This legislation provided for the setting up of a corrris- sion em;powered to establish rules for the cutting of tim- ber on commercial private lends and to sanction combina- tions among lumbermen for the limitation of output. Opposition made itself heard within the profes- sion from the very start of this campaign. There was re- sentment in some quarters because the findings and recom- mendations had been issued to the press before they -were published in the Journal. It seemed to some members of the Society that a small group was taking it upon itself to speak for the whole body of foresters, without properly consulting everybody concerned. More important, however, was the opposition that was voiced to the spirit and the details of the Committee's plan. Professor Tomey of Yale who had served on the Committee signed the report only with -16- a number of important reservations. try:" Donald Bruce wanted industry to be consulted and not held responsible for the conditions described in the report, -which were rather the result of unwise land laws. Many of the objectors to the plan urged caution and more deliberation. It was a big job to tackle all at once and the avenues of co-operation and education had been insufficiently explored. As Professor Tourney had maintained, a plan for federal regulation was an invasion of the rights of the states and of doubtful constitutionality. oreover, private industry could not be forced into enterprises that showed little promise of profit. That was the way the eco- nomic system that had made our country great was set up. As Philip T. Coolidge put it: As to the production of timber for ordinary cormner- cial use, the theories generally advanced by those op- posed to the radical changes proposed are essentially correct, because they are based upon unchangeable eco- nomic laws. Adam Smith might be dead and long since buried, but his soul went marching on in the ranks of the conservative wing of American forestry. A detailed referendum was held in which all the members of the Society voted on the Devastation report, clause by clause. A majority was found to be in favor of every important provision. But when a short time later, another ballot was held on the issue of whether regulation should be in the hands of the federal government or the states the supporters of state regulation won the day. This second referendum may have been the turning point in -17- the whole campaign. A program that did not have the whole-hearted support of the profession could hardly be successful in Congress. W. B. Greeley, who succeeded Graves as Forester, was in favor of regulation by the states. More erphasis was placed on the co-operative and educational aspects of the program and an effort was made to conciliate the lum- bermen after their rough treatment at the hands of the Pin- chot-Olmsted faction. A National Forestry Program Comit- tee was established in 1920 in which all the forestry in- terests of the country, -including private industry, were represented. The Committee put forward the Snell bills, based on the prineiple of grants-in-aid to states enacting legislation controlling cutting. Leanwhile, Pinchot and his group continued to press for federal legislation, their efforts being embodied in a series of Capper bills. try:" These bills unhappily came at a time when the Supreme Court, in its Child Labor Act decisions, was limiting the interpre- tation of the interstate commerce and taxation powers of Congress. With each decision, a change had to be made in the bill, giving aid and comfort to the opponents of federal regulation. The Clarke-UcNary bill which finally emerged from the whole controversy in~1924 was a victory for the school of thought that had urged moderation and co-opera- tion. Increased governmental aid to the states in fire con- trol was provided for, studies in forest taxation and forest insurance were authorized, the terms for the purchase of -18- lands for inclusion in national forests were liberalized, but nothing whatever was said about the regulation of cutting on private lands. The decade of the twenties was on the whole a time of confusion and little faith within the profession. Research workers at the Experiment Stations made new and important contributions to American forestry knowledge, bu the destruction of existing forests went on almost as be- fore, with foresters seemingly powerless to do anything about it. Some gains were recorded in the field of pri- vate, or industrial forestry, as it was called, but even its most enthusiastic supporters could not pretend that the steps taken represented more than'a token effort on the part of the industry. The private forestry point of view grew stronger within the profession during these years, as idealism, public and private, suffered a decline during the years of the scramble for "the big iioney." But the old spirit of fervor and idealism had not passed altogether from the scene. Efforts were made from time to time to re-vitalize the enthusiasm of the profession for a real national program of forestry. W ard Shepard was the most eloquent and clear-sighted of these re-vitalizers, the most energetic pleader for a revival of confidence and faith, and a new assault on the obstacles in the way of American forestry. Shepard returned to the first great enthusiasm for regulation as his point of de- -19- parture: At the close of the Great War when the world swayed between the bitterness of disillusion and the drunkenness of great drea{is, forestry thoug,t in America became dynamic with a magnificent dream We all reiember that great efflorescence of thought and speech and programs; and if the garish daylight of 1926 shows less of romance and mystery than the moonlight of 1918, it has the virtue of giv- ing us sharp outlines and tangible contrasts. (58) In words recalling Pinchot he re-asserted the moral na- ture of the cause of foresters, which touched on "the weal and happiness of millions of people through :any gen- erations." He struck out forcibly at the sort of reason- ing that was making many American foresters content with the painfully slow progress that was being recorded: The doctrine of economic fatalism induces leth- argy-and excuses failure to act . .. . Its very emphasis on p-urely external, im-:)rsonal forces as the solvent of human bungling gives to its adherents a certain blindness to the inward spiritual forces of human nature and narrows their appeal to only one of the many instincts that -drive-man to his'restless striving -- the instinct of gain. The indirect approach that sought to remove the obstacles to private forestry and then relied on self-interest to do the rest was stultifying American forestry. What was need- ed was "rational and piur-osive control." (59) Shepard was one of a group that included Pin- chot and Zon who spoke out in "A Letter to Foresters" in 1930 (71) in words recalling those of 10 years earlier, though perhaps in their proposed remedy there was more em- phasis placed on government acquisition of forest land and less on regulation of that belonging to private owners. -20- There was another modification noticeable also: the pri- vate lumberman was joined by the private forester in the culprit's seat: The destruction of the forests of America has been a long-drawn out tragedy of waste. Now we face the danger of a moral tradedy also: that the foresters of America will accept that destruction and by silence condone it. There were a number of coment pub!.ished with the letter, some of them showing signs of anger and indignation. "ls Forestry a Religion?" asked Franklin Reed. parture: (54) He pointed to the large numbers of foresters for whom there was no place in the public and educational fields, but only in private industry. Amid such surroundings the forester inevitably must look upon forestry as a business proposition, to be practiced with a due regard for financial prof- it, rather than a public cause to be striven for T 7-ith something akin to a religious zeal. Amid such surroundings the forester inevitably must look upon forestry as a business proposition, b i d i h d d C. S. Smith sprang warmly to the defense of the business forester: Carloads of bulletins on forestry have been writ- ten but little actually put into practice except through personal contacts with the lumber industry, not by ministers of the gospel of s >iritual redemp- tion, but by men who could appreciate the value of a dollar and talk in terms of profit as well as love of mankind. More than this was needed to silence the old school for- esters. A few years later they were back in print with a petition that began in these terms: petition that began in these terms: A group of members of the Society . . . have come to the conclusion that the editorial rolicy of the JOURNIAL during the last few years no longer rep- resents the broad social ideals of the founders of the Society. (v. 32, p. 781) -21- -21- -21- The coming of a second Roosevelt to the presi- dency in 1933 marked the opening of a period of expansion for the national forests. The camps of the Civilian Con- servation Corps changed the faces of many existing forests and aided in the development of new ones created during the early years of the hew Deal, as facilities for protection and public recreation were greatly enlarged. The new Chief of the Forest Service, F. A. Silcox, belonged to the pub- lic regulation school of thought and his administration, terminated by his death in 1939, was accompanied by a re- vival of the old time crusading spirit and social idealism. In the two years that the National Industrial Recovery Act was in effect some steps were taken by the lumber industry under Article X of its code to set up silvicultural stan- dards. What little progress was accomplished in this di- rection was nullified by the Supreme Court decision on the Schecter brothers' chickens that declared the whole N.R.A. program unconstitutional. Alongside the renewal of the campaign for govern- ment regulation of cutting on private forest lands, there was also a steady growth in private forestry during these years, particularly in the southern states, where the ex- panding market for pulpwood combined with the growing rea- lization on the part of timberland operators that trees could be grown at a profit brought about a great advance in the practices in effect on the lands of many companies. -22- With some real successes to their credit, the private foresters spoke out with greater assurance and were heard with more respect in the councils of the -profession. One final incident will reveal the extent to which the private forestry point of view had made itself respectable by this time. At the banquet of the 1940 meeting of the Society marking the fortieth anniversary of its founding, the chief address was given by Gifford Pinchot, who took as. his subject Theodore Roosevelt's old motto--"The Public Good Comes First". He insisted that business considerations were not a firm foundation to build sustained yield forestry on, and called, as in years gone by, for a thoroughgoing program of government owner- ship or control. When the proceedings of these meetings were published in the Journal for February 1941, the edi- tor commented on Pinchot'ts banquet address in these terms: Inasmuch as Ir. CHAPTER III - 1EW Dl CTIIUXS CHAPTER III - 1EW Dl CTIIUXS 1EW Dl CTIIUXS If up to this point we have pictured the years following the first great impulse for forest regulation as a time of decline and loss of faith among American for- esters, looked at in another way this same period can just- ly be regarded as a time of growth and broadening out and encouraging new development.' If the most ardent advocates of government control of private lumbering sulked in their tents or under the domes of state capitols, there were others who had learned the lesson of that great contro- versy and were striking out into the uncharted wilderness with new courage and resolution. They returned to the woods in a spirit of huility and openness and their re- ward was rich: where before they had only seen the saw log and the stick of pulpwood and forest land waiting to be planted or lightly cut, they now becane alive to the birds and mammals that inhabited the woods, to all the possibil- ities wild lands held out for the hard-pressed and driven city dwellers of our machine civilization, to the relation- ship of the forest with other forms of land use. Like a young pine whose terminal shoot had been broken off in a storm, American forestry showed great powers of recovery: the new enthusiasm for wild life and recreation and land use planning was a side branch of the sturdy young tree swinging around to form a new leader, If up to this point we have pictured the years following the first great impulse for forest regulation as a time of decline and loss of faith among American for- esters, looked at in another way this same period can just- ly be regarded as a time of growth and broadening out and encouraging new development.' If the most ardent advocates of government control of private lumbering sulked in their tents or under the domes of state capitols, there were others who had learned the lesson of that great contro- versy and were striking out into the uncharted wilderness with new courage and resolution. -21- Pinchot dealt at considerable length with his conception of the need for public regulation, there were perhaps many in the audience who did not agree fully or perhaps even in part with his analysis of the forestry situation in the United States. CHAPTER III - 1EW Dl CTIIUXS They returned to the woods in a spirit of huility and openness and their re- ward was rich: where before they had only seen the saw log and the stick of pulpwood and forest land waiting to be planted or lightly cut, they now becane alive to the birds and mammals that inhabited the woods, to all the possibil- ities wild lands held out for the hard-pressed and driven city dwellers of our machine civilization, to the relation- ship of the forest with other forms of land use. Like a young pine whose terminal shoot had been broken off in a storm, American forestry showed great powers of recovery: the new enthusiasm for wild life and recreation and land use planning was a side branch of the sturdy young tree swinging around to form a new leader, In their reaction against the attitude that had In their reaction against the attitude that had largely dominated in the regulation debates -- the atti- -24- tude that looked upon the wood crops of the forest as the only really significant resource involved -- this newly formed group of foresters was stating in new terms the doctrine of George Perkins Marsh, fine old pioneer in American conservation thought. arsh' s important and influential trivws had been presented in his long and sur- prisingly thorough book first published as Man and Nature in 1869, in later editions as The Earth as Modified . Human Action. (5) Combining the experiences of his travels here and abroad with extensive readings in modern and classic authors, this teacher of English rhetoric, -in- ister to Italy and amateur geographer built up an impres- sive picture of the interactions of man and the earth, which, at the same time, with its learned citations of Pliny and Cicero and its long and enthusiastic foot-notes on such matters as the tapping of maple trees for syrup and the relative merits of the nuts of the vralnut and hickory trees, makes enjoyable reading. Marsh had been greatly impressed in his travels in the Mediterannean world by large areas of land in desert and uninhabitable, many of which, according to the accounts of the Roman poets, had been fertile and well populated in classic times. CHAPTER III - 1EW Dl CTIIUXS There are parts of Asia Minor, of Northern hfrica, of Greece, and even of Alpine Europe, where the opera- tion of causes set in action by nan has brought the face of the earth to a desolation alost as complete as that of the moon; and though within that brief space of time which we call "the historical period", they are known to have been covered with luxuriant woods, verd- ant pastures, and fertile meadows, they are now too far deteriorated to be reclaimable by man. (p.43) -25- -25- Nature, in an undisturbed state, seemed wonderfully bal- anced and harmonious, but human settlement overturned this balance: But man is everywhere a disturbing agent. Wher- ever he plants his foot, the narmonies of nature are t d t di d ( 33) But man is everywhere a disturbing agent. Wher- ever he plants his foot, the narmonies of nature are turned to discords. (p. 33) Marsh saw this same pattern of forest destruction and careless farming being repeated in America and was trying to arouse people to the dangers involved in this policy. The influence of Marsh's book was considerable in the years following its publication, although his original doctrine was somewhat watered doon and distorted by the admixture of an overly sentimental regard for trees and by an exaggeration of the beneficent influences of the forest. These followers of Marsh were ridiculed and la- beled "denudatics" by a sceptical public; but their main article of belief, that, in Marsh's words, we were "break- ing up the floor and wainscoting and doors and window frames of our dwelling, for fuel to warm our bodies and to seethe our pottage," was worthy of more consideration in forestry deliberations than it was to be accorded again for many years. With the coming of Fernow and German forestry to this country a growing emphasis began to be placed on timber production in American forestry thought. Fernow (3) was well aware of the influences of the forest and of the need in some localities for protection forests. But quite -26- -26- rightly he saw his "Arbor Day friends" as overly poetic and emotional, as impractical in their passion for planting trees while forests were being destroyed. Fernow took some pains to make his conception of what a true forester was clearly understood. CHAPTER III - 1EW Dl CTIIUXS He was not an arboriculturist, a botanist, a dendrologist, a landscape gardener, a lumber- man, a forest guard, or even a silviculturist ("one who knows how to produce and reproduce wood-crops;") But in the fullest sense of the term, a forester is a technically eduated man who, with the knowledge of the forest trees and their life history and of all that pertains to their growth and production, combines fur- ther knowledge which enables him to manage a forest property so as to produce certain conditions resulting in the highest attainable revenue from the soil by wood-crops. The limitation of the role of the forester to the raising of wood crops is a lesson American foresters learned from their German masters. When the National Forests came under effective administration in 1905, the policy adopted for their de- velopment was from the first one of multiple use -- manage- ment for several objectives rather than the single one Fernow had stressed. The Forests were made up of some lands better suited to grazing than to timber production, some that found their highest use in farming. Besides, economic conditions in the lumber industry and the inacces- sibility of most of the government holdings put the sort of intensive management that Fernow had in mind out of the question. -27-m -27-m This intensive management for wood crops never- theless remained the dominating objective in the minds of American foresters, as was clearly brought out in the dis- cussions over forest regulation that followed the war. While it may have been in part a matter of expediency and political strategy, the advocates of federal regulation from the first built up their case in terms of an impend- ing timber shortage. The Forest Devastation Report of Pinchot's committee (72) opened in these words: A good and continuous supply of forest products is necessary for the safety and prosperity of the United States in peace or in war. Other matters were dealt with in the body of the report and in the debate in the mronths that followed -- the social consequences of forest destuction, the regulating effect of the forest on stream flows, the recreational values of forest lands -- but all of these things were rade to seeni secondary to the major concern, that of safeguarding the nation's timber supply. To some extent this turn of events was inevitable: these so-called intangible values of the forest, important as they might be, were hard to °eacure and express in quantitative terms. .But whatever may have been the logic of this plan of cam-paign, it turned out to be an extremely unfortunate one in the long run. Once it could be shown with some degree of plausibility that there was no' real cause for alarm about the supply of timber, that the demand for almost all classes of wood products -28- was declining, that a number of substitutes for lumber were being made available and put into use, the props were knocked out from under the whole forestry argument. If some members of the profession lost faith and asked themselves ominous sounding questions like "Why Saw Logs?" and "Whither Forestry?" others found new life in the ruins of the timber shortage campaign and pro- ceeded to build anew in several fresh directions. The first of these new developments, in logical if not in his- torical sequence, was the rediscovery and re-statement of George Marsh's broad vision of the dependence of man on the earth and of the complex inter-relationships that bind together all plant and animal life and the soil that nourishes them. -27-m Aldo Leopold, member of the Forest Ser- vice in the Southwest in the early days and in more recent years a pioneer in the field of wild life management, has been one of the most clear-headed and vigorous exponents of this doctrine among foresters. Writing in 1933 (40) he states it concisely and well: A harmonious relation to land is more intricate, and of more consequence to civilization than the his- torians of its progress seem to realize. Civilization is not, as they often assume, the enslavement of a stable and constant earth. It is a state of mutual and interdependent cooperation between human animals, other animals, plants, and soils, which may be dis- rupted at any moment by the failure of any of them. Leopold calls attention again to those "contingent and un- sought results which Marsh had found to flow from changes in the economy of nature initiated by man. Leopold calls attention again to those "contingent and un- Leopold calls attention again to those "contingent and un- sought results which Marsh had found to flow from changes in the economy of nature initiated by man. sought results which Marsh had found to flow from changes in the economy of nature initiated by man in the economy of nature initiated by man. -29- -29- We inherit the earth, but within the limits of the soil and the plant succession we also rebuild the earth -- without plan, without knowledge of its properties, and without understanding of the in- creasingly coarse and powerful tools which science has placed at our disposal. Je are remodelling the Alhambra with a steam shovel. This approach to forestry through ecology has had several salutary influen1ces. It has given foresters a broader view of the meaning of their work: in addition to helping provide that "good and continuous supply of forest products" mentioned in the Devastation Report, they can also serve as guardians of the biotic balance in nature, throwing their concerted weight against all developments in land use that are likely to be violently upsetting in their effects. Ecological wisdom has also contributed to the downfall of the older silviculture, based, in Leopold's phrase, on setting out trees like cab- bages: The "cabbage brand" of silviculture, at first seemingly profitable, was found by experience to carry unforeseen biotic penalties: insect epidemics, soil sickness, declining yields, foodless deer, im- poverished flora, distorted bird population. In their new Dauerwald the hard headed Germans are now propagating owls, woodpeckers, titriice, goshaks, and other useless wild life. (41) The control over the forces of nature that Hestern man imagines he has achieved is more illusory than real. The pride of the engineer and the scientist is an encumberment for the man who works in the world of growing things: - un- less he learns Nature's ways and works with her his labors will not stand. The conservation ideal is undergoing an extension from conservation of the forest and conservation of the soil to conservation of the whole biotic pyramid-- plants and animals as well as the soil which gives them life and to which they return. The growth of interest among foresters in land use planning about this same time is a related development that touches on ecology and wild life management in some of its aspects. The aim of this form of planning is to bring about a wiser and better balanced use of land, benefitting alike the land and the people who live on it and work it. -29- It has grown up and been most highly developed in cut over regions like northern Liichigan, where logging has been succeeded by a precarious kind of farming that in many places has not added up to a living. The land use planner procedes by classifying lands according to what they are best suited for, and then seeking to have the local people use them on that basis, whether it take them into farming, forestry, or development for wild-life and recreation. Land use planning is a departure from tradition- al timber crop forestry in this respect: it questions the wisdom of trying to grow timber for coimercial use on all cut-over and abandoned lands unsuited for agriculture. Land use planning is a departure from tradition- al timber crop forestry in this respect: it questions the wisdom of trying to grow timber for coimercial use on all cut-over and abandoned lands unsuited for agriculture. Critics of this forest-it-all policy, notably P. S. Lovejoy, have maintained strenuously that much of the land in these districts is sub-marginal for intensive forestry as well as for agriculture. Yet these have been the lands in many cases that the Forest Service, with its limited budget for land acquisition, has struggled to nurse back to producing forests, starting with bare land or iorse, - lands in Love- joy's words, "so poor that they were not worth stealing while the stealing was good."t t While from the point of view of the Forest Service "a crust was better than no bread," there has been something tragically ironic about the whole business: There may yet be an element of expediency about it, but to permit the steady vrjecking and devastation of good, thrifty, self-renewing forests, while at the same time laboriously building up new forests on cull lands, surely partakes of economic idiocy rather than of expediency. (43) of expediency. At the same time, in planting and laboriously tending lands that can hardly do better than three-log Norway pine in 150 years, the potential values inhering in the aspen and birch and pin cherry coming in to re-clothe the fire- and stump-scarred earth have been disregarded. The possi- bilities for game managerent and recreational development in these types are considerable, as Lovejoy long labored to show. For much of our forest and ex-forest land there will, I think, shortly be an extensive silviculture which merges into game cover management. -29- If the recreational use of wild lands was in- volved in game management and land utilization, there was also a strong impulse in this direction on more purely social grounds. When widespread use of the automobile had first made it possible for large numbers of people to take to the National Forests to picnic and camp, the response of many foresters had not been altogether friendly. Planned improvements were hardly in existence and the new visitors caused rangers considerable annoyance by their carelessness with fire and their capacity for getting lost in the woods. -32- As time went on, however, the right of the people to use their National Forests for recreation came to be respected and even encouraged and picnic grounds, camping sites and other recreational facilities were developed. Foresters came to recognize the hunger of city people for the open spaces and the woods, and sought to minister to that hun- ger. Recreation took its place along with timber production, watershed protection and grazing as an important use of- the forest. Another phase of the recreation philosophy was involved in the setting aside of large, isolated areas of land in the western forests in which developments and im- provements of virtually every kind were expressly excluded. The creation of these Wilderness Areas for the enjoyment by those who were able of the unaltered primitive and for the preservation of the original forest types of the con- tinent was largely the work of Robert Marshall and the Wil- derness Society, of which he was one of the moving spirits while he lived. The reservation of these large areas as wilderness was justified not on the basis of the number of people likely to avail themselves of their recreational possibilities, but rather on the quality of the enjoyment these preserves made possible for the few willing to rise and meet their challenge. In the other aspects of the recreational develop- ment under Silcox and Marshall, considerations of the great- est good for the greatest number were more prominent. -29- One -33- of the dominating themes of Forest Outings, the attractive and eloquent booklet of the Forest service that covers the whole field of recreation on the National Forests, is the need for making forest outings )ossible for families with small incomes, for giving priority in the allocation of lands suitable for recreation to public camp and picnic grounds rather than to private summer homes and limited membership clubs. This policy is er bodied in the "Basic Principles Governing Recreational Lianagement on the Lation- al Forests" quoted in the booklet: Particular attentionb will ce iven to facilities for the use of those in the low-income groups who can enjoy forest recreation only if its cost is small. This means emphasis on both camping and picnicking facilities, and organization campm o;wned by the Gov- ernment and -ade available =to those sponsoring vaca- tions for low-incone groups. (p. 287) The spirit of serving the people, so strong in the Jorest Service in the first years of its existence, is still something to be reckoned -ith. QUAPLTER IV- Up to this point an attempt has been rade to trace the origin of the forestry movement in this coun- try to the general protest raised against the excesses and evils of -our business civilization; to follow the course of the reaction among foresters against this first idealism; and finally, without meaning to subscribe to the action-reaction theory of history, to describe the renewal and extension of the first generous ipulse to- wards forestry and conservation in more recent years. An effort will be made in the discussion which follows to appraise these past .ovem ents in the world of forestry ideas more fully and to relate them to present and future developments in American life. In preceding pages considerable stress has been placed on the expansion of forestry into the allied fields of ild-life management, land utilization and recreation in recent years. Fully as noteworthy a development of this same period has been the growth in numbers and influ- ence of the adherents of the private forestry point of view -- in the lmber industry, in the forestry schools of the country, in the Forest Service itself to some ex- tent. Private foresters now save their own section ,thin the Society and representatives of this group -:a-e them- selves heard at Society meetings and in the pages of the Journal. If we are to criticize the position of these private foresters of today adequately, it will be well to -35- call to mind the original. sources of the impulse for forestry in America and the ideals of the profession in the minds of its founders. It wa s the wholesale destrmc- tion of the virgin forests of this country at the hands of the old time logging industry that called umerican forestry into existence. In contrast to the logger, who sought his own maxiium profit in the short run, the for- ester undertook to preserve the right of the public, now and in future generations, to enjoy the benefits of the forest. He was content with a modest remuneration for his efforts, happy to._be free of the general scramble for money and power, satisfied that his was a useful, valuable life. This ideal of the forester as public servant, we have already seen, was questioned early in the life of the profession by such men as Austin Cary and Donald Bruce. QUAPLTER IV- By the end of the war those who shared this new attitude were well enough represented to prevent the showing of solid support aon' foresters in the strugg.lge for the fed- eral regulation of cutting on private timberlands. Since that time, and particularly Ti the last ten or fifteen years, the private forestry philosophy has made further headway among foresters generally. The first private foresters were unwilling to limit the scope of their actions to the ational lorests, whose direct contribution as timber forests was at best -36- slight. There was more to forestry, as Professor =atthevjs likes to say, than fighting fire and planting trees. ,ore- over, the appeal of the world of business with its uncer- tainties and gambles, its rewards for skillful planning and bold and well executed strokes, must rave had weight with those who went to work for the lumbermen. This ele- ,rent ink'business life ought not to be under-estimated: it is no mere accident that men speak of being in the insur- ance gee or in the printing game. bven Forestry students get a certain amount of exhilaration from drawing up log- ging plans for 30,000 acre tracts and saving their mythical employers 24 cents for every thousand feet of lumber on the property. Those foresters whom the lumber industry took in and put to work it largely made its oTnm. The forester was sometimes able to convert the lumberman; but alimost inevi- tably he underWent an equally far-reaching conversion him- self. If there was a time when the private forester hesitated between loyalty to his employer's interests and what might have appeared to him to be his professional ob- ligation, that day is past. The private forester has take his place alongside the engineer as the technical supervisor of the operations of private business -- or, at least, that is what he would like to be given the chance to do. He views many of his publicly employed colleagues as mission- aries and crusaders who are not living up to their profession- al responsibilities. QUAPLTER IV- Harris Collingwood, Forester for the National Lumber anufacturer's Association, had this to say at the Society's meetings at Jacksonville just a few months ago: - The history of our profession reveals, in the be- lief of many, a disproportionate amount of energy de- voted toward evangelism and propaganda rather than to- ward the economic responsibilities for satisfactory continuous management of forest lands. (20) Loreover, the public forester is given taxpayers' money to do all sorts of economically unjustifiable things and is held to no strict accountability like that of business. The forestry problem of the nation is on the way towards being solved -- by the private foresters and the hard- headed business men they have won over to sustained yield by showing then. that it. pays. These are, in Collingwood's words, the foresters who believe that "permanent production of timber crops is a natural corollary of enlightened self- interest." Some assessment of the achievements of these foresters who have seen their function as'that of educating the private owner to a more enlightened sort of acouisi- tiveness is in order at this point. Their task of bringing more careful and exacting and more socially responsible methods to an old and once triumphant industry, set in its ways and resistant to new ideas, has not been an easy one. The lumber industry, like the mining industry to which it was all too closely related in outlook and technique, be- longed to an earlier industrial period than that of the newer chemical and metallurgical industries, with their em- phasis on scientific r anagerient, carejul cost accounting and the utilization of by-products. Tiriberuen had made their fortunes on the unearned increment accruing to them through the growth in stumpage values that went along with the growth of the country. WVhen the day of reckoning ar- rived early in the present century and carrying charges suddenly loomed large they lacked the flexibility and business intelligence to adjust to the times. It has been the contribution of the private forester to supply this needed flexibility and business intelligence -- to a cer- tain number of operators who have been receptive to new ideas. Some private comanies wrhich have been operated on a sustained yield basis for a number of years can point to important social and conunity benefits which have fol- lowed upon their change in methods. QUAPLTER IV- Private foresters also may claim to have vron a greater respect for the Dro- fession in some quarters, particularly among lumbermen and in business circles generally. It is fair to say in answer to the claims made by private foresters for their method that its successes have up to this point been limited. In certain regions -- notably the south -- in certain timber types, where cer- tain markets exist, some really noteworthy gains have been recorded. But for the country as a whole, the old policy of forest destruction goes on as before. rMany hardwood operators in the Lake States, for example, are steadily cutting themselves out of business with the end of opera- tions in sight. Sustained yield can not always be shown to be more profitable than liquidation logging, and often when it can be, the men who make the decisions are unim- pressed. P. S. Lovejoy covered this whole matter pretty satisfactorily some years ago in these words: pressed. P. S. Lovejoy covered this whole matter pretty satisfactorily some years ago in these words: satisfactorily some years ago in these words: Shall we continue the pretense that presently, be- fore long, soon, most private timberland operators will adopt and apply some method of continuous pro- duction? (42) There is an equally important reason for hesi- tating before conceding that private forestry represents our most hopeful line of advance. This other reason lies in the domain of morals, a domain that our first foresters insisted was all-important. Judged by their uncompro- mising standards, many practices cormonly engaged in by foresters to-day, many fairly generally received articles of belief, fall considerably short of the mark. Hence the petitions to the Journal on its editorial policy and the "Letters to Foresters" lamenting the present wayward- ness of the profession. Are we to consider these periodic protests by Pinchot and some of the older foresters as querulous outbursts on the part of men whose forestry philosophy belongs to another day, or ought we rather to take their words to heart and try to restore what has been lost? A few illistrations will help bring out what it is that old forest idealists feel has been lost. It will be noted that Harris Collingwood, quoted above, is forester for the National Lumiber Lanufacturer's Association. Be- sides working for trade associations, t . QUAPLTER IV- ere seews to be an increasing number of foresters interested in timber invost- ments and timber speculations in their own right. The.se lines of endeavor are not altogether new ones for fores- ters. They were sufficiently cormon to warrant consid- eration by Olmsted in a paper he read at the annual meeting of the Society in 121.(50) His whole treatment of these forms of employmaent under the general subject of profes- sional ethics will reveal the extent of the change in outlook since that tine. duisted strongly questioned the right of men in such lines of work as timber brokerage or speculation to professional recocniLion. he called at- tention to the forester who went to work for a private company in some such capacity as scaler, grader, or woods foreman and seemed to forget all about forestry; and to the forester en-aged by associations of timber producers or lumber manufacturers, producers or lumber manufacturers, a possible case in ;hich the forester abandons forestry, in practice and in sirit, falling irto the ranks of his cor'ercial emloyes, thinking and acting as they think and act. In Olmsted's ind the issue was clear-cut and sure. As in all his public utterances his vigor of exrression was undiminished by judicial reservations. The whole matter stood this way: Finally, is there or is there not a relation be- tween the professional standing of a forester and his regard or disregard for the public welfare in Finally, is there or is there not a relation be- tween the professional standing of a forester and his regard or disregard for the public welfare in his work? his regard or disregard for the public welfare in his work? his regard or disregard for the public welfare in his work? Professor Chapman of Yale covered this same ground in an article in the Journal a year or so later. (1ike pointed out the divided allegiance that might confront -41- the private forester who owed loyalty to his employer, but could not at the same time lend his support to de- structive laws contrary to his professional ideals and the public good, even though his employer might do so. To the private forester of today this distinc- tion between professional ideals and the public good on the one hand, and the possibly unsocial interests of his employer on the other, is not real and meaningful. QUAPLTER IV- Con- sider a recent article byT A. L. Wackermann endorsed by a number of other foresters and dealing with the responsi- bilities of forest owners.(67) It is acknowledged-that ownership of forest land carries with it sorie obligation to keep the land productive and prevent its becoriing a public nuisance. But Wackerrmann makes a drastic qualifi- cation: Obli gations of forest ownership, however, are held to be no greater than the actual or implied obl igations of owners of other lands or -ronerties which contribute to the Y!ealth and well-being of American citizens. (italics nine ) (italics nine ) Not only is the destructive logger no miore to be held to account for w.hat he does than the soil-depleting farmer; but also -- and 'Nackermann says this explicitly -- his un- social actions are put on a level ,rith those of the waste- ful manufacturer. Any program imposing responsibility must be applied equally to all these classes of owners. if this is not the sort of reasoning employed by the proverbial purple-faced gentlemen at their clubs, it is a ne ar relation to it. While the private forestry movenent has been responsible for some gains in the practice of forestry on private lands, in some respects it marks a throrback to- the past. It is an attempt to inake a profession that all its life has seen the spiritual hollowness and destructive recklessness of a business civilization turn back and throw in its lot with that old order. It rests on the belief that impersonal economic forces which in years past slashed and destroyed our forests, culled out the walnut and poplar and cherry and left behind the scrubs and crooks, which everywhere left their mark in bleeding lands and ghost cor- munities, that these same economic forces will now bring about sustained yield.forestry and its attendent blessings on most of good remaining forest lands of the country. If it does not seem desirable, then, that the main body of 'American foresters join with krl. Wackernann and the private foresters, where nay they look for direc- tion and purpose in years to come? The philosophy of 01r- sted and Pinchot still has its adherents and there is still much to be said for it. QUAPLTER IV- Events of recent years have sug- gested, however, that these proposals for government con- trol and a greater measure of government ownership are in themselves not sufficient to insure that all will be well. While Dlanting programs, selective cuttings and the balancin of growth against drain all have their importance, the re- sults of all these policies in terms of the people most directly affected by them must not be overlooked. Raphael Zon, veteran member of the Forest Service now serving as chief of the Great Lakes Experiment Station, -43- has recently shown a wise concern for the present needs of people in the forest regions. (70) He has seen clearly that the little trees being set out on the National Forests of the Lake States will be of little help in meeting the economic difficulties of this region for =any years to coe and that what is needed is a concerted effort directed at developing its remaining resources and processing them at home to provide employment and a better livelihood for the people. Julian Griggs of this school has been using this saime approach to the problems of the cut-over region, with a greater emphasis on its recreational resources. Zon ur- ges that uses be found for the aspen noi rotting in the woods and proposes that, with governent help, it be used to provide better housing for people who are badly in need of it in these districts. He calls for the promotion of small forest industries that :ill do such things as salvage fuelwood from the slash of lumbering operations for the mar- ket in not too distant cities, manufacture wood souvenirs for the tourist trade and make woodcraft articles for more general distribution. has recently shown a wise concern for the present needs of people in the forest regions. (70) He has seen clearly that the little trees being set out on the National Forests of the Lake States will be of little help in meeting the economic difficulties of this region for =any years to coe and that what is needed is a concerted effort directed at developing its remaining resources and processing them at home to provide employment and a better livelihood for the people. Julian Griggs of this school has been using this saime approach to the problems of the cut-over region, with a greater emphasis on its recreational resources. QUAPLTER IV- Zon ur- ges that uses be found for the aspen noi rotting in the woods and proposes that, with governent help, it be used to provide better housing for people who are badly in need of it in these districts. He calls for the promotion of small forest industries that :ill do such things as salvage fuelwood from the slash of lumbering operations for the mar- ket in not too distant cities, manufacture wood souvenirs for the tourist trade and make woodcraft articles for more general distribution. This same view of things is taken by Lyall E. Peterson in an admirable paper read at the New Developments session of the 1940 meetings. (51) He is dissatisfied with much current forestry thought that sees all our problems in technical terms and asks: "Is it possible that the great cause of conservation is eroding away along with soil, and humanity in general?" He urges a more flexible approach -.44- on the part of foresters, asks them to think of people, their needs and their lives, whether it lead to forestry agriculture or something else: All foresters, by one -eans or another, are pled- ged to the central objective of encouraging better management and use of forest resources for the great- est good of the greatest number in the long run. This social objective,, in reality, rests on the basic family needs for food, clothing, shelter, fuel and cultural expression. Perhaps if we begin with these needs -- community by conmmunity -- instead of with the forest, it will be possible to make greater strides. This approach to American life through the region and the local coumunity that has characterized the work of the land use planners and is involved in such a development as the Tennessee Valley Authority may be one of the most hopeful signs in the current forestry picture. We have long put our trust in economic self- interest or in the restraining powers of the government to attain our ends. It is time now to recognize that the most important factors involved are human customs and folkways and beliefs; that the solution to our p roblemi lies, in L. W. Wilson's phrase, "beyond economics".. QUAPLTER IV- To the extent that community planners can bring peovple to have a greater reverence for the land they work, to f eel a deeper sense of responsibility in the use they make of it, much will be accomplished towards strengthening what Aldo Leopold has well called "the conservation ethic'". For the duration of the war not too much can be hoped for in the way of improvement in the use we make of our forest lands. The greatly enlarged market for lumber and wood products is accelerating the pace of destructive -45- logging in many parts of the country. Public forest agen- cies, depleted in man power and appropriations, can only strive to hold on to what has been won in the past. The American Forestry Association has called for a truce on the issue of governmental regulation until after the war. Foresters, however, are justified in opposing unnecessary inroads made upon the forests under color of the national emergency. The years following the war will be the decisive ones for American forestry. After the strain and sacri- fice the war will surely entail there will be a strong urge to return to the irresponsibility and carelessness of the good old days. If the sacrifices made during the war are to have any lasting meaning this tendency to go back to old ways of doing things will have to be strenuously resis- ted. Part of the meaning of the Nazi revolt against civi- lization is this: the worship of money and gadgets and material possessions that has been general in the western democracies is not an ideal worthy of imen' s faith and alle- giance. With all its evils and excesses the fascist ideal has some human meaning to it, has given men something out- side themselves to believe in and has made economic institu- tions responsive to human purposes rather than at odds witL them. In the place of the Nazi barbarism we must find a new kind of system, rich in human values and satisfactions and subject to control by us for the things we want. ;Towrds this new dispensation foresters have been looking for long years. Acting in the great tradition of American forestry -46- -46- they can play an honorable part in bringing it about and in giving it form and meaning. Books 1. Cameron, Jenks. The Development of Governmental Forest Control in the United States. Balti- more, 1928. 2. Ely, Richard T. and Wehrwein, George S. Land Economics. New York, 1940. 3. Fernow, Bernhard E. Economics of Forestry. New York, 1902. 4. Gauss, Christian. A Primer for To-morrow. New York, 1935. 5. Marsh, George P. The Earth as odified by Human Action. New York, 1885. 6. Mumford, Lewis. Technics and Civilization. New York, 1938 printing. 7. Mumford, Lewis, Faith for Living. New York, 1941 printing. 8. Pinchot, Gifford. The Fight for Conservation. New York, 1910. 9. Thirty Foresters (Russell Lord, ed.) Forest Outings. United States Government Printing Office, 1940. 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Is Silviculture Possible in America? 5 28:1110-1118:; J29: 219-224, 3.4-390 , 505-515. 60. Silcox, F. A. 1920. Forestry and Labor. J 18:316. 61. Silcox, F. A. 1939. A Federal Plan for Forest Regu- lation Within the Democratic Pattern. J 37:116-119. 62. Sterling, Ernest A. 1919. A Plea for Adjust:ent. J 17:771-772. 63. Sterling, Ernest A. 1927. Why Not Teach App1ied Forestry? J 25:574-576. 64. Stuart, R. Y. 1933. Forestry in a New Era (Co'-ents by Walter Kulford). J 31:141-146n. 65. Tinker, E. W. 1929. Federal Forests, Foresters, and Recreation. J 27:251-253. 66. Tkatchenko, M. K. 1930. Origin and .Propagation of Forestry Ideas. J 28:595-617. 67. Wackermann, A. E. 1941. Private Forestry and the Professional Attitude. J 39:114-116. 68. Wehrwein, George S. 1939. The Economists Approach to Ecology. J 37:731-734. 69. Woods, John B. 1940. Progress without Hysteria. J 38:763-767. 70. Zon, Raphael. 1939. The Human Side of Land Use. J 37:735-737. 71. A Letter to Foresters. 1930. 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Research on the Intangible Cultural Heritage: Musical Culture of Hebei Local Opera "Dizi Diao"
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Introduction The flute tune is a small folk opera passed down in the Hengshui area of Hebei Province, which is isolated and has a small audience, and its historical heritage is rather tumultuous, so unlike Beijing opera, Kunqu and other nationwide operas that have received extensive attention from the academic community, there is a relative lack of literature and research results on the flute tune. In the process of reviewing the relevant literature, the author has paid special attention to the Luo Luo cavity because it is a local genre of the Luo Luo cavity. Although it has been passed down for hundreds of years, it is rarely known to outsiders and has been neglected by cultural workers and theoretical researchers, so there is a relative lack of research data. In this paper, we examine the musical culture of the flute tune based on literature, field research, and theories related to anthropology, ethnology, culture, and musicology. This paper focuses on four aspects: the historical origin of the flute tune, its historical heritage and development status, its musical and artistic characteristics, and its social function and value. In the section of historical tracing, this chapter mainly uses the method of documentary evidence to analyze the relationship between the flute tune and the three origins of the Luo Luo cavity to further reveal the significant kinship between the flute tune and the northern string cavity and to clarify the historical origin of the Luo Luo cavity to which it belongs. In the section on historical transmission and development, this chapter mainly uses oral history and documentary and historical data to sort out, analyze and verify the history of the production and transmission of the flute tune in the Hengshui area, and investigate the current development of the flute tune association, so as to have a clearer development of the transmission of the flute tune; in the section on musical and artistic characteristics, this chapter mainly focuses on the flute tune's board style, the music of the song, the accompaniment band (accompaniment instruments, accompaniment characteristics) and the performance program (role system). In the section on social functions, this chapter focuses on the moral and ethical values of the patriarchal society, entertainment and folk rituals, artistic and aesthetic values, as well as the historical changes in promoting the spirit of the times, inheriting traditional culture and regional cultural identity values of the flute. Research on the Intangible Cultural Heritage: Musical Culture of Heb Local Opera “Dizi Diao” Research on the Intangible Cultural Heritage: Musical Culture of Hebei Local Opera “Dizi Diao” Local Opera Dizi Diao Jie MaPing, Heng Tsai Article Info Abstract Article History Received: March 05, 2023 The flute tune, also known as Guanzuo tune, is an ancient local opera genre passed down in Guanzuo Village, Rao Yang County, Hengshui City, Hebei Province, China. It was named after the flute, which was the main playing instrument in the early stage of its formation. During the Zhengde period of the Ming Dynasty, the flute tune was taught to the village children by a private school teacher who taught in Guanzuo Village, and it has been sung to this day. Flute tune singing high, men and women with the same cavity in the same tune, witty and humorous language, singing lining words, false words more, unique repertoire, rich in local characteristics, once loved by the people of Hengshui Rao Yang area. Accepted: June 06, 2023 Keywords : Flute Tune, Luo Luo Cadence, Inheritance And Development, Artistic Characteristics, Social Functions DOI: 10.5281/zenodo.8011187 Jie MaPing, Heng Tsai Jie MaPing, Heng Tsai The flute tune, also known as Guanzuo tune, is an ancient local opera genre passed down in Guanzuo Village, Rao Yang County, Hengshui City, Hebei Province, China. It was named after the flute, which was the main playing instrument in the early stage of its formation. During the Zhengde period of the Ming Dynasty, the flute tune was taught to the village children by a private school teacher who taught in Guanzuo Village, and it has been sung to this day. Flute tune singing high, men and women with the same cavity in the same tune, witty and humorous language, singing lining words, false words more, unique repertoire, rich in local characteristics, once loved by the people of Hengshui Rao Yang area. Multicultural Education Multicultural Education Volume 9, Issue 6, 2023 Introduction Analysis of the changing times of the value of the social function of the flute tune in the historical construction. In the above four chapters, the author draws on the triple analysis model of "concept-behavior-sound" in music anthropology, and combines "historical construction", "social maintenance", and "social identity", In the above four chapters, the author draws on the triple analysis model of "concept-behavior-sound" in music anthropology, and incorporates three dimensions of "historical construction,""social maintenance," and "individual creation and experience" to form an interactive and mutually feedback whole, thus revealing the musical and cultural connotations of the flute tune. 20 21 Literature on Flute Tunes The literature on the flute tune is mainly found in some opera monographs, local chronicles and compilations of local opera materials; the academic research results are only available in a master's thesis (Lei reputation, 2015), and since then, the academic research on the flute tune has basically been blank, with only a short report published in the press aiming to call the attention of the community to the development of the flute tune in Guanzao village and to save the survival of the flute tune. report (Li Xiaoguo, 2017). The musical and artistic characteristics of the flute tune. The musical and artistic characteristics of the flute tune. The main focus of the literature on the musical and artistic characteristics of the flute tune is on the music of the singing voice, the accompanying instruments, and the traditional repertoire of the flute tune. The editorial committee of the Chinese Opera Dictionary (1995), the editorial committee of the Chinese Opera Music Integration - Hebei Volume (1999), the editorial department of the Cultural Bureau of the Hengshui District Office of Opera (1986), and Lei reputation (2015) have all introduced and discussed the singing music of the flute tune. The above-mentioned literature has provided either complicated or brief descriptions of the types of flute singing music and the types of tunes, and their basic views are consistent. Lei's (2015) discussion of cantorial music is based on the above-mentioned literature, and the characteristics of the dobai of the flute tune have been described through field investigations. Accompanying instruments. The Editorial Committee of the Dictionary of Chinese Opera Types (1995), the Editorial Committee of the Chinese Opera Music Integration-Hebei Volume (1999), the Editorial Department of the Opera Journal of the Cultural Bureau of the Hengshui District Administration (1986) and Lei reputation (2015) have all documented research on the accompanying instruments of flute tunes. The core view of the relevant literature is that the accompaniment band of the flute tune is divided into Wenchang and Wuchang; at the early stage of the formation of the flute tune, the accompaniment instrument is a single wind instrument combination with the flute as the main instrument, and afterwards, under the influence of Hebei Bangzi, string instruments are added; the instrumentation of Wuchang is almost similar to that of Hebei Bangzi. Based on this basic view, Lei reputation (2015) clearly listed the number of different musical instruments in Wenchang and Wuchang through field surveys. The historical origin of the flute tune. The Editorial Committee of the Dictionary of Chinese Opera Types (1995), the Editorial Committee of the Chinese Opera Music Integration - HebeiVolume (1999), the Editorial Committee of the Chinese Opera Journal- Hebei Volume (1999), the Local History Compilation Committee of Hebei Province (2001), the Local History Compilation Committee of Rao Yang County (1998), the Editorial Department of the Opera Journal of the Cultural Bureau of the Hengshui District Administration (1986), and Lei reputation (2015) have all briefly introduced the historical origin of the flute tune. The core viewpoint of the above documents is basically the same, that is, the flute tune is an alias of Luo Luo cavity, also known as Guan Zuo tune, because at the early stage of its formation, the flute is the main instrument, "flute tune" is thus named; flute tune in Ming Zhengde years, by a private school teacher in Guan Zuo village, passed on to the local children's association, sung to this day. The historical origin of the flute tune is basically discussed by Lei (2015), which is similar to the above- mentioned literature. I expect to enrich and add to it by further examination in the thesis, which already provides a historical reference for this paper. Research data on the Lingqiu Luo Luo cadence There is a wealth of scholarly information on the Lingqiu Luo Luo cadence genre, mainly focusing on the following aspects: The historical origin and transmission history of Lingqiu Luo Luo cavity. An Fang (2016) identifies the historical origin of Lingqiu Luo Luo cavity; Hu Xiang (2010) explores the relationship between the Luo Luo cavity and the Yiyang cavity and the string cavity of the Ming dynasty through the comparison of the singing voice, tune and accompaniment; and introduces the development of the three stages of the historical inheritance of the Luo Luo cavity: the rise, the flourishing and the decline. The musical and artistic characteristics of Lingqiu Luo Luo cavity. An Fang (2016) studied the performance system, vocal system, and performance script of Lingqiu Luoluo cadence; Hu Xiang (2010) introduced the musical characteristics of the singing voice, tune, and accompaniment of Luoluo cadence; Liu Zhengang (2016) conducted an in-depth study on the types of singing tune, orchestra establishment, singing repertoire, singing characteristics, and performance characteristics of Lingqiu Luoluo cadence; Duan Wenying (2018) conducted an in-depth study on the musical characteristics of Lingqiu Luoluo cadence. Jiao Chunmei (2015) discusses the typical artistic characteristics of the Lingqiu Luo Luo cadence that have been developed over a long period of time and have an ancient cultural lineage, namely, the rhyming of the lyrics, the dialectal nature of the language, the support of the accompaniment, the structure of the cantata, the unique high eight-vocals, and the unique "back-gong sound" and "back-gong sound". "Anna Zhang and Penglong Wang (2012) focus on the unique cultural characteristics of the Luo Luo cadence in the process of development, i.e., the unique stage steps, the original country voice, and the drama that is both solemn and harmonious. The research on the cultural characteristics of the music and art of Lingqiu Luo Luo cavity accounts for a large proportion of the literature. The heritage development of Lingqiu Luo Luo cavity. Deng Jiali (2015) proposes new ideas on the inheritance development of Luoluo cavity through the large modern drama "Village Judge" created and rehearsed by the Luoluo cavity troupe, and discusses the inheritance value of combining Luoluo cavity opera with campus culture; Liu Zhengang (2016) proposes multiple approaches for the future development of Lingqiu Luoluo cavity, including "strengthening the construction of the troupe itself, drawing on the development model of two- person opera, and powerfully promoting it in various ways. Literature on the Luo Luo cadence In the process of reviewing the literature on Luo Luo cavity, I found that only a few documents have been written on the distribution of the origin of Luo Luo cavity, and the research on Luo Luo cavity is mainly focused on the old Shanxi opera "Lingqiu Luo Luo cavity", which was officially approved as the first batch of national intangible cultural heritage by the State Council in 2006. The origin and distribution of the Luo Luo cavity. Traditional repertoire. p The Editorial Committee of the Dictionary of Chinese Opera Genres (1995), the Editorial Committee of the Chinese Opera Music.The traditional repertoire of the flute tune has been introduced by the Editorial Committee of the Chinese Opera Music Integration-Hebei Volume (1999), the Local History Compilation Committee of Hebei Province (2001), the Local History Compilation Committee of Rao Yang County (1998), the Editorial Department of the Cultural Bureau of the Hengshui District Office of Opera (1986), and Lei Reputation (2015). Each of the above-mentioned literature has its own focus on the traditional repertoire of the flute tune, and the number of repertoire involved varies. Lei Reputation (2015) provides a more detailed introduction to the traditional repertoire of the flute tune and, on this basis, classifies the repertoire and indicates when the different repertoires were performed. In addition to the above research on the three main aspects of the musical art features of the flute tune, namely, the music of the singing voice, the accompanying instruments, and the traditional repertoire, the editorial committee of the Chinese Opera Dictionary (1995) provides a brief introduction to the performance program of the flute tune; Lei (2015) introduces the roles of the flute tune on the basis of a field survey, making the research richer. The existing theories on the artistic characteristics of the flute tune have laid a certain theoretical foundation for this thesis, but I believe that there is still room for further advancement on the basis of Lei's 22 richer existing research results. The existing research on the musical body of the flute tune lacks the analysis of example scores to support the study, and does not form a systematic study of "meter, tune, score and instrument". In particular, the development and revitalization of the flute tune has not been discussed by the Flute Tune Association in the 21st century. richer existing research results. The existing research on the musical body of the flute tune lacks the analysis of example scores to support the study, and does not form a systematic study of "meter, tune, score and instrument". In particular, the development and revitalization of the flute tune has not been discussed by the Flute Tune Association in the 21st century. The origin and distribution of the Luo Luo cavity. The editorial committee of the Chinese Opera Journal - Hebei Volume (1999), the editorial department of the Chinese Music Dictionary (2016), and Lei's reputation (2015) have documented the distribution of the origin of the Luoluo cantata, but with different views. The Editorial Board of Chinese Opera - Hebei Volume (1999) records that the Luo Luo cavity is an older type of opera and is related to the silk string cavity in Hebei, which is popular in the Yanbei region of Shanxi, and the silk string is also called Luo Luo cavity; the Editorial Department of Chinese Music Dictionary (2016) introduces the entry of Luo Luo cavity, which is an opera cavity and is related to the high cavity, and was popular in many areas in the Qing Dynasty. many regions, but it was lost much later. Lei reputation (2015) The origin of the Luo Luo cadence is taken from the viewpoint of the editorial committee of the Chinese Opera Journal - Hebei Volume (1999). Through the above literature, I believe that the source of the Luo Luo cadence is ambiguous in the literature, and I hope to develop a clear view on the distribution of the source of the Luo Luo cadence through further research in this study, so as to prove the connection between the flute tune and it. Theoretical foundation In this thesis, the author intends to draw on the relevant theories of fusion cultural anthropology to investigate the musical The cultural connotation of the flute tune will be studied. In this paper, I will draw on the "holistic" and "functionalist" views of cultural anthropology and integrate them into my research. In this paper, we will draw on the "holistic" and "functionalist" viewpoints of cultural anthropology, and consider culture as a whole, so that the study of any cultural element should be placed in the context of the overall culture. The study of any cultural element should be understood in the context of the culture as a whole, and should not be studied in isolation from other cultural elements. Music as an important part of culture As an important part of culture, the attitude towards music art will not be regarded as a "pure art" independent of the forest of culture. The art of music, as an important part of culture, should not be treated as a "pure art" independent of culture, but rather as a more cultural In his approach to music art, he does not regard it as a "pure art" independent of culture, but rather focuses on discovering and examining its symbiosis with other cultural subsystems, with a more cultural character, and puts his research in a holographic perspective of multidimensional and systematic observation. In order to The purpose of this study is to examine the relationship between music and human beings and the cultural context of music. The study of ethnomusicology is a multi-dimensional and systematic perspective. In the field of ethnomusicology, Alan P. Merriam, an important representative of the anthropological school of thought, has proposed "an understanding of the cultural context of music. In the field of ethnomusicology, Alan P. Merriam, an important representative of the anthropological school, proposed "the study of music in culture" (1960), "the study of music as culture" (1973), and "the study of music as culture" (1973). The study of music as culture" (1973), based on the traditional methodological categories of cultural anthropology, places music in the context of cultural facts and extends music to The study of music as culture" (1973) is based on the traditional methodological scope of cultural anthropology, which places music in the context of cultural facts and extends music to cultural categories related to human behavior and perceptions. Theoretical foundation A concise triple analysis model is devised: "concept-behavior-sound" (conceptualization of music, music-related behavior, music itself). Understanding Music as a Culture The semantics of music is no longer a matter of the sound itself, but rather a matter of the relationship between concept, act and music. The answer is to be found in the relationship between concept, act and music. "Concept, act, and sound" are an inseparable whole. All three exist simultaneously in musical performance. Merriam's threefold model of analysis established the "anthropological school" (musical anthropology). Merriam's triple analysis model laid the foundation for the "anthropological" (musical anthropology) model of analysis, which became the classic research model. Later, the scholar Timothy Rice (Timothy Rice further elaborated on Merriam's model, and defined "historical composition, social maintenance, individual application and experience" anthropological framework with Merriam's "concept-act-voice" triple Merriam's "conceptual- behavioral-phonological" triad of analysis is combined. Merriam's and Rice's modes of analysis provide important insights for the author's research on this topic. In this paper, I hope to be able to provide the author with some insights into this topic. In this paper, I hope to trace the origins of the flute genre, to clarify the historical origin of the Lolo cadence to which it belongs, and to explore the musical art of the flute. In this paper, I hope to trace the origins of the flute genre, to clarify the historical origins of the Luo Luo cadence to which it belongs, and to investigate what musical and artistic characteristics of the flute are directly related to the origins of the Luo Luo cadence. The history of the flute tune is examined and its development status is investigated and researched, with the aim of discovering the history of the flute tune since its formation in Rao Yang County during the Ming dynasty. The purpose of this study is to discover the supporting information about the development of the flute tune in Rao Yang County during the Ming Dynasty until the 1950s, and to The purpose of this study is to find out the evidence of the development of the flute tune in Hengshui Rao Yang County from the Ming dynasty to the 1950s. The above is a clearer picture of the development of flute tunes. The above discussion will be based on the "historical construction" of the flute tune. The above discussion is based on the "Historical Construction" section of the flute tune. Research data on the Lingqiu Luo Luo cadence Liu Zhengang (2016) put forward feasible suggestions for the future development of Lingqiu Luo Luo chuan from multiple perspectives, such as "strengthening the construction of the troupe itself, learning from the development model of two-person opera, and powerful propaganda in various ways. The research results of Lingqiu Luoluo cavity have been published by other scholars, so I will not list them here. The old Shanxi opera "Lingqiu Luoluo cavity" and the flute tune belong to the same Luoluo cavity. The above- mentioned research results on the historical origin, musical and cultural characteristics, and inheritance and 23 development of Lingqiu Luo Luo cavity provide a rich theoretical reference and basis for this paper to study the historical inheritance and artistic characteristics of the flute tune. Theoretical foundation In this paper, we analyze and study the musical and artistic characteristics of the flute tune, such as the characteristics of the singing voice The paper analyzes the musical and artistic characteristics of the flute tune, such as the characteristics of the singing voice, modal features, performance procedures, and the accompanying orchestra, in order to reveal the common features of the Luo Luo cavity and the unique characteristics of the The interpretation of the musical characteristics is inseparable from the performers. The interpretation of the artistic characteristics of the music is inseparable from the performers, and in this section we will analyze and explain the "personal creativity and experience" of the artists. With the change of the times, the development of society, the modernization the development of society and the intensification of the modernization process, the author needs to re-examine and explore the cultural value of the flute tune in modern 24 The cultural value of the flute tune in modern society, highlighting the changing times in the historical construction of the social function of the flute tune. In this In this section, it is the functional value of the flute that has been "socially maintained" that has allowed the flute to The four parts of the discussion above are In the above four parts of the discussion, the author has put three dimensions of the three dimensions throughout the analysis, so that they form a mutually integrated, interactive, and mutually feedback whole, and in the process of In the process of analysis, the author draws on the triple analysis model of "concept-behavior-acoustics" in music anthropology to reveal the role of the flute tune as a traditional Chinese opera. In the analysis, the authors will draw on the triple analysis model of "concept-act-acoustics" in music anthropology to reveal the musical and cultural connotations of the flute tune as a cultural heritage of traditional Chinese opera. Local opera is an important part of local culture. The growth of any opera genre is a combination of its own internal development The growth of any opera genre is the result of the combined forces of its own internal development and external social and cultural forces. In this regard, it is important to examine the resources, environment, state, and laws of the existence and development of local opera art by applying the theories of cultural anthropology. Practical and Theoretical Implications p As we can see from the relevant literature, there is still a lack of documented research on flute tunes. The research results on the flute tune are relatively Since there are few research results about the flute tune, this thesis provides a large research space. However, it also means that there is a challenge to conduct an in-depth and systematic study of flute tunes. The relatively abundant research results of the Lingqiu Luoluo cadence The research results of the Lingqiu Luo Luo cadence provide a rich literature reference for this thesis, which can help to clarify the history of the transmission of the flute tune and analyze the history of the flute tune. It provides an important reference for clarifying the history of the transmission of the flute tune and analyzing whether it is homologous and similar to the Lingqiu Luo Luo cadence, which is also in the Luo Luo cadence family. Theoretical foundation To conduct a comprehensive, holistic and systematic study of the folkloric opera genre Dizi Tun and its cultural environment, revealing the The study will reveal the cultural basis of the flute genre, break through the general description and artistic aesthetic study of the flute genre, and focus on the special artistic form of the flute genre and its profound artistic form. The study of the cultural foundations of the flute genre will break through the general description of the genre and the artistic aesthetic research, and develop a comprehensive, extensive, complete and in-depth research perspective on the special artistic form and profound cultural connotation of the genre. The study of flute tunes is not limited to the content, form, and aesthetics of flute tunes, but rather to a deeper cultural understanding of flute tunes. The study will not only provide a realistic picture of the survival of the flute genre, but will also be able to explore the regularity of the sustainable development of local opera. The study not only depicts the survival of the genre, but also shows the inheritance and transformation of the artistic and social functions of the flute in the midst of historical transition, thus revealing the relationship between the cultural environment and and social functions during the historical transformation, thus revealing the symbiotic relationship between the cultural environment and the art form, and providing inspiration and lessons for the inheritance and protection of the flute tune. The study also provides inspiration and reference for the preservation of the flute. Conclusion Under the impact of today's mass media and new entertainment industries, the traditional folk opera genre of flute tune is facing the existential crisis of extinction. The call for saving, preserving and inheriting the flute tune has become stronger and stronger. In the field During the survey, although I can feel from the words and mannerisms of artists such as Fan Fanchangzha, Fan Shanli, Fan Xiyan, Fan Shaojiong, etc. I can feel their deep hope and love for the flute tunes, but, based on the current conditions, can still see the president and However, based on the current conditions, it is still possible to see some of the difficulties and helplessness of the president and members. The author hopes that this study will provide some theoretical results for the study of the local folk I hope that through this study, I can provide some theoretical results for the study of local folk opera, and provide some theoretical support for the inheritance, revitalization, and sustainable development of the flute tune. It is also hoped that the study will bring the development of the flute tune as an intangible cultural heritage to the attention of the academic community. References Chase, Gilbert (1976). Musicology, History and Anthropology: Current Thoughts. in John W. Grubb. Grubb.Current Thought in Musicology.Austin:University of Texas Press. Austin: University of Texas Press, 231-246. Helene Iswolsky (1984).Rabelais and His World (Bakhtin, M. M.).Indiana University Indiana University Press.(Original work published 1965) Jie MaPing International College, Krirk UniversityThailand Heng Tsai International College, Krirk UniversityThailand References References Chase, Gilbert (1976). Musicology, History and Anthropology: Current Thoughts. in John W. Grubb. Grubb.Current Thought in Musicology.Austin:University of Texas Press. Austin: University of Texas Press, 231-246. Helene Iswolsky (1984).Rabelais and His World (Bakhtin, M. M.).Indiana University Indiana University Press.(Original work published 1965) 25 Marcus,George E(2015).Ethnography in/of the World System: the Emergence of Annual Review of Anthropology,(24),95-117. p gy ( ) Timothy Rice (1987).Toward the Remodeling of Ethnomusicology. Ethnomusicology, 31(3),469-488. y g gy gy Timothy Rice(2003).Time,Place and Metaphor in Musical Experience and Ethnography.Ethnomusicology,47(2),151-179. Author Information Author Information Heng Tsai International College, Krirk UniversityThailand
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Superconducting quantum interference at the atomic scale
Nature physics
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Superconducting quantum interference at the atomic scale Sujoy Karan   1, Haonan Huang   1, Ciprian Padurariu   2, Björn Kubala   2,3, Andreas Theiler4, Annica M. Black-Schaffer   4, Gonzalo Morrás   5, Alfredo Levy Yeyati   5, Juan Carlos Cuevas   5, Joachim Ankerhold   2, Klaus Kern   1,6 and Christian R. Ast   1 ✉ Sujoy Karan   1, Haonan Huang   1, Ciprian Padurariu   2, Björn Kubala   2,3, Andreas Theiler4, Annica M. Black-Schaffer   4, Gonzalo Morrás   5, Alfredo Levy Yeyati   5, Juan Carlos Cuevas   5, Joachim Ankerhold   2, Klaus Kern   1,6 and Christian R. Ast   1 ✉ A single spin in a Josephson junction can reverse the flow of the supercurrent by changing the sign of the superconducting phase difference across it. At mesoscopic length scales, these π-junctions are employed in various applications, such as finding the pairing symmetry of the underlying superconductor, as well as quantum computing. At the atomic scale, the counterpart of a single spin in a superconducting tunnel junction is known as a Yu–Shiba–Rusinov state. Observation of the supercurrent rever- sal in that setting has so far remained elusive. Here we demonstrate such a 0 to π transition of a Josephson junction through a Yu–Shiba–Rusinov state as we continuously change the impurity–superconductor coupling. We detect the sign change in the critical current by exploiting a second transport channel as reference in analogy to a superconducting quantum interference device, which provides our scanning tunnelling microscope with the required phase sensitivity. The measured change in the Josephson current is a signature of the quantum phase transition and allows its characterization with high resolution. T T wo superconductors that are connected by a weak link can sustain a supercurrent, which is carried by Cooper pairs— the well-known Josephson effect1. Inserting a single spin into the junction may completely change its behaviour by revers- ing the direction of the supercurrent2, which is the result of a π shift in the phase across the junction. Such π-junctions have been used in finding the pairing symmetry in unconventional super- conductors3–7, and they have been proposed as building blocks for energy-efficient quantum computing or high-speed memory8–10. At mesoscopic length scales (~10 to 100 nm), π-junctions may be realized by singly occupied quantum dots or ferromagnetic inter- layers11–18. At the atomic scale (~0.1 nm), a single magnetic impu- rity, which is exchange-coupled to a superconductor, induces a spin nondegenerate superconducting bound state, a Yu–Shiba–Rusinov (YSR) state19–21. Superconducting quantum interference at the atomic scale By tuning the magnetic exchange coupling, the YSR state can be driven through a quantum phase transition (QPT) with a concomitant π shift22–26. tunnelling microscope (STM) requires a rudimentary phase sensi- tivity through an additional transport channel15,18. In this Article we demonstrate a supercurrent reversal in an atomic-scale Josephson junction through a YSR state as we move across the QPT. We produce a magnetic impurity at the apex of a superconducting vanadium tip (Fig. 1a), which is approached to a superconducting V(100) sample. As we approach, the atomic forces pull on the impurity27,28,32–35, which reduces the impurity–supercon- ductor coupling Γ along with the magnetic exchange coupling (also see Supplementary Section A). This concomitantly allows the YSR state to pass from the strong scattering regime (screened spin) to the weak scattering regime (free spin), as outlined in Fig. 1b. The two scenarios are schematically illustrated in Fig. 1c, where the total spin in the free spin regime is Stot = 1 2. In the screened spin regime, a Cooper pair is broken to screen the impurity spin, changing the overall parity of the system (indicating whether the total number of particles is even or odd) as well as the total spin to Stot = 0. The hallmark of this QPT in YSR states is a discontinuous change in the total spin of the respective ground states: a previously free impurity spin turns into a screened spin when the magnetic exchange coupling increases beyond a critical value. Consequently, a reversal in the flow of Cooper pairs through a YSR state has been predicted26. Experimentally, the QPT can be identified by a zero energy crossing of the YSR state in differential conductance spectra27–31. However, the actual consequences for the fundamental Josephson effect remain elusive in atomic-scale junctions. In our experiment, the detection of the supercurrent reversal is only possible by exploiting the parallel presence of a second trans- port channel featuring a conventional superconducting Bardeen– Cooper–Schrieffer (BCS) gap without any YSR state as a reference channel (Fig. 1a and Methods). The sign change in the supercurrent through the YSR state manifests itself as a step in the measured net Josephson current, resulting from the changeover of a constructive to a destructive interference of the two transport channels across the QPT. 1Max-Planck-Institut für Festkörperforschung, Stuttgart, Germany. 2Institut für Komplexe Quantensysteme and IQST, Universität Ulm, Ulm, Germany. 3Institute of Quantum Technologies, German Aerospace Center (DLR), Ulm, Germany. 4Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden. 5Departamento de Física Teórica de la Materia Condensada and Condensed Matter Physics Center (IFIMAC), Universidad Autónoma de Madrid, Madrid, Spain. 6Institut de Physique, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. ✉e-mail: c.ast@fkf.mpg.de Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics https://doi.org/10.1038/s41567-022-01644-6 https://doi.org/10.1038/s41567-022-01644-6 Superconducting quantum interference at the atomic scale j The observation of a YSR state-based π-junction is experimen- tally challenging, because detecting such a phase shift between superconducting ground states requires a reference channel. At mesoscopic length scales, this is typically solved by employing a superconducting quantum interference device (SQUID) loop geom- etry14–17. To reach similar conditions at the atomic scale, a scanning The evolution of the YSR state as a function of the normal state conductance GN is shown in Fig. 1d. The YSR state moves across the QPT when the YSR energies are closest to each other. Because both tip and sample are superconducting, in the spectrum the tip YSR states appear at voltages V shifted by the sample gap Δs, that is, eV = ε + Δs with the YSR state energy ε varying with the normal 1Max-Planck-Institut für Festkörperforschung, Stuttgart, Germany. 2Institut für Komplexe Quantensysteme and IQST, Universität Ulm, Ulm, Germany. 3Institute of Quantum Technologies, German Aerospace Center (DLR), Ulm, Germany. 4Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden. 5Departamento de Física Teórica de la Materia Condensada and Condensed Matter Physics Center (IFIMAC), Universidad Autónoma de Madrid, Madrid, Spain. 6Institut de Physique, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. ✉e-mail: c.ast@fkf.mpg.de Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics 893 Articles NATuRe PHYSicS Free spin –20 –4 0.5 1.0 1.5 QPT Conductance (µS) 2.0 2.5 –2 0 Voltage (mV) 2 4 0 20 40 Normalized dI/dV dI/dV (a.u.) 60 80 100 Impurity–superconductor coupling b d c a Screened spin Free spin Tip Impurity Sample YSR BCS ε = 0 Level asymmetry –4 10–4 10–3 Conductance GN/G0 10–2 B = 1.5 T 0 0 1 2 6 4 2 0 –4 –2 0 Voltage (mV) 2 4 TK (K) 3 4 50 100 e f Normalized dl/dV Josephson –2 0 Δt + Δs Δt + Δs –(Δt + Δs) –(Δt + Δs) Voltage (mV) GN = 0.009G0 2 4 Screened spin Γ Fig. 1 | Atomic YSR state. a, Schematic of the tunnel junction. The YSR impurity is at the tip, with two transport channels (BCS and YSR) indicated as dashed lines. b, Phase diagram of the YSR system as function of impurity–superconductor coupling and level (particle–hole) asymmetry. c, Schematic of the free spin and the screened spin regime. In the screened regime, a Cooper pair is broken, changing the overall parity of the system. Superconducting quantum interference at the atomic scale Assuming a harmonic current– phase relation in the DCB (that is, I(φ) = ICsinφ, where IC is the critical current), the Josephson current is predicted to scale with the square of the critical current (Supplementary Section C), that is, I(V) ∝I2 C ∝G2 N (refs. 37–40). It can be directly seen in the data that this square dependence is not fulfilled in the dataset in Fig. 2a. In particular, the region indicated by the horizontal bracket shows notable deviations, even a slight decrease in the Josephson current with increasing conductance. The conductance at which the QPT occurs is indicated by a vertical dashed line, which falls directly into the region of the horizontal bracket. state conductance GN. Interestingly, there are no distinct coherence peaks visible at the sum of the tip gap and the sample gap ±(Δt + Δs), which indicates that a second transport channel through an empty gap (that is, without any YSR state and hence with coherence peaks) has a much weaker, but still finite transmission compared to the YSR state. This can be more directly seen in a single spectrum near the QPT, which is shown in Fig. 1e. The coherence peaks at eV = ±(Δt + Δs) are greatly reduced, and the YSR peaks are promi- nently enhanced by a factor of almost 100. y y To confirm that the impurity–superconductor coupling (in our case the impurity–tip coupling) decreases with increasing con- ductance, we measured the Kondo effect in the same junction by quenching the superconductivity in a magnetic field of 1.5 T. The Kondo spectra are shown in the inset of Fig. 1f, from which we extract the Kondo temperature TK by fitting the data to numerical renormalization group (NRG) theory calculations (for details see Supplementary Section B). As the Kondo temperature is directly related to the magnetic exchange coupling, we conclude that the impurity–superconductor coupling decreases with decreasing tip–sample distance (that is, increasing junction conductance). Physically, the impurity is pulled away from the tip by the attractive atomic forces of the approaching sample substrate34. This has been observed in a number of different experiments on YSR states intro- ducing a tunability of the YSR energy27,28,32–34. For a more quantitative analysis we plot the current maxima IS (switching current) for each conductance as a blue line in a double logarithmic plot in Fig. 2b. Superconducting quantum interference at the atomic scale d, Differential conductance spectra as a function of bias voltage (x axis) and conductance (y axis). The prominent peaks are the YSR states, and the coherence peaks are only barely visible. The vertical dashed lines indicate the position of the sample gap, Δs. e, Horizontal line cut through d to show the prominent YSR peaks. f, Kondo temperature of the Kondo effect in the same junction at a magnetic field of 1.5 T, when superconductivity is quenched. The Kondo spectra are shown in the inset. –20 –4 0.5 1.0 1.5 QPT Conductance (µS) 2.0 2.5 –2 0 Voltage (mV) 2 4 0 20 40 Normalized dI/dV 60 80 100 d Δt + Δs –(Δt + Δs) d b Level asymmetry a b Impurity Conductance (µS) dI/dV (a.u.) 10–4 10–3 Conductance GN/G0 10–2 B = 1.5 T 0 1 2 6 4 2 0 –4 –2 0 Voltage (mV) 2 4 TK (K) 3 4 f –4 0 50 100 e Normalized dl/dV Josephson –2 0 Δt + Δs –(Δt + Δs) Voltage (mV) GN = 0.009G0 2 4 f Fig. 1 | Atomic YSR state. a, Schematic of the tunnel junction. The YSR impurity is at the tip, with two transport channels (BCS and YSR) indicated as dashed lines. b, Phase diagram of the YSR system as function of impurity–superconductor coupling and level (particle–hole) asymmetry. c, Schematic of the free spin and the screened spin regime. In the screened regime, a Cooper pair is broken, changing the overall parity of the system. d, Differential conductance spectra as a function of bias voltage (x axis) and conductance (y axis). The prominent peaks are the YSR states, and the coherence peaks are only barely visible. The vertical dashed lines indicate the position of the sample gap, Δs. e, Horizontal line cut through d to show the prominent YSR peaks. f, Kondo temperature of the Kondo effect in the same junction at a magnetic field of 1.5 T, when superconductivity is quenched. The Kondo spectra are shown in the inset. blockade (DCB) regime, in which the STM operates36, the typical voltage-biased measurement shows a negative current peak fol- lowed by a positive current peak of equal size near zero bias voltage. The evolution of the Josephson effect as a function of conductance is shown in Fig. 2a. Each spectrum is shown in a bias voltage range of ±60 μeV and offset horizontally. Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics Superconducting quantum interference at the atomic scale 0.0025 0.0050 0.0075 0.0100 0.0150 0.0200 0.0300 –20 –10 Current (pA) 0 10 20 QPT Conductance GN/G0 a IS 0.005 1 10 Switching current, ls (pA) 0.010 QPT ∝ G2 N Conductance GN/G0 0.015 0.020 0.030 0 0.1 0.2 0.3 b ε/Δt b QPT Conductance GN/G0 0.8 1.0 1.2 1.4 √ISRN 1.6 c 0.005 0.010 0.015 YSR junction Reference 0.020 0.030 Switching current, ls (pA) ε/Δt Fig. 2 | Josephson effect. a, Josephson spectra I(V) in a range of ±60 μeV, shifted horizontally by the conductance at which they were measured. The horizontal bracket indicates the region where the evolution deviates from the conventional Ambegaokar–Baratoff formula. The QPT is indicated by the vertical dashed line. b, The switching current IS, which is the current maximum indicated in a as function of the normal state conductance GN (blue line). The square dependence at low and high conductance is indicated by dashed lines (labelled ∝G2 N). The YSR energy as a function of conductance is shown as a blue line. The minimum indicates the QPT (vertical dashed line). The YSR state energy (red line) is plotted for comparison. The minimum indicates the QPT. c, √ISRN (blue line) of the data in b, which is proportional to ICRN for a harmonic energy–phase relation (RN, renormalized resistance (see text); IC, critical current). A reference junction without any YSR states is shown as an orange line, indicating the expected evolution according to the Ambegaokar–Baratoff formula. order parameter (Supplementary Section F), are shown in red and blue, respectively. To calculate the energy–phase relation, we apply a constant phase difference φ across the tunnel junction, but no bias voltage. A Fourier expansion of the energy–phase relation reveals that the most relevant contribution to the Josephson effect is the harmonic term proportional to cos(φ). Zooming in to both chan- nels (cf. Fig. 3b), we estimate that the ratio of the channel transmis- sions is about 4:1 (YSR:BCS). This results in a significantly smaller amplitude for the energy–phase relation of the BCS channel (red) than in the YSR channel (blue) (Fig. 3b, ‘Individual channels’). The coherent superposition of these two channels (Fig. 3b, ‘Channel sum’) leads to an overall sign change, as well as different amplitudes, when the channels are in phase (Fig. 3b, upper row) or out of phase (Fig. 3b, lower row). Superconducting quantum interference at the atomic scale In the measurement, we are only sensitive to the change in amplitude I(V) ∝(EYSR + 2EBCS)2 though, which results in the obvious step in Fig. 2c. We attribute the width of the step to the finite temperature in our experiment. To put the evolution of the switching current in reference to other Josephson junctions, we calculate √ISRN, which is shown in Fig. 2c (RN is the normal state tunnelling resistance). This quantity is pro- portional to the product ICRN for a harmonic energy–phase relation. In this way, the overall conductance dependence is eliminated such that the measurement appears like a step in Fig. 2c, with a sizeable reduction in height almost by a factor of two across the QPT. We will show in the following that this is due to a supercurrent reversal in the YSR channel, which leads to a crossover from a constructive to a destructive interference between the two transport channels. To compare the experimental data to the theory, we have to renormalize the normal state resistance RN for the YSR spectra due to the enhanced density of states from Kondo correlations (Supplementary Section D). The reference spectra (Fig. 2c, orange line) are measured for a Josephson junction without any YSR states—the √ISRN is constant, as expected from the Ambegaokar– Baratoff formula41–43. To understand the behaviour of the Josephson effect in Fig. 2, the most convincing and consistent explanation is to assume the pres- ence of a second transport channel that does not pass through a YSR state (BCS channel) and, therefore, is phase-insensitive. In fact, even a weak residual BCS channel is sufficient to provide a consistent analysis of our data. Thus, we first have a look at the energy–phase relations far away from the QPT at high and low conductance. In Fig. 3a, the energy–phase relations for the BCS channel and the YSR channel, which are calculated from a mean field Anderson impu- rity model (Supplementary Section E) and a non-selfconsistent Because the temperature in our experiment (10 mK) is still non-zero, we expect fluctuations due to thermal excitations close to the QPT. The probability for the system to be in the ground state (blue) or the excited state (orange) is indicated in Fig. 3c using an effective temperature of 75 mK. This will broaden the expected sharp features associated with the quantum phase transition. Superconducting quantum interference at the atomic scale The expected square dependence on the conductance (IS ∝I2 C ∝G2 N) can be clearly seen for very small and very large conductances. In the transition region (indicated by the horizontal bracket), the behaviour of the switching current IS strongly changes. For comparison, we plot the experimentally extracted energies of the YSR state (red line), which has a minimum at the QPT (vertical dashed line; see Methods for details). This indi- cates a drastic change in the behaviour of the Josephson effect across the QPT. The supercurrent, which is carried by tunnelling Cooper pairs (Josephson effect), is visible throughout the range of con- ductance values (Fig. 1e, red arrow). In the dynamical Coulomb Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics 894 Articles NATuRe PHYSicS 0.005 0.0025 0.0050 0.0075 0.0100 0.0150 0.0200 0.0300 1 10 –20 –10 Current (pA) 0 10 20 Switching current, ls (pA) 0.010 QPT QPT ∝ G2 N QPT Conductance GN/G0 Conductance GN/G0 Conductance GN/G0 0.015 0.020 0.030 0 0.8 1.0 1.2 1.4 √ISRN 1.6 0.1 0.2 0.3 c b a 0.005 ε/Δt 0.010 0.015 YSR junction Reference 0.020 0.030 IS Fig. 2 | Josephson effect. a, Josephson spectra I(V) in a range of ±60 μeV, shifted horizontally by the conductance at which they were measured. The horizontal bracket indicates the region where the evolution deviates from the conventional Ambegaokar–Baratoff formula. The QPT is indicated by the vertical dashed line. b, The switching current IS, which is the current maximum indicated in a as function of the normal state conductance GN (blue line). The square dependence at low and high conductance is indicated by dashed lines (labelled ∝G2 N). The YSR energy as a function of conductance is shown as a blue line. The minimum indicates the QPT (vertical dashed line). The YSR state energy (red line) is plotted for comparison. The minimum indicates the QPT. c, √ISRN (blue line) of the data in b, which is proportional to ICRN for a harmonic energy–phase relation (RN, renormalized resistance (see text); IC, critical current). A reference junction without any YSR states is shown as an orange line, indicating the expected evolution according to the Ambegaokar–Baratoff formula. Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics Superconducting quantum interference at the atomic scale For the Josephson current only the oscillation is relevant, but not the energy offset. b, A zoom-in to the oscillation for the two channels. The upper two panels in b show the in-phase oscillation in the screened spin regime. The lower two panels in b show the out-of-phase oscillation in the free spin regime, which is indicative of the supercurrent reversal. The STM is not sensitive to the sign of the supercurrent, but the concomitant change in magnitude is clearly observable. c, Probabilities for the system to be in the ground state (blue) or the excited state (red) as a function of YSR state energy. d, The √ISRN product comparing experimental data with a theoretical fit. The only free parameters are the temperature, which defines the width of the QPT, and the relative channel transmission, which defines the step height. (virtual) state instead of the continuum. It is this switching between transport regimes when crossing the QPT that we observe experi- mentally. Recalling from Fig. 1f that the impurity–substrate cou- pling reduces on increasing the conductance, we move from the screened spin regime across the QPT to the free spin regime as the tip approaches the sample. This is consistent with the evolution of the Josephson current from an in-phase superposition (zero junction) to an out-of-phase superposition (π-junction) as the conductance increases. are the effective temperature Teff = 75 mK, which is determined by the width of the transition, and the ratio of the two channel trans- missions, which is determined by the step height. For a best fit, we find that the YSR channel contributes 78.4% and the BCS reference channel contributes 21.6% to the total conductance relevant to the Josephson effect, which is consistent with the prominent YSR states and the strongly reduced coherence peaks in the quasiparticle spec- tra (Fig. 1e). All other parameters are given by the experimentally extracted values. In this way, we demonstrate that the supercurrent through an atomic-scale YSR state reverses on crossing the QPT, which can be detected in the STM by means of a BCS reference channel, in analogy to a SQUID geometry (for other YSR tips see Supplementary Section H). At the QPT, a system typically becomes very sensitive to external parameters, such as temperature. Here we note that the width of the QPT step in Fig. 3d depends only on temperature, but expe- riences no broadening from voltage noise. Superconducting quantum interference at the atomic scale Taking the excitation probability due to the finite temperature into account, we can calculate the expected Josephson current in the DCB regime (Supplementary Section G). The fit is shown in Fig. 3d, which shows excellent agreement with the data. The only free parameters Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics 895 Articles NATuRe PHYSicS 0.005 0.8 1.0 1.2 1.4 1.6 d c b a –50 T = 75 mK 0 Energy 0 –Δ Δ Phase, ϕ π/2 3π/2 0 0.5 Probability 1.0 0 50 Ground Individual channels EYSR YSR BCS EYSR + 2EBCS EYSR + 2EBCS 2EBCS 2EBCS EYSR Channel sum Data Fit State occupation Excited 0.010 0.015 0.020 0.030 Conductance GN/G0 YSR energy ε (µeV) √ISRN QPT π 2π In phase Out of phase ϕ ϕ ϕ ϕ Fig. 3 | Supercurrent reversal. a, Energy–phase relation for the BCS channel (red) and the YSR channel (blue). For the Josephson current only the oscillation is relevant, but not the energy offset. b, A zoom-in to the oscillation for the two channels. The upper two panels in b show the in-phase oscillation in the screened spin regime. The lower two panels in b show the out-of-phase oscillation in the free spin regime, which is indicative of the supercurrent reversal. The STM is not sensitive to the sign of the supercurrent, but the concomitant change in magnitude is clearly observable. c, Probabilities for the system to be in the ground state (blue) or the excited state (red) as a function of YSR state energy. d, The √ISRN product comparing experimental data with a theoretical fit. The only free parameters are the temperature, which defines the width of the QPT, and the relative channel transmission, which defines the step height. a 0 Energy 0 –Δ Δ Phase, ϕ π/2 3π/2 YSR BCS π 2π b Individual channels EYSR EYSR + 2EBCS EYSR + 2EBCS 2EBCS 2EBCS EYSR Channel sum In phase Out of phase ϕ ϕ ϕ ϕ a Channel sum In phase Energy Out of phase 0.005 0.8 1.0 1.2 1.4 1.6 d Data Fit 0.010 0.015 0.020 0.030 Conductance GN/G0 √ISRN QPT d d c –50 T = 75 mK 0 0.5 Probability 1.0 0 50 Ground State occupation Excited YSR energy ε (µeV) c Fig. 3 | Supercurrent reversal. a, Energy–phase relation for the BCS channel (red) and the YSR channel (blue). Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics Articles Rev. Lett. 97, 177003 (2006).f 14. Shimizu, Y., Horii, H., Takane, Y. & Isawa, Y. Multilevel effect on the Josephson current through a quantum dot. J. Phys. Soc. Jpn 67, 1525–1528 (1998). 15. van Dam, J. A., Nazarov, Y. V., Bakkers, E. P. A. M., De Franceschi, S. & Kouwenhoven, L. P. Supercurrent reversal in quantum dots. Nature 442, 667–670 (2006). Final 16. Estrada Saldaña, J. C. et al. Two-impurity Yu-Shiba-Rusinov states in coupled quantum dots. Phys. Rev. B 102, 195143 (2020). 17. Martín-Rodero, A. & Yeyati, A. L. Josephson and Andreev transport through quantum dots. Adv. Phys. 60, 899–958 (2011). y 18. De Franceschi, S., Kouwenhoven, L., Schönenberger, C. & Wernsdorfer, W. Hybrid superconductor-quantum dot devices. Nat. Nanotechnol. 5, 703–711 (2010). Fig. 4 | Cooper pair tunnelling. The tunnelling process in the free spin and screened spin regimes is shown from the initial to the final state via an intermediate (virtual) state in the excitation picture. The order is set by the numbered red arrows. The total spin Stot describes the total spin of the YSR system, including the spin of the impurity. a, In the free spin regime, the order of the spins is exchanged compared to the initial state, which results in the supercurrent reversal. b, In the screened spin regime, the order of the spins is retained, such that there is no sign change in the supercurrent. 19. Yu, L. Bound state in superconductors with paramagnetic impurities. Acta Phys. Sin. 21, 75–91 (1965).h y 20. Shiba, H. Classical spins in superconductors. Prog. Theor. Phys. 40, 435–451 (1968). 21. Rusinov, A. I. Superconductivity near a paramagmetic impurity. JETP Lett. 9, 85–87 (1969).f 22. Salkola, M. I., Balatsky, A. V. & Schrieffer, J. R. Spectral properties of quasiparticle excitations induced by magnetic moments in superconductors. Phys. Rev. B 55, 12648–12661 (1997). y 23. Flatté, M. E. & Byers, J. M. Local electronic structure of defects in superconductors. Phys. Rev. B 56, 11213–11231 (1997). 24. Flatté, M. E. & Byers, J. M. Local electronic structure of a single magnetic impurity in a superconductor. Phys. Rev. Lett. 78, 3761–3764 (1997). Having direct tunable access to the QPT could be exploited to enhance the sensitivity in quantum sensing applications, such as a local temperature measurement. Articles Articles NATuRe PHYSicS 1 2 0 ε Δ 0 ε Δ 0 ε Δ 0 ε Δ 0 ε Δ 0 ε Δ 3 4 1 2 3 4 Tip Tip Sample Sample Initial Intermediate Final Free spin regime Screened spin regime a b Impurity Spin Stot = 0 Stot = 0 Stot = 0 Stot = 1 2 Stot = 1 2 Stot = 1 2 Fig. 4 | Cooper pair tunnelling. The tunnelling process in the free spin and screened spin regimes is shown from the initial to the final state via an intermediate (virtual) state in the excitation picture. The order is set by the numbered red arrows. The total spin Stot describes the total spin of the YSR system, including the spin of the impurity. a, In the free spin regime, the order of the spins is exchanged compared to the initial state, which results in the supercurrent reversal. b, In the screened spin regime, the order of the spins is retained, such that there is no sign change in the supercurrent. 7. Tsuei, C. C. & Kirtley, J. R. Pairing symmetry in cuprate superconductors. Rev. Mod. Phys. 72, 969–1016 (2000). 1 2 0 ε Δ 0 ε Δ 0 ε Δ 3 4 Tip Sample Initial Intermediate Final Free spin regime a Impurity Spin Stot = 0 Stot = 1 2 Stot = 1 2 0 ε Δ 0 ε Δ 0 ε Δ 1 2 3 4 Tip Sample Screened spin regime b Stot = 0 Stot = 0 Stot = 1 2 a y ( ) 8. Gingrich, E. C. et al. Controllable 0–π Josephson junctions containing a ferromagnetic spin valve. Nat. Phys. 12, 564–567 (2016). 9. Cleuziou, J.-P., Wernsdorfer, W., Bouchiat, V., Ondarçuhu, T. & Monthioux, M. Carbon nanotube superconducting quantum interference device. Nat. Nanotechnol. 1, 53–59 (2006). 10. Feofanov, A. K. et al. Implementation of superconductor/ferromagnet/ superconductor π-shifters in superconducting digital and quantum circuits. Nat. Phys. 6, 593–597 (2010). y 11. Ryazanov, V. V. et al. Coupling of two superconductors through a ferromagnet: evidence for a π junction. Phys. Rev. Lett. 86, 2427–2430 (2001). g j y 12. Kontos, T. et al. Josephson junction through a thin ferromagnetic layer: negative coupling. Phys. Rev. Lett. 89, 137007 (2002). 13. Robinson, J. W. A., Piano, S., Burnell, G., Bell, C. & Blamire, M. G. Critical current oscillations in strong ferromagnetic π junctions. Phys. Articles Also, demonstrating the coherent superposition of different transport channels in the DCB regime introduces a rudimentary phase sensitivity in STM measurements that can be exploited in other scenarios as well, for example, in finding the symmetry of the superconducting order parameter. 25. Balatsky, A. V., Vekhter, I. & Zhu, J.-X. Impurity-induced states in conventional and unconventional superconductors. Rev. Mod. Phys. 78, 373–433 (2006). 26. Graham, M. & Morr, D. K. Imaging the spatial form of a superconducting order parameter via Josephson scanning tunneling spectroscopy. Phys. Rev. B 96, 184501 (2017). 27. Farinacci, L. et al. Tuning the coupling of an individual magnetic impurity to a superconductor: quantum phase transition and transport. Phys. Rev. Lett. 121, 196803 (2018). 28. Malavolti, L. et al. Tunable spin-superconductor coupling of spin 1/2 vanadyl phthalocyanine molecules. Nano Lett. 18, 7955–7961 (2018). Received: 8 September 2021; Accepted: 20 May 2022; Published online: 4 July 2022 Received: 8 September 2021; Accepted: 20 May 2022; Published online: 4 July 2022 Received: 8 September 2021; Accepted: 20 May 2022; Published online: 4 July 2022 33. Kezilebieke, S., Žitko, R., Dvorak, M., Ojanen, T. & Liljeroth, P. Observation of coexistence of Yu-Shiba-Rusinov states and spin-flip excitations. Nano Lett. 19, 4614–4619 (2019). Superconducting quantum interference at the atomic scale This is in contrast to conventional scanning tunnelling spectroscopy, where temperature broadening is typically obscured by voltage noise as well as interac- tions with the environment36. Hence, YSR-tip functionalization may open new developments for low-temperature thermometry with high spatial resolution, where measuring the slope of the QPT step accesses the temperature (Supplementary Section I). To better understand the origin of this supercurrent reversal and to illustrate the crucial role of the impurity spin, we discuss the Cooper pair tunnelling process in Fig. 4 using the excitation picture. Zero energy denotes the ground state, ε is the energy of the excited YSR state and Δ marks the beginning of the quasiparticle contin- uum. The order is given by the numbered red arrows. Figure 4a describes the free spin regime, where the total spin is Stot = 1 2 (refs. 22,25). The Cooper pair transfer process involves a swap between two fermions, one associated with the impurity and one associated with the Cooper pair, as depicted by arrows 3 and 4 in Fig. 4a. Formally, this appears as an exchange of fermion opera- tors inducing a negative sign (π shift)15,44. By contrast, Fig. 4b shows the screened spin regime, which has a ground state with total spin Stot = 0. Here the Cooper pair transfers conventionally, as in an empty BCS gap, except that the YSR state is used as an intermediate In summary, the experimental results directly reveal the con- sequences of the discrete parity change across the QPT in YSR states as well as the role of the impurity spin, which manifests itself in the supercurrent reversal. This allows for directly deter- mining the ground state of the YSR state; that is, the supercur- rent reveals whether the system is in the free or the screened spin regime. Our results establish an important connection to meso- scopic π-junctions, providing the perspective to transfer some of their concepts, for example, as sensing tools, to the atomic scale. Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics 896 Online content Any methods, additional references, Nature Research report- ing summaries, source data, extended data, supplementary infor- mation, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/ s41567-022-01644-6. 29. Franke, K. J., Schulze, G. & Pascual, J. I. Competition of superconducting phenomena and Kondo screening at the nanoscale. Science 332, 940–944 (2011 30. Bauer, J., Pascual, J. I. & Franke, K. J. Microscopic resolution of the interplay of Kondo screening and superconducting pairing: Mn-phthalocyanine molecules adsorbed on superconducting Pb(111). Phys. Rev. B 87, 075125 (2013). 31. Kamlapure, A. et al. Correlation of Yu-Shiba-Rusinov states and Kondo resonances in artificial spin arrays on an s-wave superconductor. Nano Lett. 21, 6748–6755 (2021). 32. Huang, H. et al. Quantum phase transitions and the role of impurity-substrate hybridization in Yu-Shiba-Rusinov states. Commun. Phys. 3, 199 (2020). References 1. Josephson, B. D. Possible new effects in superconductive tunnelling. Phys. Lett. 1, 251–253 (1962).h 1. Josephson, B. D. Possible new effects in superconductive tunnelling. Phys. Lett. 1, 251–253 (1962).h 34. Ternes, M. et al. Interplay of conductance, force and structural change in metallic point contacts. Phys. Rev. Lett. 106, 016802 (2011). 2. Kulik, I. Magnitude of the critical Josephson tunnel current. J. Exp. Theor. Phys. 22, 841–843 (1966).l 35. Bork, J. et al. A tunable two-impurity Kondo system in an atomic point contact. Nat. Phys. 7, 901–906 (2011). 3. Tsuei, C. C. et al. Pairing symmetry and flux quantization in a tricrystal superconducting ring of YBa2Cu3O7 − δ. Phys. Rev. Lett. 73, 593–596 (1994). y 36. Ast, C. R. et al. Sensing the quantum limit in sc spectroscopy. Nat. Commun. 7, 13009 (2016).f y 36. Ast, C. R. et al. Sensing the quantum limit in scanning tunnelling spectroscopy. Nat. Commun. 7, 13009 (2016).f 4. Kirtley, J. R. et al. Symmetry of the order parameter in the high-Tc superconductor YBa2Cu3O7 − δ. Nature 373, 225–228 (1995). spectroscopy. Nat. Commun. 7, 13009 (2016).f 37. Senkpiel, J. et al. Single channel Josephson effect in a high transmission atomic contact. Commun. Phys. 3, 131 (2020). p 5. Van Harlingen, D. J. Phase-sensitive tests of the symmetry of the pairing state in the high-temperature superconductors—evidence for dx2−y2 symmetry. Rev. Mod. Phys. 67, 515–535 (1995). 38. Ingold, G., Grabert, H. & Eberhardt, U. Cooper-pair current through ultrasmall Josephson junctions. Phys. Rev. B 50, 395–402 (1994).f 6. Wollman, D. A., Van Harlingen, D. J., Giapintzakis, J. & Ginsberg, D. M. Evidence for dx2−y2 pairing from the magnetic field modulation of YBa2Cu3O7-Pb Josephson junctions. Phys. Rev. Lett. 74, 797–800 (1995). 6. Wollman, D. A., Van Harlingen, D. J., Giapintzakis, J. & Ginsberg, D. M. Evidence for dx2−y2 pairing from the magnetic field modulation of YBa2Cu3O7-Pb Josephson junctions. Phys. Rev. Lett. 74, 797–800 (1995). 39. Devoret, M. H. et al. Effect of the electromagnetic environment on the coulomb blockade in ultrasmall tunnel junctions. Phys. Rev. Lett. 64, 1824–1827 (1990). Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics 897 Articles NATuRe PHYSicS Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics Acknowledgements We gratefully acknowledge stimulating discussions with R. Drost, J. Fransson, B. Jäck and F. Tafuri. We dedicate this manuscript to the memory of F. Portier for the many inspirational discussions that the authors had with him. This work was funded in part by the ERC Consolidator Grant AbsoluteSpin (grant no. 681164; C.R.A.) and by the Center for Integrated Quantum Science and Technology (IQST). J.A. and C.P. acknowledge funding from the DFG under grant no. AN336/13-1, the IQST and the Zeiss Foundation. A.L.Y. and J.C.C. acknowledge funding from the Spanish Ministry of Science and Innovation (grant no. PID2020-117671GB-I00 and PID2020-114880GB-I00) and from the María de Maeztu Programme for Units of Excellence in Research and Development (grant no. CEX2018-000805-M). A.M.B.-S. acknowledges funding from the Knut and Alice Wallenberg Foundation through the Wallenberg Academy Fellows programme. Some of the computations were enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC) at the High Performance Computing Center North (HPC2N) partially funded by the Swedish Research Council through grant agreement no. 2018-05973. Extracting the superconducting gap. Experimentally, we extracted superconducting gaps for tip and sample of about Δt = Δs = 725 μeV (for the data in the main text). The crossing of the tip YSR state at the QPT is at eV = Δsample in the spectrum. The other gap Δtip can be extracted from the position of the coherence peaks (Δtip + Δsample) and the previously extracted Δsample. Extracting the superconducting gap. Experimentally, we extracted superconducting gaps for tip and sample of about Δt = Δs = 725 μeV (for the data in the main text). The crossing of the tip YSR state at the QPT is at eV = Δsample in the spectrum. The other gap Δtip can be extracted from the position of the coherence peaks (Δtip + Δsample) and the previously extracted Δsample. We actually find a small gap of ~11 μeV at the YSR energy minimum, which is needed to properly fit the experimental data to the theoretical model. The existence of a small gap has actually being predicted in the context of a mean-field theory where the local variation of the order parameter close to the impurity was determined self-consistently48. This was also reproduced in our self-consistent calculation in Supplementary Section F. References 45. Koller, R. et al. The structure of the oxygen induced (1 × 5) reconstruction of V(100). Surf. Sci. 480, 11–24 (2001). 46. Kralj, M. et al. HRAES, STM and ARUPS study of (5 × 1) reconstructed V(100). Surf. Sci. 526, 166–176 (2003). g p p The tip was sputtered in ultra-high vacuum and treated with field emission as well as subsequent indentation into the vanadium substrate until the expected gap of bulk vanadium appeared in the conductance spectrum. YSR tips were designed following the method of random dipping explained in ref. 47. Although it is intrinsically not possible to know the exact composition of the tip apex, a probable scenario is that the YSR impurity on the tip consists of oxygen or carbon picked up from the surface in a way that leaves a free spin to interact with the superconductor. We purposefully chose to use YSR tips for our experiment as it gave better stability of the junction at higher conductance. Moreover, it offered better flexibility in designing and defining the junction over magnetic surface defects, which were mostly found to have a spatial extent of around 1 nm. f 47. Huang, H. et al. Tunnelling dynamics between superconducting bound states at the atomic limit. Nat. Phys. 16, 1227–1231 (2020).f 48. Salkola, M. I., Balatsky, A. V. & Schrieffer, J. R. Spectral properties of quasiparticle excitations induced by magnetic moments in superconductors. Phys. Rev. B 55, 12648–12661 (1997).h 49. Cuevas, J. C. & Scheer, E. Molecular Electronics: an Introduction to Theory and Experiment 2nd edn (World Scientific, 2017). 49. Cuevas, J. C. & Scheer, E. Molecular Electronics: an Introduction to Theory and Experiment 2nd edn (World Scientific, 2017). i 50. Žitko, R. & Pruschke, T. Energy resolution and discretization artifacts in the numerical renormalization group. Phys. Rev. B 79, 085106 (2009). Data availability y Data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request. Source data are available for this paper. Tip and sample preparation. The experiments were performed on Josephson nanojunctions built in a low-temperature STM operated at 10 mK. Approaching a superconducting vanadium tip, tailored with a spin-1 2 impurity at its apex, to a crystalline V(100) substrate, we drove the impurity-induced YSR states across the QPT and detected a reversal of the supercurrent. Code availability The V(100) substrate was cleaned by repeated cycles of Ar ion sputtering, annealing to ~925 K and cooling to ambient temperature at a rate of 1–2 K s−1. Oxygen diffused from the bulk to the surface leads to typical surface reconstructions45,46; these did not influence the characteristics of the superconducting vanadium. Surface defects mostly involve missing oxygen within the reconstruction, which appeared bright in STM topographs47. Magnetic defects were found exhibiting YSR states at arbitrary energies within the gap, as reported in ref. 47. The code that supports the conclusions within this paper and other findings of this study is available from the corresponding author upon reasonable request. NATuRe PHYSicS NATuRe PHYSicS Author contributions S.K. and H.H. conducted the experiments with support from K.K. and C.R.A. C.P., G.M., J.C.C., A.L.Y., B.K., A.T., A.M.B.-S. and J.A. provided theory support. H.H., C.P. and C.R.A. modelled and analysed the data with support from all authors. All authors discussed the results. C.R.A. wrote the manuscript with input from all authors. Theory. We modelled the Josephson effect in the dynamical Coulomb blockade regime. The interference between transport channels was calculated by a combination of the respective energy–phase relations. We exploited the concept of transport channels, which arise from the matrix diagonalization of the orbital overlap in the tunnel junction49. This is a well-known procedure in mesoscopic physics and allows us to easily connect the theoretical models to experimentally accessible quantities without knowing all the microscopic details and, therefore, retaining a certain level of generality in the conclusions. Competing interests Competing interests The authors declare no competing interests Our calculations of the Josephson effect were supplemented by self-consistent calculations of the local order parameter in a YSR state to verify that the experimentally observed step in the switching current across the QPT cannot be accounted for by a change in magnitude of the local order parameter, but must instead be attributed to the π shift of the phase in the YSR channel. p g The authors declare no competing interests Acknowledgements As we find this small gap, we extract Δsample during the conversion of the YSR energies to the corresponding impurity–superconductor coupling, which we need for the modelling. The gap can be determined by the requirement that the impurity–superconductor coupling changes smoothly as function of conductance. NATuRe PHYSicS 40. Averin, D., Nazarov, Y. & Odintsov, A. Incoherent tunneling of the cooper pairs and magnetic flux quanta in ultrasmall Josephson junctions. Phys. B Condens. Matter 165–166, 945–946 (1990). 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, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022, corrected publication 2022 41. Jäck, B. et al. Critical Josephson current in the dynamical Coulomb blockade regime. Phys. Rev. B 93, 020504 (2016). g y 42. Randeria, M. T., Feldman, B. E., Drozdov, I. K. & Yazdani, A. Scanning Josephson spectroscopy on the atomic scale. Phys. Rev. B 93, 161115 (2016).f 43. Ambegaokar, V. & Baratoff, A. Tunneling between superconductors. Phys. Rev. Lett. 10, 486–489 (1963).lf 44. Spivak, B. I. & Kivelson, S. A. Negative local superfluid densities: the difference between dirty superconductors and dirty Bose liquids. Phys. Rev. B 43, 3740–3743 (1991). Nature Physics | VOL 18 | August 2022 | 893–898 | www.nature.com/naturephysics 898 Articles Nature Physics | www.nature.com/naturephysics Funding Open access funding provided by Max Planck Society. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41567-022-01644-6. Furthermore, to extract the Kondo temperature from the experimental Kondo spectra at a magnetic field of B = 1.5 T, we used NRG theory to fit the experimental data50. Peer review information Nature Physics thanks Andreas Heinrich and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Details for all the theoretical models are provided in the Supplementary Information. Reprints and permissions information is available at www.nature.com/reprints. Nature Physics | www.nature.com/naturephysics
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A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico
Administração Pública e Gestão Social
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PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto Resumo Objetivo da pesquisa: o objetivo do ensaio é desenvolver proposições teóricas sobre a implementação de políticas públicas. Enquadramento teórico: o estudo fundamenta-se na literatura sobre os fatores influenciadores da ação dos burocratas de nível de rua na implementação de políticas públicas. Resultados: a primeira proposição defende que a discricionariedade, mais que um fator influenciador, é um pressuposto da implementação. Outra proposição deriva da percepção de que a ação da implementação das políticas públicas influencia de formas distintas os princípios que a regem — núcleo estável — e seus fatores intercontextuais — núcleo dinâmico. A última proposição sustenta que deve existir um esforço de parte dos policymakers em reconhecer a influência e conhecer os fatores que influenciam a forma como a burocracia de nível de rua implementa determinada política pública. Como resultado, o framework normativo original da política pública possuiria maior aderência à realidade observada nos contextos em que a política pública é implementada, além de trazer para o bojo da política pública a consideração da multidimensionalidade como característica basilar da burocracia de nível de rua. Adicionalmente, apresenta-se um modelo integrativo de dimensões e categorias sobre o tema. Originalidade: apresenta a discricionariedade como um pressuposto ao se estudar os fatores influenciadores da ação dos burocratas de nível de rua, diferenciando-a da ação discricionária. Além disso, o modelo proposto busca avançar no debate sobre a dinâmica existente entre a formulação e implementação de políticas públicas. Contribuições teóricas e práticas: do ponto de vista teórico, o conteúdo do ensaio suscita argumentos que contribuem qualitativamente na área de pesquisa. De uma perspectiva prática, fornece um modelo que pode servir de parâmetro para novos estudos e apresenta proposições que podem ser testadas em pesquisas empíricas futuras. Palavras-chave: Burocracia de Nível de Rua, Fatores influenciadores da ação, Framework normativo, Dimensões e categorias. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico Laerti Boldrin, Duljon da Rocha Soares Ferreira, Vicente Laerti Boldrin, Duljon Laerti Boldrin, Duljon da Rocha Soares Ferreira, Vicente , A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensa Teórico A Discricionariedade como um Pressuposto da Implement Teórico Administração Pública e Gestão Social, vol. 16, núm. 2, 2024 Universidade Federal de Viçosa bl ç Disponible en: https://www.redalyc.org/articulo.oa?id=351577342009 Esta obra está bajo una Licencia Creative Commons Atribución-NoComercial-SinDerivar 4.0 Internacional. Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico Discretion as an Assumption for the Implementation of Public Policies: a Theoretical Essay La discrecionalidad como una premisa para la implementación de políticas públicas: un ensayo teórico Duljon Laerti Boldrin Universidade Federal de Goiás, Brasil duljon@gmail.com Vicente da Rocha Soares Ferreira Universidade de Brasília, Brasil vicenterocha@ufg.br Recepción: 28 Diciembre 2022 Aprobación: 07 Noviembre 2023 Publicación: 25 Abril 2024 Acceso abierto diamante Acceso abierto diamante Abstract Originality: presents discretion as an assumption when studying the factors influencing the action of street-level bureaucrats, differentiating it from discretionary action. In addition, the proposed model seeks to advance on the debate about existing dynamics between the formulation and implementation of public policies. Theoretical and practical contributions: from a theoretical point of view, the content of the essay raises arguments that ontrib te q alitati el to the resear h area From a pra ti al perspe ti e it pro ides a model that an ser e as a parameter for y g g j p Originality: presents discretion as an assumption when studying the factors influencing the action of street-level bureaucrats, differentiating it from discretionary action. In addition, the proposed model seeks to advance on the debate about existing dynamics between the formulation and implementation of public policies. Th i l d i l ib i f h i l i f i h f h i h Originality: presents discretion as an assumption when studying the factors influencing the action of street-level bureaucrats, differentiating it from discretionary action. In addition, the proposed model seeks to advance on the debate about existing dynamics between the formulation and implementation of public policies. Theoretical and practical contributions: from a theoretical point of view, the content of the essay raises arguments that contribute qualitatively to the research area. From a practical perspective, it provides a model that can serve as a parameter for new studies and presents propositions that can be tested in future empirical research. Keywords: Street-level bureaucracy, Factors influencing the action, Normative framework, Dimensions and categories. PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto Abstract Objective: the objective of this essay is to develop theoretical propositions about public policies implementation. h l k h d b d h l b h f h fl h f h Objective: the objective of this essay is to develop theoretical propositions about public policies implementation. Theoretical Framework: the study is based on the literature about the factors that influence the action of the Objective: the objective of this essay is to develop theoretical propositions about public policies implementation. Theoretical Framework: the study is based on the literature about the factors that influence the action of the street-level bureaucrats in the implementation of public policies. j j y p p p p p p Theoretical Framework: the study is based on the literature about the factors that influence the action of the street-level bureaucrats in the implementation of public policies. Results: The first proposition argues that discretion, more than an influencing factor, is an assumption of implementation. Another proposition derives from the perception that the action of implementing public policies influences in different ways the principles that govern it — stable core — and its intercontextual factors — dynamic core. The last proposition argues that there must be an effort of policymakers to recognize the influence and to know the factors that influence the way the street-level bureaucracy implements a given public policy. As a result, the original normative framework of public policy would have greater adherence to the reality observed in the contexts in which public policy is implemented, in addition to bringing the 1 PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 1 Administração Pública e Gestão Social, 2024, vol. 16, núm. 2, Abril-Junio, ISSN: 2175-5787 consideration of multidimensionality to the bases of public policy as a basic characteristic of street-level bureaucracy. Additionally, an integrative model of dimensions and categories about the subject is presented. consideration of multidimensionality to the bases of public policy as a basic characteristic of street-level bureaucracy. Additionally, an integrative model of dimensions and categories about the subject is presented. Additionally, an integrative model of dimensions and categories about the subject is presented. Resumen etivo de este ensayo es desarrollar proposiciones teóricas sobre la implementación de políticas públicas. Objectivo: el objetivo de este ensayo es desarrollar proposiciones teóricas sobre la implementación de políticas públicas. Marco Teórico: el estudio es basado en la literatura sobre los factores que influyen en la acción de los burócratas de nivel de calle en la implementación de políticas públicas. Marco Teórico: el estudio es basado en la literatura sobre los factores que influyen en la acción de los burócratas de nivel de calle en la implementación de políticas públicas. Resultados: la primera proposición defiende que la discrecionalidad, más que un factor influyente, es una premisa de la implementación. Otra proposición se deriva de la percepción de que la acción de implementar políticas públicas influye de diferentes maneras en los principios que la rigen — núcleo estable — y sus factores intercontextuales —núcleo dinámico. La última proposición argumenta que debe haber un esfuerzo por parte de los formuladores de políticas para reconocer la influencia y conocer los factores que influyen en la forma en que la burocracia de nivel calle implementa una determinada política pública. Como consecuencia, el marco normativo original de política pública tendría mayor apego a la realidad observada en los contextos en los que se implementa la política pública, además de llevar la consideración de la multidimensionalidad al núcleo de la política pública como característica básica de la burocracia de nivel de calle. Adicionalmente, se presenta un modelo integrador de dimensiones y categorías sobre el tema. y g Originalidad: presenta la discrecionalidad como un supuesto al estudiar los factores que influyen en la acción de los burócratas de nivel de calle, diferenciándola de la acción discrecional. Además, el modelo propuesto busca avanzar en el debate sobre las dinámicas existentes entre la formulación y la implementación de políticas públicas. y p p p Contribuciones teóricas y prácticas: desde el punto de vista teórico, el contenido del ensayo plantea argumentos que contribuyen cualitativamente em la area de investigación. Desde una perspectiva práctica, proporciona un modelo que puede servir como parámetro para nuevos estudios y presenta proposiciones que pueden probarse en futuras investigaciones empíricas. Palabras clave: Burocracia de nivel de calle Factores que influyen en la acción Marco normativo Dimensiones y categorias servir como parámetro para nuevos estudios y presenta proposiciones que pueden probarse en futuras investigaciones empíricas. PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de a 1 Introdução O campo da implementação investiga a razão do sucesso ou insucesso das políticas públicas (Barrett, 2004; Ferreira et al., 2020; Hjern & Porter, 1981). Constata-se empiricamente que nem todas elas alcançam os objetivos inicialmente formulados e, de um ponto de vista teórico, as razões para que isso aconteça. Em vista disso, a identificação dos fatores que influenciam esse resultado é central nas análises de implementação (P. Sabatier & Mazmanian, 1980; Scharpf, 1977) A implementação é a ação que materializa o que foi planejado na esfera política como alternativa para resolução de problemas públicos independentemente da origem dessas ações serem públicas ou privadas (Hill & Hupe, 2002; P. Sabatier & Mazmanian, 1980; Van Meter & Van Horn, 1975). Outrossim, é o produto da interação de diversos atores, não necessariamente alinhados em termos de interesses ou equiparados em termos de influência ou recursos (Lipsky, 2010; Scharpf, 1977). Além das ações, o entendimento da implementação passa pelo comportamento dos atores envolvidos e pela inação — quando intencional —, não sendo possível prever seus resultados antes de sua implementação, indissociável do problema e do contexto específico que a política pública busca produzir resultados. Nesse sentido, não existem garantia de que o propósito das políticas públicas se mantenham conforme as intenções dos policymakers—aqueles burocratas costumeiramente localizados no alto e médio escalação da burocracia pública — antes da ação ser iniciada, admitindo que sejam definidos ou influenciados no sentido inverso, a partir da implementação e das idiossincrasias encontradas durante esse processo (Hill & Hupe, 2002). Esse olhar a partir da implementação rompe com a ideia clássica de que políticas públicas são ações desenvolvidas de maneira sequencial, lógica e objetiva (Hill & Hupe, 2002). A teoria da burocracia de nível de rua analisa a implementação de políticas públicas a partir dos implementadores, os chamados burocratas de nível de rua — professores, médicos, policiais, entre outros. São esses servidores que encontram os cidadãos e representam o poder público. Sendo assim, estes agentes são os responsáveis pela implementação das políticas públicas em seu nível operacional. Eles, diante de imprecisões, ambiguidades — ou subjetividades — dos critérios estabelecidos no planejamento e valendo-se da discricionariedade que sua atuação requer, vão além da implementação, tornando-se também formuladores (Arretche, 2001; Bronzo et al., 2022; Ferreira & Medeiros, 2016; Lipsky, 2010; G. S. Lotta et al., 2018; Nouman & Cohen, 2023). Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico 1 Introdução Apesar da existência de muitos estudos empíricos, ainda existe a necessidade do desenvolvimento de modelos integradores que permitam avançar na evolução e consistência teórica dos estudos sobre as ações dos burocratas de nível de rua na implementação de políticas públicas (Ferreira & Medeiros, 2016). O objetivo deste ensaio teórico é desenvolver proposições teóricas sobre a implementação de políticas públicas no sentido bottom-up a partir da teoria da burocracia de nível de rua. Para isso, a próxima seção contextualiza a implementação sob a ótica da burocracia de nível de rua, apresentando os principais aspectos teóricos e epistemológicos envolvidos. Em seguida, discute-se os fatores influenciadores da implementação de políticas públicas na perspectiva dos burocratas de nível de rua. Finalmente, nas considerações finais reafirmam-se as principais proposições desenvolvidas durante o ensaio, bem como encaminham-se sugestões para compor a agenda de pesquisas sobre o tema. PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto Resumen Palabras clave: Burocracia de nivel de calle, Factores que influyen en la acción, Marco normativo, Dimensiones y categorias. Palabras clave: Burocracia de nivel de calle, Factores que influyen en la acción, Marco normativo, Dimensiones y categorias. 2 Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de 2 Implementação de Políticas Públicas na Perspectiva dos Burocratas de Nível de R Até o final da década de 1960, a pressuposição dos formuladores das políticas públicas, comumente localizados nas esferas mais estratégicas e hierarquicamente superiores da administração pública, era de que elas eram claras, bem definidas e que a alocação de recursos — considerada suficiente para os fins aos quais se propunham — levaria os administradores e seus subordinados a executar o que lhes fora ordenado sem intercorrências, atendendo as expectativas e aos ditames da lei. O arcabouço legal não mencionava a figura dos implementadores (Hill & Hupe, 2002). A literatura da época acompanhava essa tendência. Apenas no 3 3 PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 3 Administração Pública e Gestão Social, 2024, vol. 16, núm. 2, Abril-Junio, ISSN: 2175-5787 final da década de 1960 e início da década de 1970, diante da constatação de que a legislação não produzia os efeitos esperados (Pressman & Wildavsky, 1973) e do desenvolvimento de estudos que analisavam programas públicos específicos nas mais diversas áreas da esfera pública, é que surge a preocupação com a implementação (P. Sabatier & Mazmanian, 1980). p A partir de então, pesquisas do campo da implementação começaram a ser desenvolvidas, primeiramente, preocupando-se com as diferenças encontradas entre os objetivos almejados e os resultados obtidos — a primeira geração. Posteriormente, surge o interesse sobre como os atores — individuais e coletivos — que fazem parte da interface de determinada política pública atuam na busca desses resultados — a segunda geração. Com o avanço das pesquisas, surgiu uma terceira geração de estudos, que buscava a convergência dessas perspectivas no sentido de reconhecer que ambas oferecem subsídios para a compreensão da implementação (Matland, 1995; McDonnell & Elmore, 1987). Apesar dos inegáveis avanços, a terceira geração de estudos sobre implementação não conseguiu resolver as divergências debatidas pelas duas primeiras, adicionando vieses de análise importantes sem, contudo, interromper o desenvolvimento teórico e a utilização empírica das outras abordagens (Hill & Hupe, 2002). Tratou-se, portanto, de um novo paradigma nas pesquisas de implementação, não uma quebra. Nesse ponto cabe a menção que autores como Howlett (2019) e G. Lotta (2019) defendem a existência de uma quarta geração — ou onda — de pesquisas sobre implementação, com uma maior influência de outros campos da ciência e utilização de múltiplos modelos de análise. 2 Implementação de Políticas Públicas na Perspectiva dos Burocratas de Nível de R De qualquer maneira, existem vários caminhos para se estudar a implementação de políticas públicas (Hill & Hupe, 2002). A adoção de uma dessas perspectivas teóricas é mais uma escolha do pesquisador na análise de um caso concreto e do problema que está sendo investigado do que a convicção que uma seja superior às demais. Nesse ponto, parte-se para uma análise mais aprofundada da segunda geração. Por analisar a implementação de baixo para cima — no sentido bottom-up —, ela considera que exista uma integração entre formulação e planejamento que leva a um fluxo contínuo de adaptações e revisões dos objetivos originalmente definidos na formulação da política quando efetivadas as ações necessárias para aplicá-las aos casos concretos (Barrett, 2004). Essa efetivação é uma condição sine qua nonnesse fluxo de retroalimentações entre o que se verificou na prática e o que foi formulado, adaptando este à realidade da implementação. Por essa razão, esses atores que implementam as políticas públicas são centrais nas análises de segunda geração, sendo chamados de burocratas de nível de rua. Três são as condições que afetam sobremaneira a atuação desses agentes: i) recursos inadequados; ii) ameaça psicológica e, em determinados casos, física; e iii) contradições nas metas de desempenho deles esperadas. Outrossim, denota-se sua importância política e social. Politicamente, compreender o foco do serviço governamental passa pelo conhecimento das funções e características dos servidores públicos. Do ponto de vista social, a atuação desses agentes impacta sobremaneira a vida dos cidadãos, sendo atores centrais inclusive no exercício da cidadania (Lipsky, 2010). A compreensão dos desafios advindos da atuação desses servidores que atuam na linha de frente passa por duas ideias ou conceitos centrais: i) a discricionariedade como elemento central da implementação; e ii) problemas públicos devem ser abordados no sentido bottom-up. PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 2.1 A discricionariedade como elemento central da implementação Outras pesquisas tiveram importantes contribuições no modo de conceber as políticas públicas a partir do deslocamento do foco de análise nas áreas estratégicas para as mais próximas do cidadão. Destaca-se as contribuições a seguir. A abordagem backward mapping é uma alternativa — e complementação — à visão top-down de controle e influência dos policymakers sobre o processo de implementação — forward mapping. Ela questiona a capacidade dos formuladores em influenciar a implementação e a ideia de que o sucesso da política pública passa exclusivamente por definições precisas de objetivos e responsabilidades a partir do centro. O problema a ser corrigido ou atitude a ser influenciada pela política pública está no nível mais operacional da implementação. Portanto, a formulação deve partir dele e subir até aos níveis estratégicos (Elmore, 1979). Além de analisar a política no sentido tradicional, é necessário fazer a análise no sentido contrário. Deve existir uma relação lógica entre o problema que se pretende enfrentar e a estratégia escolhida para fazê-lo. Sendo assim, cada viés de análise contempla apenas parte da análise ideal, que compreende ambas as dimensões. A abordagem top-down busca controle. A visão bottom-up entende que esse controle, se existir, é limitado a um conjunto de recursos que cada ator da implementação é capaz de influenciar (Elmore, 1985). Em razão de as políticas públicas serem, usualmente, vagas e ambíguas — e até inexistentes — ao nível dos objetivos que se pretendem alcançar, e reconhecendo que elas podem ser alteradas durante a implementação, a pesquisa em implementação deve se concentrar nos problemas estabelecidos por todos os atores relevantes no processo, não somente naqueles formalmente instituídos como formuladores (Hjern & Hull, 1982). Esse reconhecimento da pluralidade de atores, e sua capacidade de encaminharem alterações no desenho da política, introduz as discussões a respeito de como acontece o relacionamento entre eles. Além disso, se os objetivos das políticas estão em função dessa rede e são estabelecidos de acordo com as interações existentes pari passu à própria implementação, a noção de êxito ou fracasso deixa de ser puramente objetiva e estática, passando a ser subjetiva — por depender da relação de poderes entre os atores e suas percepções quanto aos resultados esperados — e dinâmica. As pesquisas do campo da implementação tendem a se centrar em aspectos do respeito às regras — law abidance. 2.1 A discricionariedade como elemento central da implementação A discricionariedade é compreendida como o exercício do julgamento que os implementadores possuem no desempenho de suas profissões (Lipsky, 2010). Ela é a margem de atuação dos servidores na aplicação das leis dentro dos limites por elas estabelecidos, sendo necessária ao se vislumbrar que a legislação costumeiramente trata de regras gerais e não detalha minuciosamente a multiplicidade de situações encontradas diuturnamente na aplicação da lei aos casos concretos (Ferreira et al., 2020; Oliveira, 2012). Dessa maneira, mecanismos que buscam controlar e guiar seu comportamento tendem a não funcionar na análise de casos concretos. Os burocratas de nível de rua, em razão da discricionariedade e dos 4 Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico rti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas m Ensaio Teórico mecanismos de resistência aos ditames advindos dos órgãos centrais, são formuladores quando se considera sua área específica de atuação (Hupe & Hill, 2007; Lipsky, 2010; Weatherley & Lipsky, 1977). Em função disso, as políticas públicas não podem ser completamente entendidas sem considerar que a implementação acontece a partir da relação entre a burocracia de nível de rua e os cidadãos. Por não ser possível que contemple todas as especificidades que são encontradas durante a implementação, a política pública carece de interpretação ao ser aplicada aos casos concretos. Nesse processo, conflitos não se dão somente a nível de atores interessados no processo de formulação da política, mas também, e sobretudo, na relação entre os implementadores e os cidadãos. Assim, a discricionariedade é inerente à função e fonte de autoridade para esses servidores (Lipsky, 2010) e, conjuntamente às suas habilidades individuais, é necessária para a solução dos problemas que as políticas públicas tentam resolver (Elmore, 1979). Então, mais que um elemento passível — ou desejável — de controle, a discricionariedade é instrumento importante na implementação das políticas públicas. É uma consequência natural à racionalidade limitada dos atores envolvidos com a política pública e a assimetria de informações existentes entre quem está próximo ao objeto e quem o conhece apenas de maneira teórica ou superficial. É elemento inerente às políticas públicas, da natureza do processo de implementação. PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 2.2 A burocracia de nível de rua e os fatores influenciadores de sua ação na implementação políticas públicas Uma das dificuldades em se estudar a implementação é que as ações dos burocratas de nível de rua nem sempre são documentadas. A natureza da relação entre o burocrata e o cidadão nem sempre se dá de maneira formal. É, por exemplo, o caso dos professores em sala de aula. Além do mais, diante de situações em que as normas não são claras ou nem mesmo as vislumbraram, ou ainda diante das próprias especificidades da função desempenhada, os burocratas de nível de rua precisam tomar decisões baseadas em sua interpretação das regras (Hill & Hupe, 2002; Lipsky, 2010), atuando nas dimensões estrutural, relacional e da ação individual (Bonelli et al., 2019). Portanto, percebendo que a implementação não é um processo linear e estático, com variáveis totalmente conhecidas e de comportamento presumível, a burocracia de nível de rua estabelece critérios de análise que permitem uma maior compreensão dos aspectos específicos que concernem à implementação. Uma de suas características é a existência da ação discricionária, em maior ou menor nível, mas sempre presente, na aplicação das regras por parte dos burocratas de nível de rua. Nessa perspectiva, é comum que os trabalhos da área estudem o comportamento desses agentes públicos (Hill & Hupe, 2002; Lipsky, 2010) pois o exercício do julgamento, necessário para sua atuação, afeta o grau de sucesso da política (Najberg & Barbosa, 2006). Então, a discricionariedade é um pressuposto da implementação. O que se trata de um produto entre as relações de interdependência dos fatores que influenciam a implementação é o julgamento do burocrata de nível de rua — que resulta na ação discricionária através do julgamento e da consideração dos diferentes contextos que afetam a implementação das políticas públicas, conforme ponderações de Arretche (2001), Saetren (2005) e G. S. Lotta et al. (2018). Sobre isso, importa caracterizar a discricionariedade na implementação de políticas públicas como pressuposto — abordado, por exemplo, como aspecto inevitável (Maynard-Moody & Musheno, 2003) ou incontornável (P. A. Sabatier, 1986) —, diferenciando-a da ação discricionária, aquela que decorre de sua existência e já abordada por autores consagrados da área (Lipsky, 2010; G. S. Lotta & Pires, 2020; Maynard-Moody et al., 1990). 2.1 A discricionariedade como elemento central da implementação Contudo, análises a respeito do julgamento realizado pelos burocratas de nível de rua na implementação de políticas públicas passa também por razões culturais — cultural abidance. A análise em relação às normas é considerada a narrativa predominante no campo — state-agent narrative —, sendo sua alternativa — citizen-agent narrative — frequentemente negligenciada. Essa trabalha em cooperação ou tensão com aquela, mas sempre próximas. A narrativa cidadão-agente se preocupa com julgamentos de ordem moral realizados pelos burocratas de nível de rua na interação dinâmica que se desenvolve em razão do atendimento dispensando aos cidadãos (Maynard-Moody & Musheno, 2003). 5 5 5 Administração Pública e Gestão Social, 2024, vol. 16, núm. 2, Abril-Junio, ISSN: 2175-5787 Nesse aspecto, discute-se até mesmo a discricionariedade e seu status incontornável atribuído pela narrativa estado-agente, afirmando que ela jamais pode ser eliminada. Mesmo diante da existência dos controles democráticos dos esforços públicos em se alterar uma realidade através da implementação de uma política pública, a discricionariedade sempre aparece em algum ponto. A narrativa state-agent prevalece em contextos em que, do ponto de vista dos burocratas de nível de rua, lei e cultura estão alinhadas. Por outro lado, quando a percepção desses servidores é de que o que deveria ser feito não é exatamente aquilo que a lei, normas e regras preveem, a narrativa citizen-agent prevalece (Maynard-Moody & Musheno, 2003). Isso posto, uma característica comum de diversas abordagens, narrativas e teorias que estudam a implementação no sentido bottom-up é o status central atribuído à discricionariedade. Um dos desdobramentos desse atributo é o interesse de compreender os fatores que influenciam a burocracia de nível de rua a utilizar a discricionariedade ao implementar as políticas públicas, que será tratado na seção seguinte. PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 2.2.1 Accountability A accountability está relacionada aos sistemas de gestão da política, com as interações entre os burocratas de nível de rua e os usuários, bem como com o exercício da discricionariedade (Ferreira & Medeiros, 2016). Importa destacar que uma maior autonomia se relaciona a um melhor desempenho mesmo em contextos de menor disponibilidade de recursos. Contudo, sem a existência de mecanismos de responsabilização, os implementadores tendem a não seguir os objetivos organizacionais, priorizando seus próprios interesses (Buta et al., 2022). Portanto, trata-se de um fator influenciador da ação diretamente ligado ao desempenho da política pública. O tempo é um fator influenciador do burocrata de nível de rua quanto a esse dever de prestar contas, afetando seu exercício, seus diversos relacionamentos, níveis de confiança e no foco em cumprir os requisitos estabelecidos, não necessariamente na prestação de um bom serviço (Hupe & Hill, 2007; Murphy & Skillen, 2015). Considerando que a accountability faz parte do planejamento em razão de sua forma, geralmente, ser prevista no policy framework, ao pautarem sua atuação eminentemente sob a pressão de um sistema de prestação de contas — por vezes exógeno à organização onde atuam —, os implementadores de políticas públicas podem deixar de lado a própria razão delas existirem: a solução de um problema de ordem pública. Fatores exógenos à implementação, como o fato de os burocratas de nível de rua serem sindicalizados, também são objetos de estudo. A sindicalização não afeta significativamente a experiência no trabalho dos burocratas de nível de rua. Existe, todavia, uma correlação fraca entre o aumento da sindicalização e diminuição da percepção do dever de prestar contas, mas sem repercussões no desempenho dos profissionais estudados (Z. Oberfield, 2021). A accountability pode se manifestar ainda de maneira informal. Ela relaciona-se a aspectos da personalidade dos burocratas de nível de rua — definidas em tipos de identidade — e da utilização da ação discricionária para, em determinadas situações, aplicar as regras de forma a satisfazer anseios igualitários. Como consequência, questiona-se a manifestação do princípio da impessoalidade diante da sugestão que a burocracia de nível de rua é influenciada por questões sociais e políticas (Pivoras & Kaselis, 2019). Essa tendência de pender as regras através de decisões individuais é um fator particularmente importante quando os implementadores possuem em suas funções precípuas o atendimento de necessidades pró- sociais. Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico rti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas m Ensaio Teórico Isso posto, considera-se a discussão sobre esse posicionamento epistemológico da discricionariedade na burocracia de nível de rua importante para o avanço da teoria, que é robusta e madura, na perspectiva aqui defendida, em relação à ação discricionária, não à discricionariedade como pré-requisito na implementação de políticas públicas. Ao considerar esses diversos contextos e suas interfaces — inter-contextual factors —conjuntamente aos princípios que regem uma política específica, — policy framework —, aqui entendidos como as normas legais, infralegais, guias e congêneres relacionados, forma-se o framework normativo da implementação dessa política pública (Ferreira et al., 2020). Somando-se isso à pluralidade teórica desenvolvida a respeito de implementação de políticas públicas, resulta-se em um campo de estudo complexo e fragmentado. Isso dificulta a compreensão satisfatória da implementação a partir da visão dos burocratas de nível de rua, sobretudo quando apenas um determinado conjunto de fatores está sob análise, outra tendência dos estudos da área (Ferreira & Medeiros, 2016). Feitas essas considerações e partindo do pressuposto que accountability, normas, percepção, valores e características individuais, relacionamento entre os implementadores e o cidadão usuário da política pública e o sistema de gestão são fatores influenciadores do comportamento do servidor de linha de frente (Ferreira & Medeiros, 2016), adentra-se, sem pretensões taxativas, na literatura sobre esses temas. 2.2 A burocracia de nível de rua e os fatores influenciadores de sua ação na implementação políticas públicas Essa questão se justifica em razão de, na literatura da burocracia de nível de rua, a discricionariedade vir sendo abordada como um fator influenciador da ação (Ferreira et al., 2020; Ferreira & Medeiros, 2016; Henderson et al., 2018; Tummers & Bekkers, 2014), uma fonte de poder decisório que influencia a vida do cidadão (Bovens & Zouridis, 2002; Carroll et al., 2019; Lipsky, 2010; G. S. Lotta & Pires, 2020; Moyson et al., 2018; Nunes & Lotta, 2019), uma ferramenta de enfrentamento às regras, contextos e disponibilidade de recursos disponíveis na implementação (Balica et al., 2018; Brodkin, 2011; Lipsky, 2010; G. S. Lotta & Pires, 2020) ou uma característica do processo de implementação — se possui altos ou baixos níveis de possibilidade de ação discricionária — (Collins & Augsberger, 2021; Raaphorst & Loyens, 2020; Thunman et al., 2020), para citar algumas pesquisas e tratamentos a ela dispensada, não como um elemento sem o qual a própria discussão sobre implementação seria estéril. 6 6 PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 7 2.2.2 Normas e regulação O conjunto de normas e suas instituições reguladoras fazem parte do policy framework. Portanto, sua influência na implementação é evidente. Contudo, como discutido, existem limitações práticas na abrangência desse poder e a resultante necessidade do exercício da discricionariedade para se implementar políticas públicas. Prestadores de serviços públicos, inclusive quando esses serviços são prestados pela iniciativa privada, priorizam de maneira sistemática a resolução de problemas no fornecimento dos serviços, usualmente sem controle dos órgãos de supervisão. Isso afeta a regulação do setor. A atuação dos órgãos de controle é limitada pela falta ou assimetria de informação e em razão deles próprios não as buscarem com frequência (Steenhuisen & van Eeten, 2013). O cumprimento das regras e conduta ética dos servidores é ainda dependente da cultura, seja ela organizacional ou social. Decisões individuais que buscam resolver situações fora dos padrões estabelecidos, mesmo diante de justificativas de cunho cultural, são destrutivas para o alcance de objetivos de longo prazo. Por outro lado, regramentos que não consideram a cultura da implementação também tendem a falhar (Cohen, 2018). Existem ainda repercussões das decisões judiciais — derivações da existência de normas — na atuação dos burocratas de nível de rua. Em situações em que esses servidores não possuem conhecimento jurídico especializado, o mecanismo desenvolvido pelos implementadores é o da construção de uma interpretação conjunta da lei em cada unidade organizacional — que não necessariamente está de acordo com a lei e com as prescrições dos tribunais. Essa instância local de interpretação legislativa resulta no aumento das incertezas dos cidadãos que buscam aquele serviço, pois recusas de solicitações são realizadas com base nessas interpretações. Dessa forma, as decisões judiciais são capazes de influenciar as rotinas administrativas dos implementadores (Mascia, 2020). 2.2.1 Accountability Nesses casos, as regras estabelecidas parecem ser ainda mais incapazes de prever seu comportamento, sendo o clima organizacional, personalidade e experiência do servidor mais capazes de fazê-lo (Borry & Henderson, 2020). 7 7 Administração Pública e Gestão Social, 2024, vol. 16, núm. 2, Abril-Junio, ISSN: 2175-5787 A influência da solidariedade — ou falta dela — na tomada de ação discricionária e a estrutura social do locusde implementação — o tipo de welfare state que está presente no contexto — também se relacionam com o dever de prestar contas, sobretudo o informal. Desses fatores, surgem duas tipologias de burocratas de nível de rua: a pessoa de estado — statesperson — e o profissional. O servidor considerado pessoa de estado possui características pró-cidadão, ao passo que o profissional segue seus valores particulares sem ultrapassar os limites impostos pela legislação e regras. Essas classificações emergem como uma forma de responder à pressão do ambiente onde estão inseridos (Møller & Stensöta, 2019). PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto PDF generado a partir de XML JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto PDF generado a partir de XML-JATS4R por Redalyc é f Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico rti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas m Ensaio Teórico — direto ou indireto — com o cidadão (Jewell & Glaser, 2006; Keiser, 2010) e o julgamento dos pares (Raaphorst & Loyens, 2020) são capazes de influenciar a decisão individual. — direto ou indireto — com o cidadão (Jewell & Glaser, 2006; Keiser, 2010) e o julgamento dos pares (Raaphorst & Loyens, 2020) são capazes de influenciar a decisão individual. De maneira complementar, variáveis como trajetória profissional e perfil relacional — inclusive em relação à utilização de redes sociais — também possuem a capacidade de influenciar a ação discricionária dos implementadores. A maneira como esses agentes se aproximam dos cidadãos por meio da utilização de sua linguagem, terminologias, atitudes e especificidades, revela as dificuldades enfrentadas na implementação e, ainda, as características possuídas por aqueles que conseguem estabelecer um relacionamento prático com os diversos atores que compõem o policy process (G. Lotta, 2018). Indo além, ao se abordar temas como preconceito, questões de gênero e minorias, denota-se que esses fatores também influenciam os servidores de linha de frente na implementação de políticas públicas. Aparência, comportamento, moradia e grupo social são variáveis consideradas pelos burocratas de nível de rua para medir a confiabilidade. Cidadãos com menores níveis de educação e condições econômicas, ao prestarem informações de baixa qualidade, são considerados menos confiáveis do que quando cidadãos com mais alto status social apresentam o mesmo tipo de informação. Portanto, a postura dos profissionais varia conforme a classe social do cidadão atendido (Raaphorst & Groeneveld, 2018). Outro caso estudado demonstra que a forma como os latinos norte-americanos recebem os serviços educacionais não é influenciada apenas pela organização, mas também pelo contexto político e seu poder de moldar a representatividade dos professores. Mesmo em ambientes onde os latinos possuem maior acesso a posições dentro da burocracia escolar, aspectos políticos e partidários atuam no sentido de limitar seu avanço acadêmico. Essa limitação se dá pelos membros de conselhos, atores que são capazes de guiar as políticas educacionais locais (Molina, 2020). No Paquistão, as Khawaja Sira, um gênero marginalizado pela sociedade, não conseguem usufruir, de maneira plena, de serviços públicos em razão ao tipo de tratamento a elas dispensado. Marcas em seus corpos e zombarias fazem parte de seu cotidiano ao se relacionarem com os servidores de linha de frente. 2.2.4 Interações implementador-usuário Importa destacar que as interações entre a burocracia de nível de rua e os usuários do serviço público — os cidadãos — acontecem dentro de um contexto. Essa indissociabilidade entre implementação e contexto já foi abordada anteriormente. Em países como o Brasil, vastos em termos geográficos e culturais, a implementação de políticas públicas é particularmente complexa ao se verificar a diversidade de necessidades e anseios dos cidadãos. Isso acarreta diferentes formas de se implementar uma mesma política pública quando considerados os seus diversos contextos de aplicação (Ferreira et al., 2020). Abordando a relação implementador-usuário de forma direta, verifica-se que o tratamento dispensado pelo burocrata de nível de rua tem menor influência no quesito obediência do que a intenção do próprio cidadão de estar em conformidade. Ao se analisar os feedbacks e comentários feitos online por usuários atendidos presencialmente por servidores de linha de frente, observa-se que o estilo de enforcement — a atitude dispensada ao cidadão no momento do atendimento ao se fazer cumprir uma norma — possui a capacidade de gerar mais comentários positivos, quando o cidadão se sente bem atendido, do que negativos, quando o cidadão se sente desrespeitado (de Boer, 2020). Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico A atitude da burocracia tende a utilizar a coerção para marginalizar as minorias. Ressalta-se que a lei não prescreve o tratamento dado às Khawaja Sira. Sendo assim, a discricionariedade é exercida para oprimir esse grupo em atenção aos anseios das classes dominantes (Nisar, 2020). Esses achados alinham-se com a discussão sobre a influência recíproca entre a discricionariedade, e suas bases constitutivas, e questões de gênero (Durose & Lowndes, 2023). 2.2.3 Percepção, valores e características individuais Como a discricionariedade pressupõe o exercício da interpretação de normas dentro de um contexto, aspectos psicológicos e características individuais da burocracia de nível de rua influenciam sua tomada de decisão. Em aplicação empírica, verificou-se que os aspectos de maior relevância para se compreender as atitudes dos servidores de linha de frente são os cognitivos, afetivos — positivos e negativos — e comportamentais (Keulemans & Van de Walle, 2018). Esses resultados vão ao encontro, inclusive, de observações de como traços da personalidade (Pivoras & Kaselis, 2019), características individuais e culturais (Nouman & Cohen, 2023) influenciam o burocrata na interpretação das regras. Outro fator que não pode ser descartado é o autointeresse. Porém, ao se condicionar a ação da burocracia de nível de rua inevitavelmente aos preceitos clássicos do modelo principal-agente, perde-se aderência empírica com implementações realizadas sob condições altruístas por agentes públicos vocacionados e comprometidos com determinados problemas, especialmente sociais (Ferreira & Medeiros, 2016). Se, de um lado, espera-se que existam decisões tomadas para atendimento de interesses particulares, por outro, existe o desprendimento. Incorporando à análise aspectos interpessoais e sociais e suas ascendências sobre a individualidade, demonstra-se, no estudo das dinâmicas sociais em que a implementação acontece, que até o tipo de contato 8 Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 2.2.5 Sistemas de gestão da política O sistema de gestão da política pública é o fator influenciador mais alinhado aos preceitos clássicos da implementação. É nele que os servidores de linha de frente se encontram com os burocratas de médio e alto 9 9 Administração Pública e Gestão Social, 2024, vol. 16, núm. 2, Abril-Junio, ISSN: 2175-5787 escalão através do emaranhado de normas legais, infralegais, cadeia de comando, sistema de prestação de contas, objetivos, metas e demais obrigações que vêm à esteira do exercício da função pública de implementar. Diversas são as variáveis — ou, no contexto desse trabalho, subcategorias — identificadas na literatura que influenciam o comportamento da burocracia de nível de rua, tais como: carga de trabalho (Jewell & Glaser, 2006), papel e função desempenhados (Jensen, 2018; Jewell & Glaser, 2006), colaboração entre equipes de implementadores, normas profissionais, estrutura e recursos adequados — essa, inclusive, já apontada por Lipsky (2010) em seu trabalho clássico como característica do trabalho realizado pela burocracia de nível de rua (Ferreira et al., 2020) —, organização, ambiguidades, conflitos, diferentes níveis de comprometimento, aspectos de satisfação no serviço (Z. W. Oberfield, 2012) e liderança dos gestores (Alcadipani et al., 2020; Jewell & Glaser, 2006; Keiser, 2010). Mesmo em casos mais específicos, como na implementação de políticas de humanização, verificam-se características em comum com políticas de outras áreas. Lima e D’Ascenzi (2017) observam, por exemplo, que elas são interpretadas de maneiras distintas entre diferentes equipes de implementadores. A principal razão apontada para a divergência quanto à interpretação de conceitos contidos na política é a falta de capacitação dos servidores. Essa situação transforma a interpretação da política pública de acordo com a localidade em que ela é aplicada, deixando o planejamento formal apenas um elemento orientador do processo de implementação. A falta de treinamento e capacitação pode também ser considerada uma falta de recurso. Ferreira et al. (2020) afirmam que essa disponibilidade de insumos para a implementação das atividades influencia e limita fortemente as próprias opções do burocrata de nível de rua diante de fatos encontrados diuturnamente. Igualmente, G. S. Lotta (2014) aponta que fatores relacionais, institucionais, normativos — inclusive normas informais — e estruturais influenciam a forma como os implementadores se relacionam com os cidadãos, pares e superiores e, por consequência, a própria política pública. Situações de calamidade pública constituem outra fonte de ascendência sobre a conduta do servidor de linha de frente e evidenciam essa costumaz falta de recursos. PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto PDF generado a partir de XML-JATS4R por Redalyc PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 2.2.5 Sistemas de gestão da política A política, a cultura ocupacional e a disponibilidade de material afetam a capacidade dos servidores responderem às demandas urgentes e imprevisíveis geradas em cenário de tragédia, como o da pandemia da COVID-19. Além disso, nessas situações que demandam a utilização de protocolos restritos, afeta-se a discricionariedade, que fica em segundo plano. Embora sempre presente na atuação dos implementadores, em razão inclusive dos recursos públicos estarem concentrados fortemente no combate à pandemia — serviços de saúde —, decisões discricionárias podem levar a um desperdício de recursos que não são suficientes nem em caso de uma utilização eficiente (Alcadipani et al., 2020). Ainda no âmbito da pandemia da COVID-19, evidencia-se que essas crises alteram as práticas e formas de implementação das políticas públicas (Brodkin, 2021). Sua influência passa pelos níveis de discricionariedade (Collins & Augsberger, 2021; Davidovitz et al., 2021; Gofen & Lotta, 2021; Malandrino & Sager, 2021) e accountabilityaplicáveis (Collins & Augsberger, 2021), por aspectos relacionados ao profissionalismo de determinadas categorias atuantes na crise (Collins & Augsberger, 2021), nas restrições de recursos disponíveis para solução dos problemas emergenciais (Collins & Augsberger, 2021; Gofen & Lotta, 2021), pelo desenvolvimentos de soluções através da resistência, inovação e improvisação (Cox et al., 2021), pelo aumento da ambiguidade normativa (Davidovitz et al., 2021; Gofen & Lotta, 2021), riscos presentes (Davidovitz et al., 2021) e da demanda de trabalho (Gofen et al., 2021; Gofen & Lotta, 2021) exigida dos implementadores, bem como pelo aumento da motivação da burocracia de nível de rua mobilizar-se politicamente e socialmente (Gofen et al., 2021). O último tópico destacado é o da utilização da Tecnologia de Informação (TI), outro fator influenciador da burocracia quando considerados aqueles tipos de serviços responsáveis por muitas decisões — decision-making factories—, como o judiciário. Essa influência é capaz de transformar a street-level bureaucracy em formas mais intensivas em tecnologia: screen-level e system-level bureaucracy. Os designers, analistas e desenvolvedores dos softwares passam a ter poderes discricionários à medida que a TI assume o papel dos servidores de linha de frente e, diante disso, devem ser supervisionados inclusive quanto 10 10 Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico rti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas m Ensaio Teórico à transparência em relação aos algoritmos e processos computacionais envolvidos nas tomadas de decisão (Bovens & Zouridis, 2002). 3 Integração das dimensões e categorias com apresentação de um model As pesquisas apresentadas confirmam o caráter multidimensional da burocracia de nível de rua (Hill & Hupe, 2022). Adicionalmente, revelam a heterogeneidade e atualidade da discussão sobre a implementação a partir dos implementadores, inclusive quando utilizada conjuntamente a outras abordagens. O agrupamento de diferentes subcategorias, que podem tornar-se ainda mais numerosas ao se ampliar as bases e critérios de buscas ou necessidade do pesquisador, procura aproximar as contribuições de pesquisas desenvolvidas em contextos próprios e, geralmente, com resultados não extrapoláveis. Por se tratar de um aspecto incontornável do campo da implementação — a dependência do contexto —, faz-se mister que esses fatores sejam testados na implementação das mais diversas políticas públicas e seus cenários. Além disso, investigar as causas da ação dos implementadores permite o rompimento da tendência de se analisar a implementação somente a partir do resultado, trazendo o ponto focal das pesquisas em implementação para a compreensão de como os implementadores e suas ações discricionárias a influenciam, inclusive determinando suas bases comparativas — a distância entre o previsto e o realizado — ao alterarem os próprios objetivos da política pública. O modelo apresentado na figura 1 traz uma representação que, neste trabalho, entende-se ser a dinâmica mais aproximada existente entre a formulação e implementação de políticas públicas. Idealmente, a implementação é melhor compreendida através de estudos longitudinais (Tummers, 2011; Van Meter & Van Horn, 1975). Contudo e especialmente quando a pesquisa não busca inferências causais (Henderson et al., 2018; Moyson et al., 2018), estudos transversais também se adequam ao tema. Isso posto, desde que se considere as particularidades do contexto estudado e os objetivos da pesquisa, o modelo pode ser utilizado tanto em estudos longitudinais quanto transversais. Como já discutido nas seções anteriores, o pressuposto aplicável é a presença da discricionariedade. Sobre isso, convém novamente destacar que a discricionariedade como um pressuposto não deve ser confundida com a ação discricionária em um dado contexto. A ação é influenciada e é capaz de influenciar — no longo prazo — os princípios e os fatores intercontextuais presentes na política pública, sendo limitada por essas relações. Já o pressuposto da discricionariedade se trata de uma resposta às limitações dos atores relacionados à política pública e que funciona como um instrumento que converge as intenções de resolução de problemas públicos e as ações exercidas sobre os problemas públicos realmente encontrados nos contextos de implementação. PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto PDF generado a partir de XML-JATS4R por Redalyc dé i i fi d l d ll d b j l i i i i d 2.2.5 Sistemas de gestão da política A TI também influência a prestação de serviços públicos quando o encontro com o cidadão acontece à distância, como em estudo de caso de um call center da agência sueca de seguro social. Os resultados sugerem que organizações altamente regradas não superam a questão da imprevisibilidade intrínseca das relações sociais, pelo contrário: tornam o atendimento dos cidadãos mais complexo. Portanto, a utilização da TI para fins de padronização não leva a atendimentos mais simples, pois não se trata de uma despersonalização de atendimento e a possibilidade de ação discricionária se mantém presente para resolverem questões específicas dentro dos limites dos regramentos (Thunman et al., 2020). Embora existam evidências de que processos de automação sejam capazes de diminuir a percepção de discricionariedade por parte dos burocratas de nível de rua (de Boer & Raaphorst, 2023), a utilização de soluções tecnológicas nos processos de implementação de políticas públicas demonstra-se capaz de aumentar a segurança dos implementadores em suas ações, permitindo, adicionalmente, uma maior possibilidade de monitoramento sobre elas (Aviram et al., 2023). 3 Integração das dimensões e categorias com apresentação de um model p ç A partir dos conceitos discutidos nesse ensaio e do modelo proposto na figura 1, é possível discutir aspectos inerentes à implementação de políticas públicas. Uma questão que se faz presente é o possível descompasso existente entre os princípios que regem as políticas públicas e seus fatores intercontextuais. Isso poderia colaborar na explicação do porquê as políticas por vezes não obtêm os resultados esperados. 11 11 Administração Pública e Gestão Social, 2024, vol. 16, núm. 2, Abril-Junio, ISSN: 2175-5787 Poderia, ao menos, prover respaldo teórico das razões que as levam a serem implementadas de formas diferentes da sua concepção original. Figura 1 Modelo de dimensões e categorias de fatores influenciadores da ação do burocrata de nível de rua na implementação de políticas públicas Fonte: elaboração dos autores baseado em Ferreira e Medeiros (2016). Fonte: elaboração dos autores baseado em Ferreira e Medeiros (2016). Fonte: elaboração dos autores baseado em Ferreira e Medeiros (2016). Mesmo observando a política pública de forma integral, sem encadeamentos unidirecionais e lineares, deve ser realizado um esforço teórico de reconhecimento de cada dimensão capaz de influenciá-la. Dessa forma, se propõe que os princípios que regem uma política pública sejam considerados o núcleo estável de seu framework normativo, enquanto os fatores intercontextuais formam um núcleo dinâmico. Com o decorrer da implementação, consubstanciada nas ações dos burocratas de nível de rua, sugere-se que essas ações possuem menor capacidade de alterar o núcleo estável do que o núcleo dinâmico. Logo, quanto maior o horizonte temporal de implementação de uma política pública, maior se tornaria o distanciamento entre o framework normativo original — aquele formado a partir da primeira versão de seus núcleos formadores — e o framework normativo atual — que variou a partir da ação de implementá- la. Isso se demonstra coerente com evidências de que, em momentos de crise e com a necessidade de se acelerar os ciclos de formulação e implementação — horizontes temporais curtos —, a informação fluir de maneira intensa entre formuladores e implementadores da política pública aumenta a capacidade de resposta governamental aos problemas emergenciais (Gofen & Lotta, 2021). PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 3 Integração das dimensões e categorias com apresentação de um model Logo, quando existem circunstâncias urgentes e que demandam rápidas respostas por parte do poder público, tende-se a flexibilizar regras (Collins & Augsberger, 2021) e aumentar-se a discricionariedade dos implementadores (Collins & Augsberger, 2021; Davidovitz et al., 2021; Gofen & Lotta, 2021; Malandrino & Sager, 2021), pois a situação acarreta um aumento da ambiguidade das normas aplicáveis (Davidovitz et al., 2021; Gofen & Lotta, 2021) e diminuição do recursos disponíveis (Collins & Augsberger, 2021; Gofen & Lotta, 2021). Assim, espera-se que o núcleo dinâmico seja alterado após a implementação, independentemente do espaço temporal considerado. Diante disso, sugere-se que se encontra no nível de flexibilidade do núcleo 12 12 12 Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico rti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas m Ensaio Teórico estável a chave para resultados mais condizentes com os objetivos — naturalmente dinâmicos — da política pública. Outra possibilidade teórica é considerar que o núcleo estável da política pública, mais que uma forma de controle ou fonte de parâmetros para avaliação de resultados, se trata da base para uma inevitável evolução do núcleo dinâmico. O desenvolvimento deste, fundamentado, mas não limitado aos seus princípios basilares, seria a principal fonte no estabelecimento do framework normativo da política pública e, consequentemente, a grande força motriz que leva à implementação dessa política pública. g Essa percepção, embora sutilmente distinta da anterior, leva a encaminhamentos diferentes. Enquanto no primeiro caso a flexibilidade do núcleo estável do framework normativo ainda seria uma preocupação dos policymakers, no segundo não. Seria reconhecer a natureza evolutiva da implementação da política pública, fundamentada em bases necessárias apenas para sua criação, mas livre para estabelecer, de forma variável e dentro de seus inúmeros e distintos fatores intercontextuais, seus próprios objetivos a partir de sua implementação por parte da burocracia de nível de rua. p p p Aqui, retoma-se a ideia de se considerar a discricionariedade como pré-requisito da implementação de políticas públicas e demonstra-se uma consequência, ainda teórica, desse posicionamento ao sugerir que, de fato, o parâmetro de comparação do sucesso da implementação pode não ser aquele contido nos princípios na política pública, mas aqueles advindos do ato de implementá-la. 3 Integração das dimensões e categorias com apresentação de um model Essa linha de pensamento, já tangenciada na literatura no apontamento de que a burocracia de nível de rua também é formuladora da política pública ao efetivá-la e adaptá-la aos diferentes contextos (Arretche, 2001; Bronzo et al., 2022; Ferreira & Medeiros, 2016; Lipsky, 2010; G. S. Lotta et al., 2018; Nouman & Cohen, 2023), não vem sendo explorada suficientemente. O papel da burocracia de nível de rua na tomada de decisão realizada durante o processo de formulação da política pública é pouco estudado (Davidovitz et al., 2021). Seguir essa perspectiva possibilita perceber que os objetivos podem ser desenvolvidos durante ou até mesmo após a implementação, transformando aqueles estabelecidos inicialmente pelos policymakers em linhas gerais a serem seguidas para solução de um problema. Essa discussão pode repercutir para além do campo da implementação, permitindo desdobramentos teóricos, inclusive, no campo de avaliação de políticas públicas. Finalmente, embora impossíveis de serem totalmente previstos, sustenta-se que deve existir um esforço por parte dos policymakersem reconhecer a influência e conhecer os fatores que influenciam a forma como a burocracia de nível de rua implementa determinada política pública. Esse é um aspecto intrínseco do processo de elaboração e implementação das políticas públicas que não vem recebendo a devida atenção na literatura relacionada à burocracia de nível de rua. O conhecimento desses fatores, se analisados caso a caso e a priori, considerando ainda as peculiaridades contextuais envolvidas, possibilitam uma maior compreensão a respeito do núcleo dinâmico resultante da ação de implementar. Isso daria a eles alternativas e subsídios para harmonizar o núcleo estável — ou, alternativamente, para entendê-lo como uma base, um ponto de partida — ao que de fato está sendo buscado na implementação, dirimindo dinamicamente as incongruências do framework normativo original em relação à realidade dos contextos em que a política pública é implementada e à multidimensionalidade da burocracia de nível de rua. Dizendo de outra forma, se, nesse primeiro momento, se compreender que não é provável ou mesmo possível que o conjunto de normas legais, infralegais, guias e congêneres possa contemplar a multiplicidade de fatores e contextos que serão encontrados na implementação, abre-se espaço para que se discuta o que, realmente, a política pública busca e precisa influenciar, preocupando-se menos com a forma e mais com o resultado — analisado concomitantemente ou posteriormente à implementação e com base em objetivos dinâmicos. PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de a PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 4 Considerações finais O objetivo desse ensaio foi desenvolver proposições sobre a implementação a partir da burocracia de nível de rua. Adicionalmente, apresentou-se um modelo teórico sobre a implementação de políticas públicas, considerando seu caráter multidimensional, com a identificação de fatores que influenciam a ação do implementador de políticas públicas no exercício de suas funções. 13 13 Administração Pública e Gestão Social, 2024, vol. 16, núm. 2, Abril-Junio, ISSN: 2175-5787 A primeira proposição desenvolvida é a sustentação de que a discricionariedade, mais que um fator influenciador na forma como a burocracia de nível de rua implementa as políticas públicas, é um pressuposto da implementação. O julgamento realizado pelo implementador na ação discricionária é um produto das relações de interdependência dos fatores influenciadores da ação de implementar e não pode ser confundido com a própria discricionariedade, sempre presente. A segunda proposição apresentada advém de como as ações dos implementadores das políticas públicas resultam em diferentes ritmos de adaptação nos princípios que regem a política pública — núcleo estável — e em seus fatores intercontextuais — núcleo dinâmico. Quanto mais uma política pública é implementada, mais distante seus objetivos originais parecem se tornar em relação aos objetivos que estão efetivamente sendo perseguidos pelos implementadores e até mesmo aos seus objetivos potenciais. Apresenta-se, a partir disso, dois encaminhamentos: i) a dinamicidade do núcleo estável é central para que as políticas públicas obtenham resultados mais alinhados com os problemas que objetivamente estão sendo resolvidos em sua implementação a partir das ações dos burocratas de nível de rua; e ii) o núcleo estável da política pública, mais que uma forma de controle ou fonte de parâmetros para avaliação de resultados, se trata da base para a evolução do núcleo dinâmico. Enquanto o primeiro encaminhamento mantém o foco dos policymakers no núcleo estável, o segundo reconhece a natureza evolutiva da implementação da política pública, fundamentada em bases necessárias apenas para sua criação, mas livre para estabelecer, de forma variável e dentro de seus inúmeros e distintos fatores intercontextuais, seus próprios objetivos a partir de sua implementação por parte da burocracia de nível de rua. A última proposição debatida é a necessidade de os policymakers reconhecerem a influência e conhecerem os fatores que influenciam a forma como a burocracia de nível de rua implementa determinada política pública. PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 14 PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto Referências Alcadipani, R., Cabral, S., Fernandes, A., & Lotta, G. (2020). Street-level bureaucrats under COVID-19: Police officers’ responses in constrained settings. Administrative Theory & Praxis, 42(3), 394–403. https://doi.org/10.1080/10841806.2020.1771906 Arretche, M. T. S. (2001). Uma contribuição para avaliações menos ingênuas. In M. C. R. N. Barreira & M. do C. B. Carvalho (Eds.), Tendências e perspectivas na avaliação de políticas e programas sociais (1st ed., pp. 43–56). IEE/PUC-SP. Aviram, N. F., Correa, C., & Oliviera, R. (2023). Technology 3.0: Police Officers’ Perceptions Towards Technology Shifts. American Review of Public Administration. https://doi.org/ 10.1177/02750740231186791 Balica, D. O., Henderson, A., & Țiclău, T. C. (2018). Romanian: Street level bureaucracy: A descriptive foundation. Transylvanian Review of Administrative Sciences, 2018(2018), 5–26. https://doi.org/ 10.24193/TRAS.SI2018.1 Barrett, S. M. (2004). Implementation Studies: Time for a Revival? Personal Reflections on 20 Years of Implementation Studies. Public Administration, 82(2), 249–262. https://doi.org/10.1111/ j.0033-3298.2004.00393.x Bonelli, F., Fernandes, A. S. A., Coêlho, D. B., & Palmeira, J. da S. (2019). A atuação dos burocratas de nível de rua na implementação de políticas públicas no Brasil: uma proposta de análise expandida. Cadernos EBAPE.BR, 17(spe), 800–816. https://doi.org/10.1590/1679-395177561 Borry, E. L., & Henderson, A. C. (2020). Patients, Protocols, and Prosocial Behavior: Rule Breaking in Frontline Health Care. American Review of Public Administration, 50(1), 45–61. https://doi.org/ 10.1177/0275074019862680 Bovens, M., & Zouridis, S. (2002). From street-level to system-level bureaucracies: How information and communication technology is transforming administrative discretion and constitutional control. Public Administration Review, 62(2), 174–184. https://doi.org/10.1111/0033-3352.00168 Brodkin, E. Z. (2011). Policy work: Street-level organizations under new managerialism. Journal of Public Administration Research and Theory, 21(SUPPL. 2), 253–277. https://doi.org/10.1093/jopart/ muq093 Brodkin, E. Z. (2021). Street-Level Organizations at the Front Lines of Crises. Journal of Comparative Policy Analysis: Research and Practice, 23(1), 16–29. https://doi.org/ 10.1080/13876988.2020.1848352 Bronzo, C., Cristina Rezende Costa, E., & Guimaraes, F. (2022). A Burocracia de Nível de Rua na Implementação do Serviço de Proteção e Atendimento Integral à Família (PAIF): Percepções e Traduções de Normativas nas Políticas Públicas. Administração Pública e Gestão Social, 14. https:// doi.org/10.21118/apgs.v14i2.12434 Buta, B. O., Teixeira, M. A. C., & Fernandes, A. S. A. (2022). Quando a autonomia é necessária para o desempenho: defensoria pública do Brasil. Revista de Administração Pública, 56(4), 488–507. https://doi.org/10.1590/0034-761220220047 Carroll, K., Wright, K., & Meier, K. J. (2019). Minority Public Administrators: Managing Organizational Demands While Acting as an Advocate. American Review of Public Administration, 49(7), 810– 824. https://doi.org/10.1177/0275074019859942 Cohen, N. (2018). 4 Considerações finais A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico PDF generado a partir de XML-JATS4R por Redalyc Proyecto académico sin fines de lucro, desarrollado bajo la iniciativa de acceso abierto 4 Considerações finais Isso daria a eles condições, alternativas e subsídios para harmonizar o núcleo estável — ou percebê-lo como a base — ao que de fato está sendo buscado na implementação. Como resultado, o framework normativo original da política pública possuiria mais aderência em relação à realidade dos contextos em que a política pública é implementada e a multidimensionalidade da burocracia de nível de rua. São limitações do ensaio teórico a quantidade de estudos aqui considerados na discussão sobre os fatores influenciadores da ação dos burocratas de nível de rua na implementação de políticas públicas, além da necessidade de testar as proposições desenvolvidas por meio de pesquisas empíricas para dar suporte e novas evidências para o debate. Pesquisas futuras podem avançar na busca de outras contribuições sobre o modo de agir da burocracia de nível de rua, algo que parece fundamental para a análise da implementação na linha de frente das políticas públicas. Outra possibilidade de pesquisa futura é trazer os conceitos de organizações de nível de rua (Brodkin, 2011) ao modelo apresentado, pois ele abarca as ações dos indivíduos que atuam em nível de rua, não suas organizações. Defende-se também uma agenda de estudos sobre como as crises, como da pandemia da COVID-19, afetam a implementação de políticas públicas, um assunto ainda pouco explorado na literatura e que possui potencial de levar a importantes desdobramentos teóricos e práticos (Brodkin, 2021; Gofen & Lotta, 2021), sobretudo diante da previsão de que, após a pandemia, se reverteriam muitas das mudanças nela observadas. Isso, somado à falta de recursos financeiros causada pelos dispêndios emergenciais de recursos públicos, levaria a burocracia de nível de rua a operar em condições ainda mais restritas e desafiadoras (Cox et al., 2021). Finalmente, observa-se a necessidade de se desenvolver pesquisas sobre o tema em âmbito nacional, especialmente na análise de casos empíricos, e avançar em pesquisas comparativas (Bonelli et al., 2019; Gofen & Lotta, 2021) em nível internacional. Políticas da área da saúde e da assistência social são exemplos de áreas estudadas nacionalmente, mas não são suficientes para a compreensão do modelo de implementação de um país tão diverso e desigual quanto o Brasil. 14 14 Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico Duljon Laerti Boldrin, et al. A Discricionariedade como um Pressuposto da Implementação de Políticas Públicas: um Ensaio Teórico Duljon Laerti Boldrin, et al. Referências How culture affects street-level bureaucrats’ bending the rules in the context of informal payments for health care: The Israeli case. American Review of Public Administration, 48(2), 175–187. https://doi.org/10.1177/0275074016665919 15 15 Administração Pública e Gestão Social, 2024, vol. 16, núm. 2, Abril-Junio, ISSN: 2175-5787 Collins, M. E., & Augsberger, A. (2021). 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https://bmcpregnancychildbirth.biomedcentral.com/counter/pdf/10.1186/s12884-022-05003-4
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Screening high-risk population of persistent postpartum hypertension in women with preeclampsia using latent class cluster analysis
BMC pregnancy and childbirth
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Abstract Background:  A significant proportion of women with preeclampsia (PE) exhibit persistent postpartum hypertension (PHTN) at 3 months postpartum associated with cardiovascular morbidity. This study aimed to screen patients with PE to identify the high-risk population with persistent PHTN. Methods:  This retrospective cohort study enrolled 1,000 PE patients with complete parturient and postpartum blood pressure (BP) profiles at 3 months postpartum. The enrolled patients exhibited new-onset hypertension after 20 weeks of pregnancy, while those with PE superimposed upon chronic hypertension were excluded. Latent class cluster analysis (LCCA), a method of unsupervised learning in machine learning, was performed to ascertain maternal exposure clusters from eight variables and 35 subordinate risk factors. Logistic regression was applied to calculate odds ratios (OR) indicating the association between clusters and PHTN. Results:  The 1,000 participants were classified into three exposure clusters (subpopulations with similar character‑ istics) according to persistent PHTN development: high-risk cluster (31.2%), medium-risk cluster (36.8%), and low-risk cluster (32.0%). Among the 1,000 PE patients, a total of 134 (13.4%) were diagnosed with persistent PHTN, while the percentages of persistent PHTN were24.68%, 10.05%, and 6.25% in the high-, medium-, and low-risk clusters, respectively. Persistent PHTN in the high-risk cluster was nearly five times higher (OR, 4.915; 95% confidence interval (CI), 2.92–8.27) and three times (OR, 2.931; 95% CI, 1.91–4.49) than in the low- and medium-risk clusters, respectively. Persistent PHTN did not differ between the medium- and low-risk clusters. Subjects in the high-risk cluster were older and showed higher BP, poorer prenatal organ function, more adverse pregnancy events, and greater medication requirement than the other two groups. Conclusion:  Patients with PE can be classified into high-, medium-, and low-risk clusters according to persistent PHTN severity; each cluster has cognizable clinical features. This study’s findings stress the importance of controlling persistent PHTN to prevent future cardiovascular disease. Screening high‑risk population of persistent postpartum hypertension in women with preeclampsia using latent class cluster analysis Yuan‑Yuan Li1,2,3, Jing Cao1,2, Jia‑Lei Li1,2, Jun‑Yan Zhu1,2, Yong‑Mei Li1,2, De‑Ping Wang1,2, Hong Liu3, Hai‑Lan Yang4, Yin‑Fang He4, Li‑Yan Hu5, Rui Zhao6, Chu Zheng7, Yan‑Bo Zhang7* and Ji‑Min Cao1,2* *Correspondence: sxmuzyb@126.com; caojimin@sxmu.edu.cn 1 Key Laboratory of Cellular Physiology at Shanxi Medical University, Ministr of Education, Taiyuan, China 7 Division of Health Statistics, School of Public Health, Shanxi Medical University, Taiyuan, China Full list of author information is available at the end of the article Li et al. BMC Pregnancy and Childbirth (2022) 22:687 https://doi.org/10.1186/s12884-022-05003-4 Li et al. BMC Pregnancy and Childbirth (2022) 22:687 https://doi.org/10.1186/s12884-022-05003-4 Open Access Study populationh This retrospective multicenter cohort study recruited patients from the First Hospital of Shanxi Medical Uni- versity (FHSMU) between June 2017 and May 2020 and Shanxi Children’s Hospital and Women Health Center (SCWHC) from September 2019 to May 2020 with a confirmed diagnosis of PE or who developed PE after admission. A study flowchart is shown in Fig. 1. The diagnosis of PE conformed to the 2018 definition of International Society for the Study of Hypertension in Pregnancy and ACOG 2019 [1, 2]. PE is characterized by new-onset of hypertension (systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg) and exhibits at least one of the following new-onset symptoms during or after 20 weeks of gestation: 1) proteinuria (24-h urinary protein ≥ 300 mg/day or dipstick reading ≥ 2+); 2) other mater- nal organ dysfunction, such as acute kidney injury (cre- atinine ≥ 90 μmol/L or 1.0 mg/dL), hepatic dysfunction (alanine aminotransferase or aspartate aminotransferase > 40 IU/L, with or without epigastric abdominal or right upper quadrant pain), neurological dysfunction (such as eclampsia and change in mental status), or hema- tologic complications (blood platelet count < 150,000/ μL, disseminated intravascular coagulation, or hema- tolysis); and 3) uteroplacental complications (abnormal Doppler waveform of the umbilical artery, fetal growth restriction, or stillbirth). Patients who were diagnosed with PE superimposed upon chronic hypertension, pre- pregnancy hypertension, or hypertension that occurred within the first 20 weeks of pregnancy were not included in the study. PE is associated with multiple risk factors [2], and its risk assessments require methods that can integrate them. Current PE models mostly involve the early pre- diction of adverse pregnancy outcomes and long-term cardiovascular disease risk [19–21]. Prognostic data on BP profiles at short-term follow-up during the postpar- tum period in patients with PE are scarce, although some other risk factors of PHTN have been suggested, includ- ing preexisting hypertension before pregnancy and a higher body mass index (BMI) [11], older age, smoking, pre-pregnancy obesity, comorbidities such as thyroid dis- orders [12], and decreased serum placental growth factor [22]. Few studies to date have reported the clustered and combined effects of multiple risk factors on BP recovery during the 3-month or longer period after delivery in patients with PE. It remains unclear whether PE increases the occurrence of PHTN through the known risk factors The study flowchart and patient information are pre- sented in Fig. 1. Keywords:  preeclampsia, postpartum hypertension, cardiovascular disease, latent class cluster analysis Keywords:  preeclampsia, postpartum hypertension, cardiovascular disease, latent class cluster analysis Keywords:  preeclampsia, postpartum hypertension, cardiovascular disease, latent class cluster analysis © 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Li et al. BMC Pregnancy and Childbirth (2022) 22:687 Page 2 of 10 Introduction associated with PE and CVD. The present study aimed to identify the categories of PE patients using latent class cluster analysis (LCCA) by combining multiple risk fac- tors. Unlike traditional single-factor approaches, LCCA is a machine learning method and has certain advantages; for example, it can explore the interrelationships among multiple risk factors and classify similar objects into groups, and thus can be applied for screening high-risk populations [23, 24]. This study may help identify high- risk clusters in the PE population and provide appro- priate treatment strategies for those who may develop persistent PHTN and CVD. Preeclampsia (PE) is a common and severe complica- tion of pregnancy that manifests as new-onset hyperten- sion and proteinuria and can progress to severe PE with multi-organ involvement [1–3]. An estimated 2-8% of pregnant women worldwide suffer from PE [4, 5]. In the last decade, mounting evidence has suggested that preec- lamptic women are susceptible to developing cardiovas- cular disease (CVD) later in life [6–8]. Maternal hypertension and proteinuria usually disap- pear in most PE patients within the first week postpar- tum; until 3 months, blood pressure (BP) mostly returns to normal [1, 9, 10]. However, about 20% of patients with PE exhibit persistent postpartum hypertension (PHTN), and the occurrence of some forms of PHTN, includ- ing sustained hypertension, masked hypertension, and white-coat hypertension, could be as high as 41.5% at 1 year after discharge in patients with severe PE [11–13], who require long-term antihypertensive medication. One proposed risk factor for cardiovascular morbidity is the persistence of hypertension in the postpartum period [14, 15]. Hypertensive women who experience PE are reportedly at a two-fold risk of developing CVD in the next decades compared to those with PE but become normotensive after delivery [16]. Notably, postpartum follow-up of PE is inadequate, with reported rates of 20-60%, and a large proportion of obstetricians neglect to follow up postpartum BP in PE patients [13, 15, 17, 18]. Therefore, early screening for those at high risk of devel- oping persistent PHTN helps clinicians provide accurate postpartum BP monitoring and timely intervention for in such patients. Defining persistent PHTNh The recruited PE patients were followed up by telephone interviews to confirm persistent PHTN at 3 months post- partum. BP was measured twice a day by trained nurses in community clinics and/or by trained family members at home using mercurial or electronic arm cuff BP meters after a 5-min rest. BP measurements were repeated three times within 10 min, with a 1-min interval between measurements. Mean systolic and diastolic BP values were recorded. In the present study, patients with per- sistent PHTN were defined as those who experienced PE and still showed hypertension (average systolic BP ≥ 140 mmHg; average diastolic BP ≥ 90 mmHg) within 3 months postpartum and the requirement for cardiovas- cular consults for further investigation and medication. Data collectionh The data and diagnoses of the enrolled patients were col- lected, including maternal demographic characteristics and relevant clinical laboratory tests performed within 7 days prior to the end of pregnancy. If the index visit involved multiple tests, the worst value was selected. A total of 35 candidate risk factors, including laboratory test results, were entered from the literature reviews [1, 2, 20, 25–27]. Laboratory indicators were converted from continuous to categorical variables based on whether they were outside the normal range (Table 1). Study populationh Between June 2017 and May 2020, a total of 722 PE patients who were admitted to the FHSMU and ended their pregnancies were included, excluding Li et al. BMC Pregnancy and Childbirth (2022) 22:687 Page 3 of 10 Fig. 1  Study flowchart outlining the composition of final PE cohort using the datasets from the First Hospital of Shanxi Medical University (FHSMU) and Shanxi Children’s Hospital and Women Health Center (SCWHC) Fig. 1  Study flowchart outlining the composition of final PE cohort using the datasets from the First Hospital of Shanxi Medical University (FHSMU) and Shanxi Children’s Hospital and Women Health Center (SCWHC) 16 without follow-up and 16 without postpartum BP measurement data. Thus, 690 of the 722 PE patients treated at the FHSMU were included; the other 32 were excluded. Of the 690 patients from the FHSMU, the BP data of 94 (13.62%) remained higher at 3 months post- partum. Among patients treated at the SCWHC between September 2019 and May 2020, 328 with PE ended their pregnancies, excluding 16 patients without follow-up and two without postpartum BP measurement data; the remaining 310 were included in the study. Among the 310 patients in the SCWHC group, the BP values did not return to normal by 3 months postpartum in 40 (12.90%). Thus, 1,000 (690 + 310) PE patients from the two hospi- tals were included, while 50 were excluded due to lack of postpartum BP data or phone interview failure. The end date of follow-up was October 2020. Patients with BP that did not return to normal by 3 months post- partum were included as outcome events. Patients who could not be reached by telephone for follow-up and whose BP was not monitored within 3 months postpar- tum were excluded from the study cohort. Latent class cluster analysish The 35 candidate risk factors were categorized into eight important indicator variables, including maternal deliv- ery age, mean arterial pressure (MAP = diastolic BP + 1/3 pulse pressure difference; maximum MAP levels during pregnancy were used in the study), drug use dur- ing pregnancy, medical history, adverse pregnancy out- comes, blood cell and coagulation tests performed within 7 days before delivery, altered liver and renal functions within 7 days before delivery, and elevated blood myo- cardial enzymes and electrolyte disbalance within 7 days before delivery. Among the 35 risk factors, no categorical Li et al. BMC Pregnancy and Childbirth (2022) 22:687 Page 4 of 10 Table 1  Description of the eight indicator variables for LCCA and the baseline characteristics of the 1,000 enrolled PE patients Indicator variable Risk factors YES NO MIN MAX Persistent PHTN (134) No PHTN (866) P References 1. Maternal delivery age Maternal delivery age 16 46 32 (28,36) 30 (27,33) < 0.001 [1, 2, 20] 2. Mean arterial pressure Mean arterial pressure (MAP) (mmHg) 91 180 126.67 (116.67,137.33) 117.33 (110,126.67) < 0.001 [1, 2, 20] 3. Drugs use Beta blockers 1 0 0 4 59 (44.03) 218 (25.17) < 0.001 [1, 2, 20] Calcium antagonist 1 0 29 (21.64) 89 (10.28) < 0.001 Dexamethasone 1 0 11 (8.21) 42 (4.85) 0.106 MgSO4 1 0 30 (22.39) 77 (8.89) < 0.001 4. Medical history Body mass index (BMI) before pregnancy ≥ 25 1 0 0 12 63 (47.01) 329 (37.99) 0.046 [1, 2, 20, 25–27] Prior preeclampsia 1 0 9 (6.72) 55 (6.35) 0.872 History of heart / kidney disease 1 0 2 (1.49) 7 (0.81) 0.435 Family history of hyper‑ tension 1 0 25 (18.66) 124 (14.32) 0.189 Number of abortions 1-3 0 0.201  0 64 (47.76) 489 (56.47)  1 49 (36.57) 248 (28.64)  2 13 (9.70) 90 (10.39)  ≥ 3 8 (5.97) 39 (4.5) Number of births 1-3 0 0.177  0 68 (50.75) 523 (60.39)  1 54 (40.30) 286 (33.03)  2 11 (8.21) 49 (5.66)  ≥ 3 1 (0.75) 8 (0.92) ≥ 10 years from the previous birth 1 0 30 (22.39) 94 (10.85) < 0.001 PE was diagnosed before 32 weeks of gestation 1 0 61 (45.52) 295 (34.06) 0.010 5. Latent class cluster analysish Adverse pregnancy outcome Preterm birth 1 0 0 6 90 (67.16) 459 (53) 0.002 [1, 2, 20] Postpartum hemorrhage 1 0 7 (5.22) 42 (4.85) 0.852 Pericardial or pleural effusion 1 0 15 (11.19) 23 (2.66) < 0.001 Placental abruption 1 0 18 (13.43) 94 (10.85) 0.378 Low birth weight infants / fetal growth restriction 1 0 24 (17.91) 130 (15.01) 0.387 HELLP syndrome 1 0 20 (14.93) 50 (5.77) < 0.001 6. Blood cell and coagu‑ lation test Platelet count (< 100×109/L) 1 0 0 5 12 (8.96) 53 (6.12) 0.215 [1, 2, 20, 27] Neutrophil count (> 6.3×109/L) 1 0 83 (61.94) 472 (54.50) 0.107 Monocyte count (> 0.6×109/L or < 0.1×109/L) 1 0 51 (38.06) 283 (32.68) 0.219 PT% (> 130% or < 70%) 1 0 57 (42.54) 311 (35.91) 0.139 INR (>1.15 or < 0.85) 1 0 26 (19.40) 150 (17.32) 0.556 Table 1  Description of the eight indicator variables for LCCA and the baseline characteristics of the 1,000 enrolled PE patients Li et al. BMC Pregnancy and Childbirth (2022) 22:687 Page 5 of 10 MAP Mean arterial pressure, expressed as median (25% quartile, 75% quartile), i.e., [M (P25, P75)]. BMI Body mass index, HELLP syndrome Hemolysis, elevated liver enzymes, low platelets syndrome, PT% prothrombin activity (%), INR International normalized ratio, AST Aspartate aminotransferase, ALT Alanine aminotransferase, ALB Albumin Table 1  (continued) Indicator variable Risk factors YES NO MIN MAX Persistent PHTN (134) No PHTN (866) P References 7. Liver and renal func‑ tion AST (> 40 U/L) 1 0 0 9 29 (21.64) 128 (14.78) 0.042 [1, 2, 20, 27] ALT (> 40 U/L) 1 0 13 (9.70) 69 (7.97) 0.496 ALB (< 30 g/L) 1 0 65 (48.51) 346 (39.95) 0.061 Proteinuria 1-4 0 0.308  0 20 (14.93) 123 (14.22)  1+ 26 (19.40) 237 (27.40)  2+ 29 (21.64) 149 (17.23)  3+ 49 (36.57) 307 (35.49)  4+ 10 (7.46) 49 (5.66) Serum creatinine (> 1.0 mg/dL) 1 0 11 (8.21) 49 (5.66) 0.247 Serum urea nitrogen (> 7.6 mmol/L) 1 0 9 (6.72) 45 (5.20) 0.469 8. Latent class cluster analysish Blood myocardial enzyme and electrolyte test Serum creatine kinase (> 200 U/L) 1 0 0 4 14 (10.45) 69 (7.97) 0.333 [1, 2, 27] Serum lactate dehydro‑ genase (> 250 U/L) 1 0 47 (35.07) 263 (30.37) 0.273 Serum potassium (> 5.5 mmol/L or < 3.5 mmol/L) 1 0 7 (5.22) 35 (4.04) 0.525 Serum calcium (< 2.11 mmol/L) 1 0 60 (44.78) 297 (34.30) 0.018 MAP Mean arterial pressure, expressed as median (25% quartile, 75% quartile), i.e., [M (P25, P75)]. BMI Body mass index, HELLP syndrome Hemolysis, elevated liver enzymes, low platelets syndrome, PT% prothrombin activity (%), INR International normalized ratio, AST Aspartate aminotransferase, ALT Alanine aminotransferase, ALB Albumin e, expressed as median (25% quartile, 75% quartile), i.e., [M (P25, P75)]. BMI Body mass index, HELLP syndrome Hemolysis, elevated liver drome, PT% prothrombin activity (%), INR International normalized ratio, AST Aspartate aminotransferase, ALT Alanine aminotransferase, is called latent profile analysis (LPA). The eight indicators we studied were continuous variables, and the basic prin- ciple of LPA was to suppose that the probability density function of the P-dimensional continuous manifest variable vector yi can be expressed as the following equation 1: is called latent profile analysis (LPA). The eight indicators we studied were continuous variables, and the basic prin- ciple of LPA was to suppose that the probability density function of the P-dimensional continuous manifest variable vector yi can be expressed as the following equation 1: variable data were missing. Some continuous variable data were missing, including up to 2.5% of those for serum creatine kinase and serum lactate dehydrogenase, while data for prothrombin activity (%), international normalized ratio, albumin, serum creatinine, serum urea nitrogen, and serum potassium were missing for fewer than five cases; instead, mean values were used. Each of the eight indicator variables except maternal age and MAP included multiple risk factors. These variables were aggregated and assessed as total risk factor scores, with a dimensionality reduction of 0-N (Table 1) [28]. All eight indicators were considered continuous variables and standardized by the LCCA. Statistical analysis Continuous variables in the baseline information are expressed as median and quartile [M ­(P25, ­P75)], and com- parisons between PE patients with versus without persis- tent PHTN were made using the Mann-Whitney U test. Categorical variables are expressed as count and percent- age, and the chi-squared test was used to compare PE patients with versus without persistent PHTN. Standard- ized characteristics of clusters are expressed as mean ± standard deviation (SD), while cluster comparisons were performed using analysis of variance. The logistic regres- sion analysis was performed to explore the association between exposure clusters and persistent PHTN. The statistical analysis was performed using SPSS 22.0, and statistical significance was set at P < 0.05. Bonferroni cor- rection was used to adjust the P values for multiple tests. Latent class cluster analysish (1) f  yi  = K  k=1 ηkfk  yi|µk, k (1) (1) Here ηk denotes the latent class probabilities and K is the number of clusters (= 1, 2, …, K); yi is the score of object i on a set of manifest variables, assuming that within the cluster k, yi came from an independent multivariate normal distribution; μk is the mean vector; and ∑k is the variance- covariance matrix. After model establishment using Bayes- ian theory, the posterior probability of assigning patients to class k was calculated using the following equation 2: LCCA, a model-based clustering approach, was con- ducted to analyze the eight indicator variables using R 3.6.1 software. It assumes that heterogeneous populations are a mixture of populations; that is, a latent class is used to clas- sify populations. This method classifies the population by probability; that is, the individual belongs to a cluster with a certain probability, and the individual is ultimately assigned to the cluster with the highest posterior probability [29]. LCCA for categorical indicator variables is called latent class analysis, while that for continuous indicator variables (2) P k yi = ηkfk yi|µk, k K k=1 ηkfk yi|µk, k (2) LPA with the mclust package was used to define clus- ters of participants with similar clinical profiles. We used LPA with the mclust package was used to define clus- ters of participants with similar clinical profiles. We used Li et al. BMC Pregnancy and Childbirth (2022) 22:687 Page 6 of 10 mclustBIC to observe the Bayesian Information Criterion (BIC) for different profiles and the integrated completed likelihood (ICL) to penalize the model’s instability to sta- bilize the number of obtained models. Finally, PE patients were classified into different latent classes. medication. Maternal delivery age, MAP, and prenatal use of antihypertensive drugs in the 134 patients with PHTN were significantly higher than those in the 866 patients without PHTN (P < 0.001, Mann-Whitney U test and chi-squared test). Compared with normotensive sub- jects at 3 months postpartum, patients who developed persistent PHTN showed PE features earlier (earlier than 32 weeks’ gestation), longer intervals between births (/ pregnancies) (> 10 years), a higher incidence of adverse pregnancy outcomes, and worse laboratory results. LCCA results Table S1 presents the analytical results of the five models assessed for goodness of fit. The BIC model 2- and model 3-clusters were smaller, while the model 3-cluster was more suitable for screening high-risk study populations. According to the model 3-cluster, three clusters of maternal exposure were identified and the standardized values of the eight indicator variables were compared (Table 2). The risk factors among the three clusters are shown in Table S2. The numbers (percentages) of 1,000 PE patients distributed in the three clusters are as fol- lows: cluster 1 (low-risk), 320 (32.0%); cluster 2 (medium- risk), 368 (36.8%); and cluster 3 (high-risk), 312 (31.2%). st = Myocardial enzyme and electrolyte test. c Mye and Elec test = Myocardial enzyme and electrolyte test. c Mye and Elec test = Myocardial enzyme and electrolyte test. Baseline characteristics of PE patients BMC Pregnancy and Childbirth (2022) 22:687 Page 7 of 10 Fig. 2  Standardized means of the indicator variables for different risk clusters. Cluster 1 indicated the low-risk cluster, cluster 2 was the medium-risk cluster, and cluster 3 was the high-risk cluster Fig. 2  Standardized means of the indicator variables for different risk clusters. Cluster 1 indicated the low-risk cluster, clust cluster, and cluster 3 was the high-risk cluster Fig. 2  Standardized means of the indicator variables for different risk clusters. Cluster 1 indicated the low-risk cluster, cluster 2 was the medium-risk cluster, and cluster 3 was the high-risk cluster Each patient with PE was assigned to the most likely cluster based on the parameters constructed by the tri- chotomous model. Table S3 describes the standard- ized values and LCCA cluster assignments for selected patients with PE. higher than that in the low- and medium-risk popula- tions, respectively. The probability of persistent PHTN was 1.677 times higher in the medium- versus low-risk population, but the difference was statistically insignifi- cant (Table 3). Baseline characteristics of PE patients Cluster 3 exhibited the highest levels of the eight indi- cator variables. Thus, cluster 3 was characterized as a high-risk cluster. Cluster 2 did not differ from cluster 1 in maternal delivery age and medical history indicators and had a lower drug use indicator level, but higher lev- els of the remaining five indicators than cluster 1. Con- sequently, cluster 2 was characterized as a medium-risk cluster, while cluster 1 was a low-risk cluster. Figure  2 shows the normalized mean values of the eight indicator variables in the three clusters. There were statistically sig- nificant differences among the eight indicator variables in the high-, medium-, and low-risk clusters. Indicator variables and patients’ baseline characteristics are presented in Table 1. Except for the first and second indicator variables (maternal delivery age and MAP, respectively), all indicator variables consisted of 33 risk factors that were aggregated into a score for each indica- tor variable according to the presence or absence of the corresponding risk factor. The scoring standards (a series of yes/no questions) are shown in the Data collection sec- tion of the Methods section. Among the 1,000 PE subjects, 134 developed persis- tent PHTN and required transfer to a cardiovascular department for further evaluation and antihypertensive Table 2  Comparison of standardized characteristics among the three clusters of PE patients a Outcome = Adverse pregnancy outcome. b B and C test = Blood cell and coagulation test. c Mye and Elec test = Myocardial enzyme and electrolyte test. Indicator variables Low-risk cluster (cluster 1, 320) Medium-risk cluster (cluster 2, 368) High-risk cluster (cluster 3, 312) F P Multiple comparisons Maternal delivery age -0.05±0.88 -0.06±1.01 0.13±1.09 3.376 0.024 3 > 2 Mean arterial pressure -0.63±0.58 -0.06±0.88 0.72±1.01 203.341 < 0.001 3 > 2 > 1 Drugs use -0.43±0.47 -0.57±0.31 1.12±1.02 622.598 < 0.001 3 > 1 > 2 Medical history -0.24±0.87 -0.10±0.90 0.36±1.13 33.610 < 0.001 3 > 2, 3 > 1 Outcome a -0.71±0.56 0.25±1.06 0.43±0.89 160.049 < 0.001 3 > 2 > 1 B and C test b -0.21±0.87 -0.01±1.04 0.22±1.04 14.452 < 0.001 3 > 2 > 1 Liver and renal function -0.55±0.80 0.04±0.93 0.52±0.97 110.936 < 0.001 3 > 2 > 1 Mye and Elec test c -0.90±0.00 0.36±0.95 0.50±0.95 316.834 < 0.001 3 > 2 > 1 Table 2  Comparison of standardized characteristics among the three clusters of PE patients Li et al. Strengths and limitations Th d ’ The present study’s main strengths were as follows: 1) over 1,000 PE patients were screened; and 2) the LCCA assessment model had good accuracy, could be applied to evaluate persistent PHTN in PE patients, and may help establish early and precise guidance for managing per- sistent PHTN and reducing the risk of future CVD. The study also had some limitations, including: 1) its relatively shorter follow-up time duration (3 months); 2) potential bias caused by its relatively small cohort size; and 3) its retrospective study design inevitably leading to incom- plete medical history data, such as routine testing of blood pro-brain-type natriuretic peptide, anticardiolipin antibodies, proteinuria within the follow-up period, and blood lipids, especially in the week before delivery. This situation may prevent some of the predictive variables from being included in the model. In future studies, we may expand the cohort size, prolong the follow-up dura- tion, and perform prospective validation, which might compensate for the limitations of the current study. PE is a severe hypertensive disorder of pregnancy (HDP). The follow-up of HDP patients for 5-21 years after delivery demonstrated that they had a four-fold higher risk of developing PHTN than those without HDP [6, 8, 21]. One-year follow-up studies showed that PHTN occurred in 17-29% of patients with PE after discharge and that the probability of postpartum CVD was signifi- cantly higher in the PE group than in those with normal pregnancies; among those with PE, the BP returned to normal [12, 13, 22, 30]. The 13.4% incidence of PHTN among the 1,000 PE subjects here reflects the morbid- ity of persistent PHTN in Taiyuan City, Shanxi Province, China. An appropriate approach may help improve evaluations of PHTN severity and subsequent cardiovascular events in pregnant women after delivery. A combination of maternal and fetal parameters may reportedly detect the higher risk of PE [31]. The evaluation and management of PHTN requires a multidisciplinary approach [10]. Stud- ies of cardiovascular risk factors before versus after preg- nancy reported that half of the increased risk of future hypertension in women with PE is attributable to pre- natal risk factors [32]. LCCA, an unsupervised machine learning method, can be used to screen high-risk popu- lations. LCCA assumes that a heterogeneous group consists of a mixture of aggregates, and a latent class variable determines the optimal model. Discussionh The present study investigated the trends in BP changes and other cardiovascular risk factors in 1,000 PE patients at 3 months postpartum, mainly using the LCCA method. We found that these PE patients showed differ- ential risks of persistent PHTN and could be subdivided into high-, medium-, and low-risk clusters according to persistent PHTN severity as indicated by the eight indi- cator variables and the subordinate 35 risk factors shown The event rate of persistent PHTN was significantly higher in the high-risk cluster (24.68%) than in the medium-risk (10.05%) and low-risk (6.25%) clusters, and all statistical differences were significant, but the differ- ences in the latter two were statistically insignificant (Table 3). In addition, the probability of persistent PHTN in the high-risk population was 4.915 and 2.931 times Table 3  Event rates of PHTN in different clusters and associations of PHTN with risk clusters * P < 0.001 vs. low-risk cluster. # P < 0.001 vs. medium-risk cluster. △ P < 0.001. a Referent (ref) was low-risk cluster. b Referent (ref) was medium-risk cluster. Clusters N Event (%) χ2 OR (95% CI)a OR (95% CI)b High-risk cluster (cluster 3) 312 77 (24.68) *# 51.854 4.915 (2.92, 8.27) △ 2.931 (1.91, 4.49) △ Medium-risk cluster (cluster 2) 368 37 (10.05) 1.677 (0.95, 2.95) 1 (ref) Low-risk cluster (cluster 1) 320 20 (6.25) 1 (ref) Total 1000 134 (13.4) Li et al. BMC Pregnancy and Childbirth (2022) 22:687 Page 8 of 10 in Table 1. Overall, approximately 13.40% of patients with PE exhibited persistent PHTN at 3 months postpartum. The incidence of persistent PHTN in the high-risk cluster was nearly five and three times higher than those in the low-risk and medium-risk clusters, respectively. Those who developed persistent PHTN were usually older; had higher BMI and MAP values; used antihyperten- sive drugs before delivery; and had abnormal pregnancy features (including earlier onset of PE and higher abor- tion rate), larger birth numbers, longer intervals between births, and worse laboratory results. These findings may help clinicians and patients realize the severity of persis- tent PHTN, thus strengthening the importance of follow- up, timely intervention, and improved patient awareness of postpartum BP self-regulation. As a result, such moni- toring and treatment may reduce the incidence of long- term CVD in women. evaluating the risks of pregnancy-associated diseases in later life. Strengths and limitations Th d ’ With the help of goodness-of-fit evaluation metrics, LCCA may achieve “dimensionality reduction” at the variable level and clus- ter at the individual level. The latent classes extracted by LCCA both reflect the comprehensive effects of differ- ent influencing factors and aid the further analysis of the characteristics of different population clusters. No single current test can reliably predict the risk of PE. As a mul- tidisciplinary approach that integrates multiple risk fac- tors, LCCA has certain advantages over single tests for Conclusionh This study leveraged LCCA, screened a subpopula- tion of PE patients at high risk of persistent PHTN, and identified some related risk factors, including older age, higher BMI, earlier PE onset, longer interval between births, higher incidence of abnormal pregnancy, and worse laboratory results. This study’s findings may help clinicians realize the severity of persistent PHTN, encourage patients to actively seek early medical advice, facilitate the early identification of high-risk PE women, and encourage precise monitoring and management of postpartum BP. Discussionh LCCA has been used to identify high-risk popu- lations requiring clinical treatment and identify distinct subgroups within the clinical risk population [33, 34]. LCCA has also been used effectively to screen popula- tions at high risk of birth defects [35, 36]. Using LCCA, we successfully clustered the subpopulations of patients with PE in terms of persistent PHTN and identified the characteristics of each clustered population. This assess- ment model can be applied to assess the risk of persistent PHTN. BMI: Body mass index; BP: Blood pressure; CVD: Cardiovascular disease; HDP: Hypertensive disorder of pregnancy; LCCA​: Latent class cluster analysis; LPA: Latent profile analysis; MAP: Mean arterial pressure; OR: Odds ratios; PE: Preec‑ lampsia; PHTN: Postpartum hypertension. Ethics approval and consent to participate The study was approved by the Ethics Committee of First Hospital of Shanxi Medical University and the Institutional Review Board of Shanxi Children’s Hospital and Women Health Center, and followed the principles of the Decla‑ ration of Helsinki. The protocol numbers were 2020-K083 and KYYN-2021-002, respectively. Informed consents were obtained from all subjects and/or their legal guardian(s) in the study. 13. Levine LD, Nkonde-Price C, Limaye M, Srinivas SK. Factors associated with postpartum follow-up and persistent hypertension among women with severe preeclampsia. J Perinatol. 2016;36(12):1079–82. https://​doi.​org/​10.​ 1038/​jp.​2016.​137. 14. Edlow AG, Srinivas SK, Elovitz MA. Investigating the risk of hypertension shortly after pregnancies complicated by preeclampsia. Am J Obstet Gynecol. 2009;200(5):e60–2. https://​doi.​org/​10.​1016/j.​ajog.​2008.​10.​012. 14. Edlow AG, Srinivas SK, Elovitz MA. Investigating the risk of hypertension shortly after pregnancies complicated by preeclampsia. Am J Obstet Gynecol. 2009;200(5):e60–2. https://​doi.​org/​10.​1016/j.​ajog.​2008.​10.​012. Availability of data and materials The original datasets of the present study are available from the correspond‑ ing author on reasonable request. 11. Benschop L, Duvekot JJ, Versmissen J, van Broekhoven V, Steegers E. Roeters van Lennep JE. Blood Pressure Profile 1 Year After Severe Preec‑ lampsia. Hypertension. 2018;71(3):491–8. https://​doi.​org/​10.​1161/​HYPER​ TENSI​ONAHA.​117.​10338. 11. Benschop L, Duvekot JJ, Versmissen J, van Broekhoven V, Steegers E. Roeters van Lennep JE. Blood Pressure Profile 1 Year After Severe Preec‑ lampsia. Hypertension. 2018;71(3):491–8. https://​doi.​org/​10.​1161/​HYPER​ TENSI​ONAHA.​117.​10338. Declarations 12. Chen KH, Chen LR. Provoking factors for postpartum chronic hyperten‑ sion in women with preceding gestational hypertension/preeclamp‑ sia: A longitudinal cohort study of 22,798 pregnancies. Int J Med Sci. 2020;17(4):543–8. https://​doi.​org/​10.​7150/​ijms.​39432. 12. Chen KH, Chen LR. Provoking factors for postpartum chronic hyperten‑ sion in women with preceding gestational hypertension/preeclamp‑ sia: A longitudinal cohort study of 22,798 pregnancies. Int J Med Sci. 2020;17(4):543–8. https://​doi.​org/​10.​7150/​ijms.​39432. Acknowledgements The authors thank FHSMU and SCWHC for providing the registration informa‑ tion of patients. 6. Bellamy L, Casas JP, Hingorani AD, Williams DJ. Pre-eclampsia and risk of cardiovascular disease and cancer in later life: systematic review and meta-analysis. BMJ. 2007;335(7627):974. https://​doi.​org/​10.​1136/​bmj.​ 39335.​385301.​BE. Supplementary Information Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12884-​022-​05003-4. Additional file 1: Table S1. Goodness-of-fit indicators for the five differ‑ ent class models. Table S2. Comparison of risk factors among the three clusters of PE patients. Table S3. Class assignment for a minority of PE patients. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12884-​022-​05003-4. 3. Steegers EA, von Dadelszen P, Duvekot JJ, Pijnenborg R. Pre-eclampsia. Lancet. 2010;376(9741):631–44. https://​doi.​org/​10.​1016/​S0140-​6736(10)​ 60279-6. Additional file 1: Table S1. Goodness-of-fit indicators for the five differ‑ ent class models. Table S2. Comparison of risk factors among the three clusters of PE patients. Table S3. Class assignment for a minority of PE patients. 4. Khan KS, Wojdyla D, Say L, Gülmezoglu AM, Van Look PF. WHO analysis of causes of maternal death: a systematic review. Lancet. 2006;367(9516):1066–74. https://​doi.​org/​10.​1016/​S0140-​6736(06)​ 68397-9. 5. Duley L. The global impact of pre-eclampsia and eclampsia. Semin Peri‑ natol. 2009;33(3):130–7. https://​doi.​org/​10.​1053/j.​sempe​ri.​2009.​02.​010. Consent for publication Not applicable (No such identifying information/images of participants are provided). Not applicable (No such identifying information/images of participants are provided). 15. Berks D, Steegers E, Molas M, Visser W. Resolution of hypertension and proteinuria after preeclampsia. Obstet Gynecol. 2009;114(6):1307–14. https://​doi.​org/​10.​1097/​AOG.​0b013​e3181​c14e3e. 15. Berks D, Steegers E, Molas M, Visser W. Resolution of hypertension and proteinuria after preeclampsia. Obstet Gynecol. 2009;114(6):1307–14. https://​doi.​org/​10.​1097/​AOG.​0b013​e3181​c14e3e. 1. Brown MA, Magee LA, Kenny LC, et al. Hypertensive Disorders of Preg‑ nancy: ISSHP Classification, Diagnosis, and Management Recommenda‑ tions for International Practice. Hypertension. 2018;72(1):24–43. https://​ doi.​org/​10.​1161/​HYPER​TENSI​ONAHA.​117.​10803. Author details 17. Traylor J, Chandrasekaran S, Limaye M, Srinivas S, Durnwald CP. Risk perception of future cardiovascular disease in women diagnosed with a hypertensive disorder of pregnancy. J Matern Fetal Neonatal Med. 2016;29(13):2067–72. https://​doi.​org/​10.​3109/​14767​058.​2015.​10815​91. 1 Key Laboratory of Cellular Physiology at Shanxi Medical University, Ministry of Education, Taiyuan, China. 2 Department of Physiology, Shanxi Medical University, Taiyuan, China. 3 Department of Critical Care Medicine, The First Hospital of Shanxi Medical University, Taiyuan, China. 4 Department of Mater‑ nity, The First Hospital of Shanxi Medical University, Taiyuan, China. 5 Depart‑ ment of Obstetrics Gynecology, Shanxi Children’s Hospital and Women Health Center, Taiyuan, China. 6 Department of Clinical Laboratory, Shanxi Children’s Hospital and Women Health Center, Taiyuan, China. 7 Division of Health Statis‑ tics, School of Public Health, Shanxi Medical University, Taiyuan, China. 18. Mito A, Arata N, Sakamoto N, et al. Present status of clinical care for postpartum patients with hypertensive disorders of pregnancy in Japan: findings from a nationwide questionnaire survey. Hypertens Pregnancy. 2015;34(2):209–20. https://​doi.​org/​10.​3109/​10641​955.​2014.​10019​02. 19. Perales A, Delgado JL, de la Calle M, et al. sFlt-1/PlGF for prediction of early-onset pre-eclampsia: STEPS (Study of Early Pre-eclampsia in Spain). Ultrasound Obstet Gynecol. 2017;50(3):373–82. https://​doi.​org/​10.​1002/​ uog.​17373. Received: 20 January 2022 Accepted: 25 August 2022 Received: 20 January 2022 Accepted: 25 August 2022 Received: 20 January 2022 Accepted: 25 August 2022 20. von Dadelszen P, Payne B, Li J, et al. Prediction of adverse maternal outcomes in pre-eclampsia: development and validation of the fullPIERS model. Lancet. 2011;377(9761):219–27. https://​doi.​org/​10.​1016/​S0140-​ 6736(10)​61351-7. Competing interests 16. Breetveld NM, Ghossein-Doha C, van Kuijk S, et al. Cardiovascular disease risk is only elevated in hypertensive, formerly preeclamptic women. BJOG. 2015;122(8):1092–100. https://​doi.​org/​10.​1111/​1471-​0528.​13057. The authors declare that they have no conflict of interests in this study. Authors’ contributions Y.Y.L., H.L.Y., L.Y.H. and J.M.C.: organized and designed study. Y.Y.L., J.C., J.L.L., J.Y.Z. and Y.M.L.: collected data. Y.B.Z.: provided suggestions of study design and supervised data analysis. Y.Y.L. and C.Z.: conducted data analysis. Y.Y.L.: wrote the manuscript. J.M.C., D.P.W., H.L., Y.F.H. and R.Z.: revised manuscript. All authors read and approved manuscript submission. 7. Chen CW, Jaffe IZ, Karumanchi SA. Pre-eclampsia and cardiovascular disease. Cardiovasc Res. 2014;101(4):579–86. https://​doi.​org/​10.​1093/​cvr/​ cvu018. 7. Chen CW, Jaffe IZ, Karumanchi SA. Pre-eclampsia and cardiovascular disease. Cardiovasc Res. 2014;101(4):579–86. https://​doi.​org/​10.​1093/​cvr/​ cvu018. 8. Leon LJ, McCarthy FP, Direk K, et al. Preeclampsia and Cardiovascular Dis‑ ease in a Large UK Pregnancy Cohort of Linked Electronic Health Records: A CALIBER Study. Circulation. 2019;140(13):1050–60. https://​doi.​org/​10.​ 1161/​CIRCU​LATIO​NAHA.​118.​038080. 8. Leon LJ, McCarthy FP, Direk K, et al. Preeclampsia and Cardiovascular Dis‑ ease in a Large UK Pregnancy Cohort of Linked Electronic Health Records: A CALIBER Study. Circulation. 2019;140(13):1050–60. https://​doi.​org/​10.​ 1161/​CIRCU​LATIO​NAHA.​118.​038080. Abbreviations BMI: Body mass index; BP: Blood pressure; CVD: Cardiovascular disease; HDP: Hypertensive disorder of pregnancy; LCCA​: Latent class cluster analysis; LPA: Latent profile analysis; MAP: Mean arterial pressure; OR: Odds ratios; PE: Preec‑ lampsia; PHTN: Postpartum hypertension. Page 9 of 10 Li et al. BMC Pregnancy and Childbirth (2022) 22:687 Li et al. BMC Pregnancy and Childbirth 2. ACOG Practice Bulletin No 202. Gestational Hypertension and Preeclamp‑ sia. Obstet Gynecol. 2019;133(1):1. https://​doi.​org/​10.​1097/​AOG.​00000​ 00000​003018. Funding Thi d This study was financed by Shanxi “1331” Project Quality and Efficiency Improvement Plan (1331KFC) and partially by Key Medical Science and Tech‑ nology Program of Shanxi Province (2020XM01) and National Natural Science foundation of China (82170523). 9. Lowe SA, Bowyer L, Lust K, et al. The SOMANZ Guidelines for the Manage‑ ment of Hypertensive Disorders of Pregnancy 2014. Aust N Z J Obstet Gynaecol. 2015;55(1):11–6. https://​doi.​org/​10.​1111/​ajo.​12253. 9. Lowe SA, Bowyer L, Lust K, et al. The SOMANZ Guidelines for the Manage‑ ment of Hypertensive Disorders of Pregnancy 2014. Aust N Z J Obstet Gynaecol. 2015;55(1):11–6. https://​doi.​org/​10.​1111/​ajo.​12253. y p g j 10. Sibai BM. Etiology and management of postpartum hypertension- preeclampsia. Am J Obstet Gynecol. 2012;206(6):470–5. https://​doi.​org/​ 10.​1016/j.​ajog.​2011.​09.​002. 10. Sibai BM. Etiology and management of postpartum hypertension- preeclampsia. Am J Obstet Gynecol. 2012;206(6):470–5. https://​doi.​org/​ 10.​1016/j.​ajog.​2011.​09.​002. References 1 B M Orri M, Pingault JB, Rouquette A, et al. Identifying affective personal‑ ity profiles: A latent profile analysis of the Affective Neuroscience Personality Scales. Sci Rep. 2017;7(1):4548. https://​doi.​org/​10.​1038/​ s41598-​017-​04738-x. 34. Orri M, Pingault JB, Rouquette A, et al. Identifying affective personal‑ ity profiles: A latent profile analysis of the Affective Neuroscience Personality Scales. Sci Rep. 2017;7(1):4548. https://​doi.​org/​10.​1038/​ s41598-​017-​04738-x. 35. Cao H, Wei X, Guo X, et al. Screening high-risk clusters for developing birth defects in mothers in Shanxi Province, China: application of latent class cluster analysis. BMC Pregnancy Childbirth. 2015;15:343. https://​doi.​ org/​10.​1186/​s12884-​015-​0783-x. 36. Pei L, Zeng L, Zhao Y, Wang D, Yan H. Using latent class cluster analysis to screen high risk clusters of birth defects between 2009 and 2013 in Northwest China. Sci Rep. 2017;7(1):6873. https://​doi.​org/​10.​1038/​ s41598-​017-​07076-0. References 1 B M 21. Stuart JJ, Tanz LJ, Cook NR, et al. Hypertensive Disorders of Preg‑ nancy and 10-Year Cardiovascular Risk Prediction. J Am Coll Cardiol. 2018;72(11):1252–63. https://​doi.​org/​10.​1016/j.​jacc.​2018.​05.​077. 22. Neuman RI, Figaroa A, Nieboer D, et al. Angiogenic markers during preeclampsia: Are they associated with hypertension 1 year postpartum. Page 10 of 10 Li et al. BMC Pregnancy and Childbirth (2022) 22:687 Pregnancy Hypertens. 2021;23:116–22. https://​doi.​org/​10.​1016/j.​preghy.​ 2020.​11.​011. Pregnancy Hypertens. 2021;23:116–22. https://​doi.​org/​10.​1016/j.​preghy.​ 2020.​11.​011. 23. Funderburk JS, Maisto SA, Sugarman DE, Wade M. The covariation of multiple risk factors in primary care: a latent class analysis. 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Notes on evaluating the ecology of rehabilitation praxis
Scandinavian journal of disability research
2,003
cc-by
5,767
By Thomas A. Schwandt Abstract: Rehabilitation services are typically regarded as an intervention, delivered by a professional service provider, designed to achieve the outcome of functional improvement in the life of the client or patient The primary evaluation question is whether that intervention is an effective and efficient means to the given end of functional improvement I suggest an alternative way of thinking of both rehabilitation and evaluation: The phrase 'ecology of rehabilitation praxis' is used to emphasize that rehabilitation is a matter of a situated, lived, technical, moral, and political interaction of people with their environment, with each other, and with the artifacts of their environment Three conceptual tools are discussed for understanding the ecology of rehabilitation praxis: community-based practice philosophy, community of practice, and the discursive nature of a practice. When rehabilitation is conceived as ecology, and not simply a treatment, then a different way of thinking about evaluation is required. The provision of rehabilitation as a treatment, regardless of the specific nature of individual rehabilitation services, involves three, and possibly four, kinds of care services which are thought to contribute to the outcome of functional improvement: (a) technical care—applying medical science and technology; (b) interpersonal care— treating patients/clients with con- sideration, respecting patient/client autonomy, listening to patient/client concerns, and so on; (c) amenities of care—providing services in a clean, comfortable, and aesthetically pleasing setting (Eldar, 2000). In the current ideology of providing medical services, The provision of rehabilitation as a treatment, regardless of the specific nature of individual rehabilitation services, involves three, and possibly four, kinds of care services which are thought to contribute to the outcome of functional improvement: (a) technical care—applying medical science and technology; (b) interpersonal care— treating patients/clients with con- sideration, respecting patient/client autonomy, listening to patient/client concerns, and so on; (c) amenities of care—providing services in a clean, comfortable, and aesthetically pleasing setting (Eldar, 2000). In the current ideology of providing medical services, Notes on Evaluating the Ecology of Rehabilitation Praxis A common approach to evaluating rehabilitation services follows the logic of a biomedical model of clinical research. Rehabilitation services are regarded as a treatment or intervention, delivered by some professional service provider, designed to achieve the particular outcome or effect of functional improvement. The aim of evaluation is to determine whether, in fact, the treatment is producing that effect, and if so, to what degree. SJDR - Volume 5, No.l - 2003 93 Thomas A. Schwandt interpersonal care, the amenities of care, and the imparting of confidence to patients, but this is considered to be largely a methodological problem, not a serious threat to the logic of the model. we might even add a fourth kind of 'care' that involves imparting competence and confidence to disabled individuals to participate in their rehabilitation. These four attributes of the professional service are regarded as measurable (and ideally, quantifiable). we might even add a fourth kind of 'care' that involves imparting competence and confidence to disabled individuals to participate in their rehabilitation. These four attributes of the professional service are regarded as measurable (and ideally, quantifiable). This way of thinking of the relationship between evaluation and rehabilitation focuses our attention on the efficacy and strength of the intervention. Evaluation here is a technical task: It is a matter of using research tools to assess the means-end relationship or to measure performance against a standard. In either case, the evaluator plays a role similar to that of the clinical medical researcher, using the procedures of social scientific research to examine this relationship between the provision of rehabilitation services and the outcome of functional improve- ment. To briefly summarize: The object of interest to evaluation in the treatment-effect model is the provision of rehabilitation sen ice as an inter- vention into the lives of patients/ clients. The primary question about that object is whether it is an effective means to the given end of functional improvement. To evaluate the relationship between treatment (care services) and outcome, two approaches are employed. In one approach, different kinds of treatment are compared to one another to determine whether the outcome is attributable to the treatment. For example, the treatment-effect relation- ship can be examined in an institutional setting versus a community-placed setting. Community-Based Practice Community-Based Practice In my judgment, the Norwegian way of approaching the provision of health care services to its citizens reflects a general philosophy of rehabilitation as community-based practice (NOU, 1998, 2001; Sosial- og Helsedepartementet, n.d.). Community-based practice is not simply a strategy for relocating services from centralized institutional settings to more local community-placed settings; rather, it is a way of redefining the technical, political, and moral dimensions of the very idea of rehabilitation. It is a shorthand way of referring to a different ecology of relations between pro- fessionals, citizens, and society. This way of thinking is characterized by several key ideas (Kendall, Buys, and Lamer, 2000): Notes on Evaluating the Ecology of Rehabilitation Praxis These kinds of comparison are relevant because rehabilitation services are delivered in complicated contexts where resources of time and money, professional competence, types of patient problems, and so on vary and might affect the treatment-outcome relationship. Likewise, follow-up studies can be conducted to determine whether the outcome of functional improvement is long-lasting; different combinations of professional services can be evaluated for their contribution to the outcome, and so on. In what follows. I discuss how we might go about thinking of evaluating not an object but a lived, situated practice; something that is always unfolding as a performance, always in the process of being accomplished or achieved. I have chosen the phrase ecology of rehabilitation praxis to emphasize the fact that the provision of In a second approach, standards are developed for each attribute and performance is measured against the standard. Of course, standards for technical care are often more easily established, and more precisely measurable, than standards for SJDR - Volume 5, No.l - 2003 94 Evaluating the Ecology of Rehabilitation Praxis rehabilitation services is a matter of the situated, lived interaction of people with their environment, with each other, and with the artifacts of their environment. (One might say the principal difference here is between rehabilitation as an outcome and rehabilitating as a process.) Moreover, that interaction has technical, moral, and political dimensions. My central thesis is that when rehabilitation is conceived as ecology, and not simply an object, then a different way of thinking about evaluation is required. Thus, my purpose is to two-fold: First, I sketch an understanding of the ecology of rehabilitation praxis in Norway, and second discuss a way of thinking about and conducting evaluation that matches this ecology. ecology of praxis is from evaluating it as an object, I explore briefly three conceptual tools for understanding the ecology of rehabilitation praxis: com- munity-based practice ideology, com- munity of practice, and the discursive nature of a practice. Towards an Ecology of Praxis Model In a model assuming the ecology of rehabilitation praxis, evaluation focuses on the discursive, positioned enactment of rehabilitation in all of its com- plexity1. The primary questions addres- sed in this way of thinking about evaluation are not technical, means-end questions, but value-rational questions about the ways rehabilitation practices are and should be organized and conducted. These kinds of questions simultaneously take up issues of both the means and ends of a practice. First, it emphasizes the competence and participation of citizens in decisions influencing their existence as members of society. This is a complex idea that holds together two potentially paradoxical notions. On the one hand, it emphasizes the empowerment of the individual citizen; the right and capacity of an individual to be an independent, autonomous participant in all aspects of To understand how different the matter of evaluating rehabilitation as an SJDR - Volume 5, No.l - 2003 95 Thomas A. Schwandt social life, and capable of mastery and control over her or his own life. The notion of citizen is key here because it marks a shift away from thinking of the individual who receives a particular kind of social or health service as a consumer or buyer. Empowerment also refers to something like a state of being, a capacity that all citizens have—not some right or capability that is bestowed upon them by others. To be empowered is to be able to exercise one's capacity for self-determination. On the other hand, the key to such empowerment is connectedness and interdependence with others in a local community. Empowerment thus does not mean that an individual is left to her or his own devices; rather that he or she works together, in consort with, other members of a local community. custodial and exclusively restorative approach to rehabilitation. Rehabilitation becomes not a matter of 'healing' the sick but of providing services that allow people to realize their capacities to develop. Fourth, the philosophy promotes the development of a different identity and way of working for rehabilitation professionals. A common fear is that this philosophy of community-based practice challenges, and even threatens to destroy, the identity of medical care and health service professionals. Towards an Ecology of Praxis Model On the contrary, what is emphasized here is the creation of a new, additional identity, new models for the use of best practice technical medical care, and a new ethic of professional-citizen relations for those who collectively consider themselves rehabilitation professionals. Second, this philosophy emphasizes inclusion of all citizens within a community. Inclusion here refers to the shift from a custodial and medical care model of health services design and delivery to a community development model. This is more than a notion of community-placed services for it refers to the combined efforts of disabled people, their families and communities, and the appropriate professional service providers to orchestrate rehabilitation. Community of Practice y f A second conceptual tool that helps us develop an understanding of the ecology of rehabilitation praxis is drawn from the work of Etienne Wenger (1998). A community of practice is a social group engaged in the sustained pursuit of a shared purpose. The group is chara- cterized by mutual engagement; it is a negotiated enterprise; and, it draws on a shared repertoire of resources (routines, sensibilities, artifacts, vocabulary, styles, and so on). Practice is a way of negotiating meaning through social action. Wenger identifies five chara- cteristics of a community of practice. Third, the philosophy challenges the model of restorative care. Community- based practice signals a holistic and individualized orientation to rehabili- tation and a move away from a SJDR - Volume 5, No.l - 2003 96 Evaluating the Ecology of Rehabilitation Praxis to reflect within themselves a deep understanding of different ways of seeing a phenomenon such as rehabilitation. In fact the closer one is to the center of a community of practice (i.e., the more one is considered a full member) the less likely one is to develop a heterogeneous and varied understanding of different ways to think about rehabilitation. Each community, be it that of primary care physicians, nurses of various kinds, physiotherapists, social workers, municipal officials, and so on has its own way of seeing and its own tools and procedures for making sense of the phenomenon. Thus, part of the pedagogical task of understanding and evaluating the ecology of rehabilitation praxis is learning how to educate each community of practice in the views of others. Members of such a community: (a) interact more intensively with and know more about others in the community than those outside the community; (b) hold actions accountable more to the community's joint enterprise than to some other enterprise; (c) are more able to evaluate actions of other members of their community than the actions of those outside the community; (d) draw on locally produced resources and artifacts to negotiate meaning more so than resources and artifacts imported from outside the community; and, (e) share an identity. Community of Practice Members of such a community: (a) interact more intensively with and know more about others in the community than those outside the community; (b) hold actions accountable more to the community's joint enterprise than to some other enterprise; (c) are more able to evaluate actions of other members of their community than the actions of those outside the community; (d) draw on locally produced resources and artifacts to negotiate meaning more so than resources and artifacts imported from outside the community; and, (e) share an identity. Communities of practice are local and discrete and not coterminous with professional groups. For example, the primary care physicians in a particular hospital share a common institutional environment, have a shared history, and interact with one another around important issues they face regarding patient care in that setting. While they share the professional identity and goal of physician and thus are broadly related to other physicians, they share membership in this particular, discrete, local community of practice. Taken collectively, these various discrete communities of practice constitute a constellation of communities of practices. Precisely because multiple communities of practice are involved in rehabilitation as an orchestrated social process there will inevitably be complex value conflicts and conflicting practice norms. While these norms and practice vocabularies do pose limitations on negotiation, a full understanding of the ecology of rehabilitation practice suggests that what might be the object of evaluation is not simply the provision of a professional treatment or intervention but the development of different models for professional work and the negotiation of an ethic of relationship between service providers and between service providers and citizens (Lindqvist and Tamm, 1999). This notion of community of practice is significant for understanding the ecology of rehabilitation praxis. Local communities of practice are not likely SJDR - Volume 5, No.l - 2003 97 Thomas A. Schwandt Thomas A. Schwandt In addition, evaluation here would involve examining how, and how effective, professionals are in opening their respective discourses to public scrutiny. Evaluation focused on discourse helps us see the importance of the provision of rehabilitation as an ethical-political relationship, not merely the delivery of a medical service—a particular way of valuing others and of envisioning the responsibilities we have towards them. An Evaluation Approach for the Ecology of Rehabilitation Praxis I turn now to the implications of this focus on the ecology of rehabilitation praxis for evaluation. Specifically, I discuss three ideas: 1) a different way of thinking about what evaluation is, 2) a different way of thinking about the questions that define the purpose of an evaluation, 3) a different way of thinking about the responsibilities, roles, and obligations of parties (stakeholders) in an evaluation. Regarded as a multifaceted set of discourses, rehabilitation is not merely a service provided by a professional but a way of seeing and thinking about phenomena (Solvang, 2000). A great deal of what is a stake in evaluating holistic rehabilitation praxis is learning how the new discourse of holistic rehabilitation praxis is intersecting with medical discourses of normalization or normality that have long dominated ways of thinking about what it means to be disabled or in need of rehabilitation. Discourse of a Practice f One additional analytic tool for making sense of the ecology of rehabilitation praxis is that of the discourse of a practice. A discourse is an institutionalized use of language and language-like sign systems that takes place at cultural, political, and professional levels. It is a public process through which meanings are progressively and dynamically achieved. Discourses can compete with one another and create distinct and incompatible visions of reality (Davies andHarre, 1990). Community of Practice Again, the evaluation task here is pedagogical, not technical; for it will involve helping various members of disparate communities of practice (nurses, physicians, social workers, physiotherapists, and so on) constitute another community of practice—a community of rehabilitation professionals organized around the joint enterprise of community-based rehabilitation practice. Thinking Differently About Evaluation The evaluation logic sketched at the beginning of this paper would have us view the social world as in some way at our disposal in a manner similar to how the physical sciences regard the natural world. What are evaluated are object- like entities (in the evaluation literature there is a special name for these kinds SJDR - Volume 5, No.l - 200S 98 Evaluating the Ecology of Rehabilitation Praxis of objects—evaluands) with properties that can be observed, described, classified, and measured. These objects (an individual educational plan, a particular social service, a medical treatment, and so on) are regarded as tangible such that it becomes possible to talk of observing their outcomes, documenting their effects, tracking changes in their behavior, as well as auditing their performance. Evaluation serves as a means—a tool, a procedure—for generating two types of knowledge about these objects: theoretical knowledge of cause and effect, or technical knowledge of effective means. doing the right thing and doing it well, their various interpretations are almost always contested, never certain, and forever subject to reinterpretation. So conceived, evaluation activity displays several fundamental characteristics: First, it is situated or embedded—a matter of specific people in distinct settings at particular times struggling to reach an understanding (an interpretation) of the value of their actions (taken or about to be taken). It is a matter of responsiveness— 'being with' or in relation to the activities one evaluates. Second, evaluation is thus a kind of accomplishment; it is something that is continuously carried out (and not some- thing simply achieved) like parenting, teaching, counseling, and so on. It is corporeal and takes up ways of acting— gestures, demeanor, style, habitations, recognition, intimacy (or the lack thereof), and so on. Now consider evaluation as another way we are in the world. Let us begin with the assumption that all evaluation activity is fundamentally engaged, concrete, indigenous, and experienced (as Erfahrung) as opposed to abstract, disengaged, or somehow removed from the erratic, contentious, uncertain, ambiguous, and generally untidy character of life itself. This means that all judgments of the value (merit, worth, or significance) of human action are undertaken within specific places and circumstances where these judgments both reflect and depend upon the thinking (including socio-economic, political, and moral values) and acting of the specific parties involved at the distinct time and place in question. Thinking Differently About Evaluation Moreover, in the particular circum- stances where some group of people is attempting to decide whether they are Third, this accomplishment is a lived reality and thus it is marked by disorder, playfulness, spontaneity, disagreement, ambiguity, uncertainty, situational specificity, contingency, a good deal of (some might say, endless) criticism, and often something like radical diversity of views about what is the right thing to do or whether one has done the right thing well. Fourth, evaluation is performed in the sense that the evaluation decisions that rehabilitation professionals and SJDR - Volume 5, No.l - 2003 99 Thomas A. Schwandt In this kind of dialogue, all parties in the ecology of rehabilitation praxis— professionals, disabled individuals and their families, and municipal officials— must learn that dialogue proceeds pedagogically and has the aim of understanding self and other, rather than unfolding technically as a method for resolving difference. Dialogue is a situated practice that can have several kinds of outcomes including: (a) not agreement, but a common under- standing in which parties do not agree but establish common meanings in which to discuss their differences; (b) not a common understanding, but an understanding of differences in which the parties do not entirely bridge these differences, but through analogies of experience or other indirect translations can understand, at least in part, each other's position; (c) little understanding, but a respect across differences, in which parties do not fully understand one another, but by each seeing that the other has a thoughtful, conscientious position, they can come to appreciate and respect even positions they disagree with. This idea of an evaluation "dialogue across differences" embraces difference, diversity, and the messiness of human life rather than seeking to resolve it (Burbules and Rice, 1991). municipal officials make (about what it is right to do) are not simply intellectual choices but embodied conditions— decisions taken make, in part, who and what the decision makers are. The lived reality of determining the merit, worth, or significance of our human actions reveals it to be a self-constitutive activity. Fifth, evaluation is dialogical, but in a special sense of that word. The dialogue that transpires among stakeholders and between stakeholders and the evaluator is not a neutral communicative proce- dure concerned only with deliberating rival claims. Dialogue in evaluation is typically presented as a deliberative procedure concerned with a search for knowledge, understanding, and (ideally at least) agreement. Thinking Differently About Evaluation In the way of thinking about evaluation presented here, dialogue is not regarded as means to "domesticate difference," that is, to regarding differences simply as variations in (diverse) viewpoints that are potentially reconcilable through deliberation (Burbules and Bruce, 2001). Rather, dialogue is regarded as embracing differences, even those that resist accommodation or assimilation into an agreement, and is understood as a situated practice (not a neutral procedure) implicated by the particulars of who, what, where, when, and how the dialogue takes place. Dialogue in evaluation is thus a moral-practical encounter of embodied agents not just of ideas or points of view. Understood in this way, the very reality of evaluation—its significance as a human undertaking—is grasped not in the application of some procedure or method to determine the effectiveness of a treatment, nor in distanced, abstract SJDR - Volume 5, No.l - 2003 100 Evaluating the Ecology of Rehabilitation Praxis always "put in the way" of evaluation experiences. We always already find ourselves in a process of valuing, of deciding whether we are doing the right thing and doing it well. contemplation of the properties and behavior of some evaluand—some object-like entity such as a program or a project—but in its very enactment as a moral-political practice. Evaluation is a performative activity; what it means unfolds in its very doing or actualization. This doing is invariably marked by ambiguity, contingency, circumstantiality, and always-contested ways of what it means to do the right thing. In facing this situation, the individual (or the group), must learn to embrace the lived reality of diverse, contested, messy evaluative decision-making. The assumption is that there is inherent, inescapable, fundamental ambiguity, diversity, and uncertainty in life. The first move here is to welcome and profoundly respect this state of affairs (including social differences, local traditions, professional obligations, and so forth) instead of working so hard to overcome, eradicate, or control these facets of every day life. At issue here is not simply the liberal tolerance of or deference toward diverse points of view, but the idea of welcoming the very idea of difference. Evaluation here means learning to struggle with questions of value rationality: Where am I (are we) going? Is this desirable? What should be done? Who gains or loses by this action? (Flyvbjerg, 2001). Thinking Differently About Evaluation They are not invited to embrace and dwell in the divergence of views as an opportunity for self-transformation and new understanding, but to reach for knowledge and procedures that will assist them in eliminating their confusion and the ambiguity and diversity of views that characterize the situation in which they find themselves. In sum, when evaluation is cast in exclusively instrumentalist terms, then individuals are led to frame the evaluative decisions they face as technical problems to be solved. They are encouraged to think that what is at issue is whether they have either the Thinking Differently About Evaluation Or, we might say the fundamental evaluative questions here are: What is it right to do and good to be in this situation? These questions are not answerable in terms of means-to- end thinking, but require a kind of thinking and acting that are inseparable from being. They require knowledge of the kind Aristotle called phronesis (wise judgment). Therefore, evaluation is less a deliberative human enterprise concerned with acquiring knowledge for the purpose of having power and control, and more like a process that happens to human beings—an event in which we must learn to participate, not a tool we must learn to apply. Evaluation is not a process in which we are in control of means to an end, but a process which itself achieves our sense of ourselves as individuals (and a society) of a particular kind. We are This way of thinking about the activity of evaluation also requires owning up to the moral and political accountability of our decisions and actions. Instrumentalist conceptions of evaluation as a means to an end encourage us to mistake technical proficiency for moral responsibility. When evaluation is conceived instrumentally, all the problems we face in deciding about how to deal with questions of what should be done (which is the central problem of providing the 'right' rehabilitation services) are regarded as technical problems that can be solved either by acquiring special knowledge that we SJDR - Volume 5, No.l - 2003 101 Thomas A. Schwandt lack or a special method or procedure of which we are unaware or only poorly follow or execute. right knowledge or procedure at hand so that they can clear up errors in their faulty reasoning. However, efforts to enhance practitioners' ability to manage and control their practice, over time, alienate them from the responsibility that inevitably befalls them for the moral-practical decisions they must make. When evaluation activity is conceived solely as a means to an end, practitioners are not encouraged to personally grapple with the inherent ambiguity, the diversity of opinion, and the contingencies that characterize everyday life as they, either individually or collectively, attempt to make the right evaluative decision at this time, in this place, under these circumstances, facing this particular person. They are not encouraged to accept responsibility for their decisions in the face of the inherent ambiguity of moral-political life. New Focusing Questions for Evaluation As noted above, the sole focusing question of a treatment-effects model of evaluation is the outcome of functional improvement—the primary issue is the efficacy of treatment. It should be apparent that although communities of rehabilitation professionals cannot ignore this question, evaluation of the ecology of rehabilitation praxis requires attending to at least three other central questions: • In what ways are new work models being developed in communities of rehabilitation professionals? • How are members of these communities developing a sense of mutual engagement around these new models? • What new routines, sensibilities, vocabularies, and so on are being developed to support these new models? • How is the issue of the ethics of relation (including notions of professional obligations and responsibilities, citizen rights, and state responsi- bilities) being discussed in the development of communities of rehabilitation practice? • How are issues of the independence and interdependence of citizens fostered in the actions of these communities? SJDR - Volume 5, No.l - 2003 102 Evaluating the Ecology of Rehabilitation Praxis officials become responsible for contributing to the collective, shared notion of the public interest or good. They are responsible for brokering existing communities of practice, fostering the development of a new community of practice of rehabilitation professionals, and serving as catalysts for community engagement. This is fundamentally an evaluative undertaking for it requires that these officials critically reflect upon their actions and justify their choices as appropriate and effective. Responsibilities of Stakeholders in an Evaluation It is typical to regard stakeholders in an evaluation simply as sources of information that the evaluator draws upon to prepare her or his judgment of the effect of a treatment. Evaluation is regarded as the responsibility of the evaluator, and stakeholders simply await the evaluator's final report. In an evaluation approach that matches the idea of ecology of rehabilitation praxis, roles and responsibilities of people involved in an evaluation change dramatically. Most significantly, all participants are responsible for evaluation, albeit in different ways. Professionals in discrete communities of practice face a daunting two-fold evaluative task: First, they must look within their own discrete communities and ask themselves the following evaluative questions: a) How do we understand the concepts disability and rehabilitation! b) What is required for us listen to the voices of disabled people and simultaneously to foster their independence and interdependence? c) How do relations of privilege and power influence our work and our sense of responsibilities to disabled people? d) How are we complicit in reproducing disabling images, language, and discriminations? Second, they must look outside their own respective communities of practice to learn how others answer the same questions. Here, their responsibilities intersect with those of municipal officials responsible for brokering the dialogue between communities of practice. Disabled citizens seeking various kinds of rehabilitation services must realistically assess their needs, ask themselves how they will achieve the mix of independence and interdependence necessary to their self- realization, decide how they will meaningfully participate with professionals and others in the efforts to meet their needs, decide when acting up or out is more effective than acting in concert, and so on. Officials in municipalities and county municipalities face new challenges to their role as public officials: They are now being called upon to serve rather than steer—to help citizens articulate and meet their shared interests rather than attempt to control or steer society in new directions (Denhardt and Denhardt, 2000). Municipal and county SJDR - Volume 5, No.l - 2003 103 Thomas A. Schwandt Finally, the evaluator in this process has the three-fold responsibility of teaching this way of thinking about evaluation, facilitating the examination of value-rational questions within different stakeholder groups, and orchestrating this evaluative undertaking across these groups. The evaluator would most likely produce a case study narrative report of this activity in a particular locale. Responsibilities of Stakeholders in an Evaluation Others can then use this case study as a pedagogical aid in examining their own specific undertakings. services by helping us think more carefully and critically about the ends and means of what we do in the name of rehabilitation, then statistical infor- mation alone will hardly be adequate for this task. We need a way of thinking about evaluation that locates this activity centrally in everyday life and communities of praxis and that helps all of us to develop the capacity to reflect on whether we are doing the right thing and doing it well. Of course, as self- interpreting beings, we must face the reality that this is a task that never ends and a responsibility that we cannot escape. References NOU (2001). "From User to Citizen: A Strategy for the Dismantling of Disabling Barriers", Summary in English of NOU 2001: 22. Burbules, N. C. and Bruce, B. C. (2001). Theory and research on teaching as dialogue. In V. Richardson (ed.), Handbook of Research on Teaching, 4th Edition. Washington, DC: American Educational Research Association. NOU (1998). "Use for Everyone" (original title: Det er bruk for alle), Short English version of NOU 1998: 18. Burbules, N. C. and Rice, S. (1991). Dialogue across differences: Continuing the conversation. Harvard Educational Review 61(4): 393-416. Solvang, P. (2000). The emergence of an Us and Them discourse in disability theory. Scandinavian Journal of Disability Research 2(1): 3-20. Davies, B. and Harre, R. (1990). Positioning: The discursive production of selves. Journal for the Theory of Social Behavior 20(1): 43- 63. Sosial- og Helsedepartementet (n.d.) "Responsibility and Empowerment: Towards a Holistic Rehabilitation Policy", Summary in English of Report no.21 to the Storting (1998-99). Sosial- og Helsedepartementet (n.d.) "Responsibility and Empowerment: Towards a Holistic Rehabilitation Policy", Summary in English of Report no.21 to the Storting (1998-99). Denhardt, R. B. and Denhardt, J. V. (2000). The new public service: Serving rather than steering. Public Administration Review 60(6): 549-59. Wenger, E. (1998). Communities of Practice: Learning, Meaning and Identity. New York: Cambridge University Press. Eldar, R. (2000). A conceptual proposal for the study of the quality of rehabilitation care. Disability and Rehabilitation 22(4): 163- 169. Notes 1 This paper is an invitation to think differently about the idea of evaluating rehabilitation. The ideas presented here preliminary and suggestive. However, there is no reason why we cannot reconstitute linguistically and practically our understandings of both evaluation and rehabilitation. Perhaps there is every reason why we should. There will always be evaluations that generate quantitative indicators on the types, extent, and functional outcomes of rehabilitation services. This way of evaluating is an indelible feature of professional practice in late modernity. This paper is not a call to abandon such ways of evaluating; rather it is a plea to place them in a perspective that more fully takes up the complex ways of experiencing evaluative questions in rehabilitation praxis. If the ultimate goal of evaluation is to improve Notes 1 The terms praxis and practice are often used interchangeably. But the two terms carry quite different meaning. The significance of the modern use of the word practice is typically determined by its connotations in connection with the application of theory and science to everyday life. In contemporary definitions, theory is the realm of thinking or reflecting and practice is the realm of doing or acting. Theory and practice are defined oppositionally, and practice is everything that theory is not. Thus, we hear talk of evaluation theory as something different than evaluation practice, or a theory of teaching as opposed to the practice of teaching. The Greek term praxis (although used in several different ways by Aristotle), however, is not related to a theory-practice distinction. Praxis is a distinct type of human activity concerned with our moral-practical-political behavior and it is distinguishable from that kind of activity called poiesis that is concerned with our ways of making or crafting a product or outcome. SJDR - Volume 5, No.l - 2003 104 Evaluating the Ecology of Rehabilitation Praxis Scandinavian Journal of Disability Research 1(2): 44-65 SJDR - Volume 5, No.l - 2003 The Author: Flyvbjerg, B. (2001). Making Social Science Matter. Cambridge, England: Cambridge University Press. Thomas A. Schwandt is professor of education at the University of Illinois, USA and part time professor at Statens kunnskaps- og utviklingssenter for helhetlig rehabilitering (SKUR) [National Center for Comprehensive Rehabilitation Research and Development] in Norway Thomas A. Schwandt is professor of education at the University of Illinois, USA and part time professor at Statens kunnskaps- og utviklingssenter for helhetlig rehabilitering (SKUR) [National Center for Comprehensive Rehabilitation Research and Development] in Norway Kendall, E., Buys, N., and Larner, J. (2000). Community-based service delivery in rehabilitation: The promise and the paradox. Disability and Rehabilitation 22(10): 435- 445. Lindqvist, R. and Tamm, M. (1999). Rehabilitation in the home—Interplay and conflicts between different parties. SJDR - Volume 5, No.l - 2003 105
https://openalex.org/W4200115383
https://journals.vilniustech.lt/index.php/Aviation/article/download/15837/10820
English
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ANALYSIS OF GENERAL AVIATION FIXED-WING AIRCRAFT ACCIDENTS INVOLVING INFLIGHT LOSS OF CONTROL USING A STATE-BASED APPROACH
Aviation
2,021
cc-by
9,999
AVIATION ISSN: 1648-7788 / eISSN: 1822-4180 2021 Volume 25 Issue 4: 283–294 https://doi.org/10.3846/aviation.2021.15837 AVIATION ISSN: 1648-7788 / eISSN: 1822-4180 2021 Volume 25 Issue 4: 283–294 https://doi.org/10.3846/aviation.2021.15837 AVIATION ISSN: 1648-7788 / eISSN: 1822-4180 2021 Volume 25 Issue 4: 283–294 https://doi.org/10.3846/aviation.2021.15837 Received 10 August 2020; accepted 7 January 2021 Abstract. Inflight loss of control (LOC-I) is a significant cause of General Aviation (GA) fixed-wing aircraft accidents. The United States National Transportation Safety Board’s database provides a rich source of accident data, but conventional analyses of the database yield limited insights to LOC-I. We investigate the causes of 5,726 LOC-I fixed‑wing GA aircraft accidents in the United States in 1999–2008 and 2009–2017 using a state-based modeling approach. The multi-year analy- sis helps discern changes in causation trends over the last two decades. Our analysis highlights LOC-I causes such as pi- lot actions and mechanical issues that were not discernible in previous research efforts. The logic rules in the state-based approach help infer missing information from the National Transportation Safety Board (NTSB) accident reports. We inferred that 4.84% (1999–2008) and 7.46% (2009–2017) of LOC-I accidents involved a preflight hazardous aircraft condi- tion. We also inferred that 20.11% (1999–2008) and 19.59% (2009–2017) of LOC-I accidents happened because the aircraft hit an object or terrain. By removing redundant coding and identifying when codes are missing, the state-based approach potentially provides a more consistent way of coding accidents compared to the current coding system. ords: General Aviation safety, General Aviation accidents, loss of control, accident modeling, NTSB database. Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.3846/aviation.2021.15837 There is a clear need to better understand the reasons for LOC-I accidents. One approach to improving our un- derstanding is by analyzing historical accident reports. In the U.S., the National Transportation Safety Board [NTSB] investigates all civil aviation accidents. After concluding their investigation, the NTSB publishes a final report, which includes a prose section with summary analysis of the accident, a discussion of the probable cause and findings, and “factual information” on the flight history, personnel, aircraft, meteorological conditions, medical and pathological information, and any tests and research the investigators conducted (NTSB, 2019b). Each acci- dent is also coded using a set of codes for occurrences, findings, and phases of flight to facilitate trend analysis (NTSB, 1998). Rao et al. (2016) provides a detailed discus- sion of this system. The NTSB coding system is based on an event-based model, where one event leads to another, but not all aspects of accidents are events. For example, an impaired pilot is better understood as a continuing condi- tion, or a state (Rao & Marais, 2020). The pilot’s impaired ANALYSIS OF GENERAL AVIATION FIXED-WING AIRCRAFT ACCIDENTS INVOLVING INFLIGHT LOSS OF CONTROL USING A STATE-BASED APPROACH Neelakshi MAJUMDAR   1, Karen MARAIS   1,*, Arjun RAO   2 1School of Aeronautics and Astronautics, Purdue University, West Lafayette, USA 2Collins Aerospace, 400 Collins RD, MS 124-319 Cedar Rapids, USA 1School of Aeronautics and Astronautics, Purdue University, West Lafayette, USA 2Collins Aerospace, 400 Collins RD, MS 124-319 Cedar Rapids, USA *Corresponding author. E-mail: kmarais@purdue.edu This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unre- stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright © 2021 The Author(s). Published by Vilnius Gediminas Technical University Introduction Such redundancy in codes can lead to inaccurate counts in accident causes. Finally, the NTSB database does not present all findings as codes, for example, in the pre-2008 coding system, there are no codes to capture improper aircraft heading. Studies using NTSB data to understand LOC-I acci- dents face similar challenges. Previous work attempted to build chains of events in accidents using occurrence codes in the NTSB database. Rao and Marais (2015) found that 13.8% of 5051 GA rotorcraft fatal accidents had LOC-I as the first occurrence. Houston et al. (2012) found that 75% of the 147 instructional LOC-I accident reports cited LOC-I as the first occurrence - thus we cannot determine what led to the LOC-I. Other studies investigated the im- pact of aircraft characteristics on accidents. Franza and Fanjoy (2012) analyzed correlations between contributing factors to accidents from 2002–2012 in Cirrus SR20 and Piper PA28-161 aircraft. They also found that pilots’ fail- ure to maintain directional control contributed to 50% of fatal accidents in both aircraft models. Ud-Din and Yoon (2018) found that poor health and impairment due to medication, followed by poor manual control and inad- equate pilot adherence to flight procedures were the most significant events for LOC during maneuvering. p p pt g Unfortunately, the prose content for GA accidents tends to be short. In 2017 (the most recent year to have completed factual reports), the average length for the 105 accidents that had LOC-I codes was just 449 words. The occurrence chains (number of occurrence codes) for these 105 acci- dents are also short (mean chain length = 3.36, SD = 1.42), albeit somewhat longer than that for all GA fixed-wing aircraft accidents (mean chain length = 2.48, SD = 1.60). 145 accidents had only a single recorded occurrence. The longest chain was 8 (for one LOC-I accident; NTSB ID: GAA17CA303). 80% of these reports included a code re- lated to crashing into terrain/water. Thus, the potentially wide range of accident stories is reduced to a small set of short stories, most of which are some variation of “the pi- lot lost control and crashed into the ground/water”. These problems are compounded by the lack of information about the cause for LOC-I. Introduction Fixed-wing General Aviation (GA) accidents comprise approximately 64% of all aviation accidents in the United States (U.S.) every year (NTSB, 2019a). Most fixed‑wing GA accidents result from inflight loss of control (LOC- I), controlled flight into terrain (CFIT), continued visual flight rules flight into instrumental meteorological condi- tions (‘continued VFR into IMC’), engine failures, and fuel exhaustion/contamination (cf. AOPA, 2018; GAJSC, 2016). In particular, inflight loss of control (LOC-I) con- tinues to be a significant cause of GA fixed-wing aircraft accidents each year. Loss of control is “a hazardous condi- tion that involves an unintended departure of an aircraft from controlled flight regime” (FAA, 2019). Nearly 50% of fixed-wing GA accidents in the last two decades in the United States (U.S.) are attributed to LOC-I (NTSB, 2019a). In 2017, 21% of fixed-wing GA accidents overall involved LOC‑I; for fatal accidents, this percentage in- creases to 57%. *Corresponding author. E-mail: kmarais@purdue.edu Copyright © 2021 The Author(s). Published by Vilnius Gediminas Technical University Copyright © 2021 The Author(s). Published by Vilnius Gediminas Technical University This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativ stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unre- stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. N. Majumdar et al. Analysis of General Aviation fixed-wing aircraft accidents involving inflight loss of control... N. Majumdar et al. Analysis of General Aviation fixed-wing aircraft accidents involving inflight loss of control... 284 used the Human Factors Analysis and Classification System (HFACS) to identify unsafe operator acts. 80% of the GA accidents were associated with at least one skill-based er- ror such as handling. While these studies uncovered part of what causes GA accidents (e.g., flight into IMC is often involved in fatal accidents), they were not able to explain how, for example, IMC leads to fatal accidents. condition makes subsequent errors more likely, and there- fore does not fit well as “only” an initiating event. Addi- tionally, multiple codes in the NTSB database have similar meanings. For example, the subject codes 24518: Altitude and 24519: Proper altitude both indicate that the pilot did not maintain the correct altitude. Introduction For instance, the most frequently used cause for fixed-wing LOC-I accidents is aircraft control not maintained - in other words, the pilot lost control because they did not maintain control (Houston et al., 2012; Franza & Fanjoy, 2012). So, we cannot easily determine why LOC-I happens, what most often causes it, or whether there have been any changes in its causes. i One way to improve understanding is by modeling accidents. Several researchers have used Bayesian net- works to identify causal factors and assess risk (Ancel & Shih, 2012; Ancel et al., 2015; Ayra et al., 2019; Xiao et al., 2020; Uğurlu et al., 2020). Ancel et al. (2015) developed an object-oriented Bayesian network (OOBN), based on HFACS, to model Part 121 and 135 LOC-I accidents. They identified organizational deficiencies as underlying flight-related and maintenance crew-related airline acci- dents. Bayesian networks are useful to visually represent a summary analysis of accidents. But they require detailed information that is often not available for GA accidents. The probability calculation for each node in a Bayesian network requires expert judgment and information from sources such as operators and aviation agencies. Further, some accident sequences have cyclic relationships, e.g., an aircraft stall may cause an LOC-I and vice versa. Since Bayesian networks are directed acyclic graphs, they cannot capture such cyclic relationships between aircraft states.i y g Several researchers have used NTSB codes to identify GA accident causes. Boyd (2015) found that failure to fol- low single engine procedures following loss of an engine was the highest factor in fatal twin-engine piston aircraft GA accidents under visual weather conditions. Fultz and Ashley’s (2016) found that 60% of weather-related fatal accidents occurred in IMC. Bazargan and Guzhva (2007) found that hazardous weather and light conditions such as IMC and dark night conditions increased the likeli- hood that accidents would be fatal. Goldman et al. (2002) found that of maintenance errors, installation errors such as using the wrong parts were most likely to cause injury or fatality. Aguiar et al. (2017) found that GA accidents in mountainous terrain and high elevation environments most commonly involved CFIT and wind gusts/shear. Other analyses used NTSB accident narratives. Boyd and Stolzer (2016) identified accident-precipitating factors and found that not following the checklist/flight manual contributed the most to fatal or serious turbine-powered GA accidents. Ballard et al. 1.1. State-based model introduction Th e state-based model consists of two core concepts: accidents are modelled as a series of states and triggers; and, states and triggers (the dictionary) are ordered and linked by rules (the grammar), as shown in Figure 1.ht Th e system comprises the aircraft and pilot(s) operat- ing the aircraft . A state is a segment of time wherein a system exhibits a particular behavior. Th e nodes in Figure 1 represent states of a notional system where the fi rst state represents the default or start state of the system and the last (end) state represents the system’s behavior in the fi nal segment of time in the accident. A system can be in only one state at any given point of time. Th ere are two types of states: nominal and hazardous. A nominal state is a state of a system that is generally accepted as suffi ciently safe by the applicable stakeholders. “Suffi ciently safe” depends on the particular context and stakeholders. For example, safe states are those where the aircraft is operating in good weather with all systems functioning and with a compe- tent and fi t-to-fl y pilot. A system is in a nominal state only when both the pilot and aircraft are in nominal states. A nominal state cannot lead directly to an accident state - it must be directly preceded by a hazardous state.f A trigger is an event that occurs at a precise instant of time, causing either the aircraft , pilot(s), or both the air- craft and pilot(s) to transition between states or remain in the same state. For example, failure of an engine can cause a system to transition from a nominal state to a hazardous state. Th e links connecting to each state in Figure 1 repre- sent triggers to each state. Th e initiating trigger, points to the default or start state of the system. Introduction (2013) considered three major risk factors for fatalities, post‑crash fires, crashes after flight in IMC, and off-airport crashes (in other words, away from emergency services), and found that fatalities were most likely to oc- cur in accidents occurring after flight in IMC contributed the most to fatal air tour accidents. Wiegmann et al. (2005) t We investigate whether additional insight into fixed- wing LOC-I can be garnered from the NTSB database by taking a different modelling approach. We extend the state-based approach developed for rotorcraft accidents by Rao and Marais (2020) to fixed-wing aircraft accidents. Section 1 presents the state-based approach and devel- ops a vocabulary of 108 state and 226 trigger definitions, along with a set of grammar rules for connecting states and triggers. We show how these rules can be used to logically infer some missing states and triggers. In Sec- tion 2, we identify and analyze GA LOC-I accidents us- ing the approach and identify an additional 1,214 LOC-I accidents that were not directly identifiable using LOC-I Aviation, 2021, 25(4): 283–294 285 a) b) c) Figure 2. Illustration of three possible scenarios where a system is in hazardous state. Examples of these states are: (a) pilot’s poor physiological condition; (b) loss of engine power; (c) pilot’s poor physiological condition during a loss of engine power (adapted from Rao & Marais, 2020) NTSB codes. Using the state-based approach, we found additional causes and diff erent cause rankings for LOC-I accidents than those resulting from conventional analyses, such as those described earlier. b) c) a) a) b) c) 1. A state-based approach for fi xed-wing aircraft accidents Figure 2. Illustration of three possible scenarios where a system is in hazardous state. Examples of these states are: (a) pilot’s poor physiological condition; (b) loss of engine power; (c) pilot’s poor physiological condition during a loss of engine power (adapted from Rao & Marais, 2020) Rao and Marais (2020) developed a state-based approach for modelling helicopter accidents by representing acci- dents as a sequence of states and triggers, rather than the event-centric current coding system. Th e state and trigger defi nitions are based on codes in the NTSB database that have been used for rotorcraft accidents (occurrence codes, fi nding codes, modifi er codes, and phase of fl ight codes). In this section, we extend the state-based model to fi xed- wing aircraft accidents. A system is in a hazardous state if either the pilot(s), the aircraft , or both the pilot(s) and aircraft are in hazard- ous states, as shown in Figure 2. We categorize hazardous states based on when they occur in an accident sequence. A prefl ight hazardous state is a hazardous state that exists before a fl ight starts, for example, prefl ight mechanical is- sue. An intermediary hazardous state occurs between a prefl ight state and an end state (in this case, an accident), for example, infl ight loss of control. Each fl ight terminates in an end state, which can be nominal (e.g., safe landing), an incident (e.g., bounced landing), or an accident (e.g., midair collision). A system is in a hazardous state if either the pilot(s), the aircraft , or both the pilot(s) and aircraft are in hazard- ous states, as shown in Figure 2. We categorize hazardous states based on when they occur in an accident sequence. A prefl ight hazardous state is a hazardous state that exists before a fl ight starts, for example, prefl ight mechanical is- sue. An intermediary hazardous state occurs between a prefl ight state and an end state (in this case, an accident), for example, infl ight loss of control. Each fl ight terminates in an end state, which can be nominal (e.g., safe landing), an incident (e.g., bounced landing), or an accident (e.g., midair collision). 1.2. Fixed-wing aircraft dictionary of hazardous states, triggers, and additional information Th e existing rotorcraft data dictionary has 84 state defi ni- tions and 182 trigger defi nitions. Here, we extend the data dictionary to fi xed-wing aircraft accidents. 1.2.1. Fixed-wing state defi nitions Building on the existing rotorcraft dictionary and using the NTSB database, we amended states and created new states, resulting in a set of 108 states that are applicable to fi xed-wing aircraft , as shown in Table 1.tft A hazardous state is an off -nominal state that may lead to an accident or an incident. For example, a pilot’s poor physiological condition is a “pilot hazardous state”, and loss of engine power is an “aircraft hazardous state”. it Fixed-wing aircraft diff er from rotorcraft in four ways relevant to accident modelling: (1) Maneuvering. Rotor- craft and fi xed-wing aircraft have diff erent maneuvering capabilities due to diff erent fl ight mechanics. For example, rotorcraft , unlike fi xed-wing aircraft , can perform maneu- vers such as hovering, and can autorotate in the event of losing engine power. So, these rotorcraft states are not applicable to fi xed-wing aircraft . (2)  Control surfaces. Fixed-wing aircraft , unlike rotorcraft , have ailerons, a rud- der, and an elevator for aerodynamic stability. For example, fi xed wing aircraft have fl aps, unlike rotorcraft . Th erefore, we created a new state for improper fl aps extended speed (VFE). (3) Takeoff and landing characteristics. Advanced Figure 1. State-based representation of a notional system Figure 1. State-based representation of a notional system N. Majumdar et al. Analysis of General Aviation fixed-wing aircraft accidents involving inflight loss of control... N. Majumdar et al. Analysis of General Aviation fixed-wing aircraft accidents involving inflight loss of control... 286 rotorcraft with wheels that can perform running takeoffs, hover taxi, and air taxi, are relatively rare in civil aviation, and therefore rotorcraft accidents associated with these maneuvers are also rare (there were no such accidents in the 34 years covered in Rao and Marais’ 2020 analysis). We therefore created new fixed-wing states such as im- proper takeoff, improper taxi speed, water loop/swerve, and aircraft hydroplaning. (4)  Airspeed factors. Fixed- wing aircraft have additional airspeeds to rotorcraft. For example, fixed‑wing aircraft have five different airspeeds that convey takeoff or rotation speed: lift-off speed (VLOF), takeoff safety speed (V2), minimum takeoff speed (V2MIN), rotation speed (VR), and maximum speed from which the airplane can stop within the accelerate-stop distance (V1). We define a new improper takeoff/rotation speed state, as shown in Table 2. 1.2.1. Fixed-wing state defi nitions Table 2 also shows the Boolean logic for the improper takeoff or rotation speed state, which serves as input to our translation code. Similarly, we created 10 additional airspeed states for fixed-wing aircraft such as improper landing gear operating/ extended speed (VLO and VLE) and improper flaps extended speed (VFE). Finally, we added several states that may also apply to rotorcraft but did not appear in any of the rotorcraft acci- dents in the database. For example, Rao and Marais (2020) defined two LOC states for rotorcraft: inflight loss of con- trol (LOC-I) and on-ground loss of control (LOC-G). Because the database does not always specify whether the LOC was inflight or on the ground, we created an unknown phase LOC state (LOC-U). Table 3 shows the definition and coding for the LOC-I state. Table 1. Summary of the states for rotorcraft and fixed-wing aircraft Rotorcraft States Fixed-wing States State Example States applicable only to rotorcraft 13 N/A Improper autorotation only occurs in rotorcraft. Rotorcraft states applicable to both rotorcraft and fixed-wing aircraft 54 Both rotorcraft and fixed-wing aircraft can experience hard landings. Rotorcraft states re-coded for fixed-wing aircraft 17 Fixed-wing aircraft have additional airspeed types, compared to rotorcraft such as minimum takeoff speed (V2MIN). New states defined for fixed-wing aircraft only N/A 37 Rotorcraft do not have flaps, unlike fixed-wing aircraft, so we created improper flaps extended speed (VFE) state for fixed-wing aircraft. Total states 84 108 Table 1. Summary of the states for rotorcraft and fixed-wing aircraft Table 1. Summary of the states for rotorcraft and fixed-wing aircraft Table 2. Improper takeoff or rotation speed state definition for fixed-wing aircraft Table 2. 1.2.2. Fixed-wing trigger definitions example, unsuitable runway PEC gives information about a runway condition but does not describe a state or a trig- ger in an accident (Table 5). Using the NTSB codes used for fixed-wing aircraft acci- dents, and combining codes that convey the same mean- ing, we defined 226 triggers (Table  4). Similar to haz- ardous states, we accounted for the differences between helicopters and fixed-wing aircraft when augmenting and creating new triggers for fixed-wing aircraft. For example, based on different speed characteristics of fixed-wing air- craft, we re-coded the rotorcraft trigger improper aborted landing/takeoff for fixed-wing aircraft by adding a subject code 24503 Abort above V1 with its modifiers. V1 is the takeoff decision speed, beyond which a flight can continue to take off even in case of an engine failure. Information code: Detail about a system that is neither a state, a trigger, nor a pre-existing condition, is defined as an information code. Information codes describe ter- rain/object(s) that an aircraft collided with and phases of flight in accidents. For example, the code 03022020: tree indicates that an aircraft collided with a tree during the accident. In the pre-2008 system, the NTSB uses subject codes 19200: terrain condition or 20200: object with modifiers to describe the type of terrain or objects. In the post-2008 system, the NTSB uses different finding codes to describe the type of terrain or objects with modifiers such as 91: contributed to outcome. Additionally, the NTSB uses a separate set of codes to describe phases of flight with each occurrence in accidents. Therefore, we defined three information code categories: information about objects, information about terrain, and information about phases of flight. 1.2.1. Fixed-wing state defi nitions Improper takeoff or rotation speed state definition for fixed-wing aircraft NTSB Codes (Pre-2008) Description 24507 AND (3101 OR 3104 OR 3107 OR 3108 OR 3109 OR 3112 OR 3115 OR 3120 OR 3122 OR 3127 OR 3129 OR 3138) Airspeed, lift off speed (VLOF) AND (“Below” OR “Delayed” OR “Exceeded” OR “Excessive” OR “Improper” OR “Inattentive” OR “Inadequate” OR “Misjudged” OR “Not attained” OR “Not maintained” OR “Not obtained” OR “Reduced”) 24568 AND (3000 OR 3011 OR 3107 OR 3127 OR 3129) Airspeed, maximum speed from which the airplane can stop within the accelerate- stop distance (V1) AND (“Above” OR “Not obtained/maintained” OR “Exceeded” OR “Not obtained” OR “Not maintained”) 24569 AND (3122 OR 3127) Airspeed, takeoff safety speed (V2) AND (“Not attained” OR “Not maintained”) 24570 AND (3011 OR 3115 OR 3122) Airspeed, minimum takeoff safety speed (V2min) AND (“Not obtained/maintained” OR “Inadequate” OR “Not attained”) Table 3. Inflight loss of control (LOC-I) state definition for fixed-wing aircraft NTSB Codes (Pre-2008) Description 250 Loss of control - in flight 110 Altitude deviation, uncontrolled 553 Descent - uncontrolled phase 24524 AND (3140) Descent AND (“Uncontrolled”) 24525 AND (3140) Proper descent rate AND (“Uncontrolled”) NTSB Codes (Post-2008) Description 240 Loss of control in flight 650 Uncontrolled Descent 01062022 Pitch control 01062023 Lateral/bank control Aviation, 2021, 25(4): 283–294 287 1.2.3. Additional information Rao (2016) used information codes to translate NTSB codes that provide additional information about the pre- vailing conditions during an accident, but do not translate to states or triggers. Here, we amend this definition by adding a fourth category, pre-existing condition (PEC), and redefining the information codes to exclude PECs. i Pre-existing Condition (PEC): A condition in the aircraft’s environment that remains true or applicable throughout a flight and is neither a state nor a trigger is defined as a pre-existing condition. We define three pre- existing conditions: unsuitable airport facilities, unsuit- able runway, and unsuitable physical environment. For 1.3. Illustrative example We demonstrate the working of fixed-wing state and trig- ger definitions and the grammar rules using an accident (NTSB ID: ERA13FA059) that happened in November 2012 in Owls Head, Maine involving a Cessna 172N. Table 4. Breakdown of rotorcraft and fixed-wing aircraft triggers Rotorcraft Triggers Fixed-wing Triggers Trigger Example Triggers applicable only to rotorcraft 43 N/A Rotor system failure can occur only in rotorcraft. Rotorcraft triggers applicable to both rotorcraft and fixed-wing aircraft 76 Both fixed-wing and rotorcraft can experience improper engine shutdown. Rotorcraft triggers re-coded for fixed- wing aircraft 63 Both rotorcraft and fixed-wing aircraft can experience improper aborted landing/takeoff, but there are additional NTSB codes that apply for fixed-wing aircraft. New triggers defined for fixed-wing aircraft N/A 87 Rotorcraft have rotors and not propellers, so they do not have a propeller control failure trigger. Total triggers 182 226 Table 5. Unsuitable runway pre-existing condition NTSB Codes (Pre-2008) Description 19201 Runway/landing area condition NTSB Codes (Post-2008) Description 03023000 Runway/land/takeoff/taxi surface - (general) 03023010 Runway/land/takeoff/taxi surface - Wet 03023015 Snow/slush/ice covered 03023020 Soft 03023025 Glassy 03023030 Choppy Table 4. Breakdown of rotorcraft and fixed-wing aircraft triggers Table 4. Breakdown of rotorcraft and fixed-wing aircraft triggers Rotorcraft Triggers Fixed-wing Triggers Trigger Example Triggers applicable only to rotorcraft 43 N/A Rotor system failure can occur only in rotorcraft. Rotorcraft triggers applicable to both rotorcraft and fixed-wing aircraft 76 Both fixed-wing and rotorcraft can experience improper engine shutdown. Rotorcraft triggers re-coded for fixed- wing aircraft 63 Both rotorcraft and fixed-wing aircraft can experience improper aborted landing/takeoff, but there are additional NTSB codes that apply for fixed-wing aircraft. New triggers defined for fixed-wing aircraft N/A 87 Rotorcraft have rotors and not propellers, so they do not have a propeller control failure trigger. Total triggers 182 226 Table 5. Unsuitable runway pre-existing condition Table 5. Unsuitable runway pre-existing condition NTSB Codes (Pre-2008) Description 19201 Runway/landing area condition NTSB Codes (Post-2008) Description 03023000 Runway/land/takeoff/taxi surface - (general) 03023010 Runway/land/takeoff/taxi surface - Wet 03023015 Snow/slush/ice covered 03023020 Soft 03023025 Glassy 03023030 Choppy N. Majumdar et al. Analysis of General Aviation fi xed-wing aircraft accidents involving infl ight loss of control... 288 Figure 3. Th ree states do not have entering triggers, because the accident report does not mention any applicable trigger related codes. During the departure roll, the aircraft collided with a ground vehicle that was crossing the runway, breaking the right elevator. 1.3. Illustrative example Th e pilot continued taking off , stalled the aircraft , and went into a low-altitude spin before hitting the ground. Th e fi rst two columns of Table 6 show the resulting NTSB codes for the accident report.i 5. Infer triggers and states based on grammar rules: Th e NTSB codes for an accident may not be suf- fi cient to identify all states and triggers in that ac- cident, as shown by the three missing triggers in Figure 3. We use the state-and-trigger sequencing rules to infer some of the missing information. 5. Infer triggers and states based on grammar rules: Th e NTSB codes for an accident may not be suf- fi cient to identify all states and triggers in that ac- cident, as shown by the three missing triggers in Figure 3. We use the state-and-trigger sequencing rules to infer some of the missing information. We model the accident in fi ve steps: 1. Identify states and triggers from the accident data: We map the fi nding codes and occurrence codes from the database with corresponding states and triggers as shown in Table 6. Figure 3 shows the states and triggers. Since there are no codes in- dicating that the pilot was impaired or the aircraft was functioning improperly, we indicate their state as nominal.l Consider for example the trigger recovery action not possible aft er loss of control. We infer this trigger when- ever an end state succeeds a loss of control state in an accident, and the accident does not include any codes re- lated to an improper remedial action or a lack of action triggers (Loss of control state AND (end state) AND NOT (“Improper remedial action” trigger OR “Lack of action” trigger). 2. Identify prefl ight, intermediary, and end states: Next, we identify the prefl ight, intermediary and end states, as shown in the last column of Table 6. 3. Sequence hazardous states: We apply the grammar rules to sequence hazardous states. Th e sequencing rules are based on fl ight physics and the sequence that the NTSB used to report accidents. See Rao and Marais (2020) for a detailed discussion of grammar rules. Figure 3 shows the accident model aft er ap- plying the sequencing rules. 2. Analysis of infl ight loss of control accidents Th is section compares conventional and state-based sta- tistical analyses of LOC-I accidents involving fi xed-wing aircraft operating under 14CFR  Part  91 that occurred from 1999–2017 and are recorded in the NTSB database. Because the NTSB coding system changed in 2008, we consider two diff erent time frames: 1999–2008 and 2009– 2017. 4. Link states and triggers: Using the grammar rules, we link triggers to the sequenced states, as shown in Table 6. NTSB Codes and Corresponding States or Triggers (NTSB ID: ERA13FA059) Finding code/ Occurrence code Modifi er/Person/Phase Code Resulting State/Trigger Prefl ight/intermediary/ end state 2041015: Incorrect action performance 44: Pilot Improper action performance trigger N/A 1062022: Pitch control 20: Not attained/maintained Infl ight loss of control state Intermediary 2041030: Lack of action 48: Airport personnel Lack of action trigger N/A 3036000: Light condition 91: Contributed to outcome Prevailing weather and light state Prefl ight 320: Runway incursion 300: Takeoff Runway incursion state Intermediary 490: Collision during takeoff / landing 300: Takeoff Collision during takeoff /landing state Intermediary 240: Loss of control in-fl ight 350: Initial Climb Infl ight loss of control state Intermediary 470: Collision with terrain/ object (non-CFIT) 650: Uncontrolled descent Infl ight collision with terrain/object state End Figure 3. State-based representation of the accident. Note: Using the grammar rules, we infer triggers to the sequenced states that have missing triggers. Th e links (and text) in blue are the inferred triggers. Th e text in red shows a missing trigger that cannot be inferred using the grammar rules. Table 6. NTSB Codes and Corresponding States or Triggers (NTSB ID: ERA13FA059) Finding code/ Occurrence code Modifi er/Person/Phase Code Resulting State/Trigger Prefl ight/intermediary/ end state 2041015: Incorrect action performance 44: Pilot Improper action performance trigger N/A 1062022: Pitch control 20: Not attained/maintained Infl ight loss of control state Intermediary 2041030: Lack of action 48: Airport personnel Lack of action trigger N/A 3036000: Light condition 91: Contributed to outcome Prevailing weather and light state Prefl ight 320: Runway incursion 300: Takeoff Runway incursion state Intermediary 490: Collision during takeoff / landing 300: Takeoff Collision during takeoff /landing state Intermediary 240: Loss of control in-fl ight 350: Initial Climb Infl ight loss of control state Intermediary 470: Collision with terrain/ object (non-CFIT) 650: Uncontrolled descent Infl ight collision with terrain/object state End Table 6. NTSB Codes and Corresponding States or Triggers (NTSB ID: ERA13FA059) Figure 3. 2.1. Conventional analysis of LOC-I accidents tained/maintained, are variations of “loss of control”. Only the second highest cause, airspeed - not attained/main- tained provides some indication of what happened during the LOC-I accident. Some of the top causes in 2009–2017 were different from the 1999–2008 findings. In 2009–2017, we found more causes related to aircraft performance (per- formance/control parameters - not attained/ maintained) and pilot actions (decision making/ judgment - pilot, in- correct action performance - pilot, and angle of attack - not attained/maintained). This difference arises from two main reasons: (1) the NTSB codes and their descriptions changed in 2008 (the NTSB built an entirely new coding system); and (2) the NTSB started using some code de- scriptions more extensively as causes in post-2008 LOC-I accidents compared to the pre-2008 accidents. For exam- ple, the NTSB used aircraft performance related finding as a cause more in the post-2008 system (1062000-20: Per- formance/control parameters - not attained/maintained) than in the pre-2008 system (17300: Aircraft performance).h Conventional analyses such as those discussed in the in- troduction, analyse the relative frequencies with which NTSB codes are cited in accident reports. Here we do such an analysis for LOC-I accidents. In the pre-2008 coding system, LOC-I accidents are indicated by 250: Loss of con- trol – in flight; in the post-2008 system, by 240: Loss of con- trol – in flight. The NTSB uses subject code and modifier combinations (and finding codes in the post-2008 system) to provide greater detail about the level of contribution each finding had to the outcome. Each combination is des- ignated as a cause or contributing factor. We identified the subject code and modifier combinations that the NTSB designated as causes (denoted as j Cause ) and calculated the presence for each subject code and modifier combina- tion as the number of times it was used at least once in an accident, normalized by the total number of accidents (cf. Sorenson & Marais, 2016). ( ) ( ) 1 | 1| . accidents j n j i i presence Cause Accident TRUE Cause Accident Total Accidents = = ≥ ∑ (1) ( ) ( ) 1 | 1| . 2. Analysis of infl ight loss of control accidents State-based representation of the accident. Figure 3. State-based representation of the accident. Figure 3. State-based representation of the accident. Note: Using the grammar rules, we infer triggers to the sequenced states that have missing triggers. Th e links (and text) in blue are the inferred triggers. Th e text in red shows a missing trigger that cannot be inferred using the grammar rules. Note: Using the grammar rules, we infer triggers to the sequenced states that have missing triggers. Th e links (and text) in blue are the inferred triggers. Th e text in red shows a missing trigger that cannot be inferred using the grammar rules. Note: Using the grammar rules, we infer triggers to the sequenced states that have missing triggers. Th e links (and text) in blue are the inferred triggers. Th e text in red shows a missing trigger that cannot be inferred using the grammar rules. Aviation, 2021, 25(4): 283–294 289 2.1. Conventional analysis of LOC-I accidents accidents j n j i i presence Cause Accident TRUE Cause Accident Total Accidents = = ≥ ∑ (1) yt The pre-2008 coding system has four separate subject codes to indicate decision making or judgment (24000: Planning/decision, 24010: Inflight planning/decision, 24031: Improper decision, and 60000: Judgment) as com- pared to only one finding code in the post-2008 system (02041520: Decision making/judgment). Out of these four subject code and modifier combinations in the pre-2008 system, 24010-3109: Improper inflight planning/decision appears the most frequently (2.15%) in 1999–2008 (cited as 12th most frequent cause in LOC-I accidents). The NTSB used decision making/judgment more frequently as a cause in the post-2008 LOC-I accidents than in the pre-2008 acci- dents (presence of 11.29%). Unlike in the post-2008 system, there are no codes in the pre-2008 system that indicate in- correct action by pilot or angle of attack and therefore these are some new causes that we identified in 2009–2017. Some of the top causes from 1999–2008 such as stall - inadvertent and stall/spin - inadvertent were not identifiable in 2009– 2017 because the post-2008 coding system does not use any stall- or spin-related finding codes as causes. However, the post-2008 system uses 241: Aerodynamic stall/spin as an occurrence code in 16.79% of LOC-I accidents. (1) We use the same method to calculate the presence of contributing factors in accidents. In this section, we dis- cuss the most frequent (top) causes and contributing fac- tors in LOC-I accidents. Table 7 shows the top ten causes for LOC-I accidents in 1999–2008. The highest cause of LOC-I is that the pilot did not maintain aircraft control (24566–3127: Aircraft control - not maintained), thus providing no indication of what caused the loss of control. Failure to maintain airspeed (24506–3127: Airspeed - not maintained) has the second highest presence, providing at least some suggestion that airspeed is an important factor in LOC-I accidents. This finding is corroborated by the other top‑ten causes related to airspeed in our analysis. Table 8 shows the top ten causes for LOC-I accidents in 2009–2017. Three of the top four causes of LOC-I, air- craft control - pilot, directional control - not attained/ maintained, and performance/control parameters - not at- Table 7. 2.1. Conventional analysis of LOC-I accidents Top ten causes for LOC-I accidents in 1999–2008 Subject code with modifier Description Presence, % 24566–3127 Aircraft control - Not maintained 27.43 24506–3127 Airspeed - Not maintained 21.97 24551–3113 Stall - Inadvertent 7.68 24511–3127 Airspeed, stall (Vs) - Not maintained 5.78 25000–3001 Reason for occurrence undetermined - No Modifier 5.60 24539–3127 Directional control - Not maintained 5.20 24026–3115 Compensation for wind conditions - Inadequate 4.22 24552–3113 Stall/spin - Inadvertent 3.93 33400–0 Spatial disorientation - No Modifier 3.49 24506–3115 Airspeed - Inadequate 2.84 Table 7. Top ten causes for LOC-I accidents in 1999–2008 N. Majumdar et al. Analysis of General Aviation fixed-wing aircraft accidents involving inflight loss of control... 290 N. Majumdar et al. Analysis of General Aviation fixed-wing aircraft accidents involving inflight loss of control... . Majumdar et al. Analysis of General Aviation fixed-wing aircraft accidents involving inflight loss of contr Table 9 shows the top contributing factors to LOC‑I accidents in 1999–2008. From 1999–2008, weather and light conditions, collision with objects, and low altitude were the top contributing factors in LOC-I accidents. Ad- verse weather conditions appear in 33.25% of LOC-I ac- cidents, out of which wind gust (6.37%) and crosswind (4.69%) are the most dangerous weather conditions for LOC-I. Further, 6.11% of LOC-I accidents involved col- lision with an object, out of which collision with tree(s) was the most frequent. Table  10 shows the top ten contributing factors in LOC-I accidents from 2009–2017. In contrast to the pre- 2008 results, terrain, object, or weather-related codes do not appear in the top ten factors. This difference in top causes is because the NTSB has tended to designate weather and object related finding code-modifier combinations as causes rather than factors in the post-2008 system. In the post- 2008 coding system, the NTSB used pilot-related codes more as factors which give more information about pilot’s decision making, actions, physical wellness, and experience. Table 8. Top ten causes for LOC-I accidents in 2009–2017 Finding code Description Presence, % 206304044 Aircraft control – Pilot 59.39 106201020 Airspeed – Not attained/maintained 21.10 106200020 Performance/control parameters – Not attained/ maintained 18.43 106202020 Directional control – Not attained/maintained 11.68 204152044 Decision making/judgment – Pilot 11.29 500000000 Not determined – No Modifier 7.03 202202544 Spatial disorientation – Pilot 6.41 106204220 Angle of attack – Not attained/maintained 5.90 204101544 Incorrect action performance – Pilot 5.62 206304046 Aircraft control – Student pilot 4.59 Table 9. Top ten contributing factors for LOC-I accidents in 1999–2008 Subject code with modifier Description Presence, % 20000–2207 Weather condition – Gusts 6.37 20000–2203 Weather condition – Crosswind 4.69 20200–2517 Object – Tree(s) 3.20 24518–3004 Altitude – Low 3.02 20000–2214 Weather condition – Low ceiling 2.91 20000–2212 Weather condition – High density altitude 2.58 20100–2305 Light condition – Dark night 2.55 20000–2204 Weather condition – Clouds 2.44 20000–2222 Weather condition – Tailwind 2.36 24551–3113 Stall – Inadvertent 1.89 Table 10. Top ten contributing factors for LOC-I accidents in 2009–2017 Table 8. Top ten causes for LOC-I accidents in 2009–2017 Table 9. 2.2. State-based analysis of LOC-I accidents Th e state-based analysis helped in identifying some new fi ndings that could not be identifi ed from a conven- tional analysis, such as abnormal runway contact and ex- ceeding aircraft performance limits. As shown in Figure 5, we identifi ed abnormal runway contact state only in 2009–2017 accidents because the NTSB’s pre-2008 coding system does not have any codes to describe abnormal con- tact of the aircraft with the runway. Similarly, the pre-2008 system contains only one code that indicates exceeding aircraft performance limits (17300: Aircraft performance), as compared to three diff erent codes in the post-2008 sys- tem. Th e NTSB did not use the pre-2008 subject code (17300) extensively in the 1999–2008 accidents and it therefore has a presence of only 0.28%. We identifi ed new fi ndings such as prefl ight mechanical issue and insuffi cient qualifi cation/training as important causes for LOC-I, with a presence of 8.13% and 10.15% respectively in 2009–2017 accidents. Prefl ight mechanical issue involves scenarios such as improper weight and balance calculations by pilot and operating an aircraft with known defi ciencies. Insuf- fi cient qualifi cation/training includes lack of experience in a type of aircraft , night or instrument fl ying, inadequate fl ight training, and the pilot not being current in their certifi cation. Th is section presents the top hazardous states and triggers using the state-based approach and compares our fi ndings to the conventional analysis. We identifi ed LOC-I acci- dents in 1999–2017 using the data dictionary to map the NTSB codes to the LOC-I state defi nition. Our conven- tional analysis identifi ed 4,512 LOC-I accidents in 1999– 2017. By mapping the NTSB codes to the LOC-I state, we found 1,214 additional LOC-I accidents, as shown by year in Figure 4. We calculated the presence of hazardous states and triggers in the LOC-I accidents using Equation (1). Figure 5 compares the top hazardous states for LOC-I accidents in 1999–2008 and 2009–2017, ranked based on the top hazardous states for the recent years (2009–2017). Figure 5 shows that in 2009–2017, most (77.8%) of the LOC-I accidents typically ended with an infl ight collision with terrain/water/object. Figure 4. Comparison of number of LOC-I accidents found using the NTSB codes vs. N. Majumdar et al. Analysis of General Aviation fixed-wing aircraft accidents involving inflight loss of control... Top ten contributing factors for LOC-I accidents in 1999–2008 Subject code with modifier Description Presence, % 20000–2207 Weather condition – Gusts 6.37 20000–2203 Weather condition – Crosswind 4.69 20200–2517 Object – Tree(s) 3.20 24518–3004 Altitude – Low 3.02 20000–2214 Weather condition – Low ceiling 2.91 20000–2212 Weather condition – High density altitude 2.58 20100–2305 Light condition – Dark night 2.55 20000–2204 Weather condition – Clouds 2.44 20000–2222 Weather condition – Tailwind 2.36 24551–3113 Stall – Inadvertent 1.89 Table 10. Top ten contributing factors for LOC-I accidents in 2009–2017 Finding Code Description Presence, % 204152044 Decision making/judgment – Pilot 4.08 203102544 Total experience with equipment – Pilot 1.87 206304044 Aircraft control – Pilot 1.08 206301544 Use of equipment/system – Pilot 0.96 201202544 Prescription medication – Pilot 0.91 500000000 Not determined – No Modifier 0.79 206151044 Preflight inspection – Pilot 0.74 201202044 Illicit drug – Pilot 0.68 204102540 Delayed action – Instructor/check pilot 0.68 201203044 OTC medication – Pilot 0.62 Aviation, 2021, 25(4): 283–294 291 2.2. State-based analysis of LOC-I accidents Some of the top triggers such as delayed action, lack of action, improper action perfor- mance, and improper angle of attack were only cited in the NTSB’s post-2008 coding system, since the pre-2008 In 19 59% of LOC-I accidents, aircraft clipped (hit) Figure 6. Comparison of presence of top triggers for LOC-I accidents in 1999–2008 and 2009–2017 Figure 7. Presence of inferred states in LOC-I accidents in 1999–2008 and 2009–2017 Figure 8. Comparison of presence of top inferred triggers in LOC-I accidents in 1999–2008 and 2009–2017 Figure 7. Presence of inferred states in LOC-I accidents in 1999–2008 and 2009–2017 Figure 8. Comparison of presence of top inferred triggers in LOC-I accidents in 1999–2008 and 2009–2017 Figure 7. Presence of inferred states in LOC-I accidents in 1999–2008 and 2009–2017 Figure 6. Comparison of presence of top triggers for LOC-I accidents in 1999–2008 and 2009–2017 Figure 7. Presence of inferred states in LOC-I accidents in 1999–2008 and 2009–2017 Figure 8. Comparison of presence of top inferred triggers in LOC-I accidents in 1999–2008 and 2009–2017 Figure 6. Comparison of presence of top triggers for LOC-I accidents in 1999–2008 and 2009–2017 (such as an unsafe to fl y aircraft or severe weather condi- tions) even before it starts. Some of the top triggers such as delayed action, lack of action, improper action perfor- mance, and improper angle of attack were only cited in the NTSB’s post-2008 coding system, since the pre-2008 coding system does not use any relevant codes to describe such pilot actions. Th e trigger Undetermined reason, al- though used extensively (8.87% in 1999–2008 and 9.50% in 2009–2017), does not provide any useful information about how LOC-I happened. (such as an unsafe to fl y aircraft or severe weather condi- tions) even before it starts. Some of the top triggers such as delayed action, lack of action, improper action perfor- mance, and improper angle of attack were only cited in the NTSB’s post-2008 coding system, since the pre-2008 coding system does not use any relevant codes to describe such pilot actions. Th e trigger Undetermined reason, al- though used extensively (8.87% in 1999–2008 and 9.50% in 2009–2017), does not provide any useful information about how LOC-I happened. Figure 8. 2.2. State-based analysis of LOC-I accidents Comparison of presence of top inferred triggers in LOC-I accidents in 1999–2008 and 2009–2017 In 19.59% of LOC-I accidents, aircraft clipped (hit) terrain or object and continued the fl ight, suggesting that the aircraft did not crash and collided with the object or terrain, thus inferring clipping of object/terrain trigger. In 3.29% of LOC-I accidents, the NTSB database did not describe how an aircraft transitioned from system failure to LOC-I state. We infer impossible/reduced authority af- ter system failure state trigger for such accidents, where no other related trigger codes were used. We inferred the trigger no/failed recovery from disoriented state (6.29% in 2009–2017) whenever a disoriented pilot directly transi- tioned into a loss of control state, with no related trigger information to describe the transition. We also inferred time spent in poor weather (4.79% in 2009–2017) as a trigger to pilot’s disoriented state when the NTSB cites prevailing weather/light or a fl ight through poor weather as the immediate former state with no related trigger in- formation. Next, we use the state-based approach to identify omis- sions in accident coding and infer the missing informa- tion using the grammar rules. Figure 7 shows the inferred hazardous states. 4.84% and 7.46% of LOC-I accidents in 1999–2008 and 2009–2017 respectively did not cite any codes related to aircraft prefl ight hazardous state (such as prefl ight mechanical issue state and prefl ight low engine fl uids states). We inferred this state by using other trigger codes that implied that the aircraft was in a hazardous state before starting the fl ight. Similarly, we inferred pre- fl ight pilot hazardous state in 1.23% and 0.20% of LOC-I accidents in 1999–2008 and 2009–2017 respectively. Fig- ure 8 shows the inferred hazardous triggers. 94.87% of the LOC-I accidents had no corresponding codes to describe how an LOC-I state leads to an accident. Using our logic rules, we inferred the trigger recovery action not possible from loss of control which represents the missing data in the accidents. 2.2. State-based analysis of LOC-I accidents state defi nitions Weather factors such as prevailing/existing weather and light conditions (31.72% in 2009–2017 and 32.88% in 1999–2008) and fl ight through poor weather (12.38% in 2009–2017 and 10.73% in 1999–2008) play a major role in LOC-I accidents. Although the number of prevailing weather-related codes increased from just two codes to 47 in the new NTSB coding system, the number of times that the NTSB cited codes describing “prevailing weather/light conditions” or “fl ying through a poor weather” in LOC-I accidents remains similar.t Aircraft stall/spin appears in only 22.3% of LOC-I accidents in 2009–2017 as compared to the high pres- ence (41.72%) in pre-2008 LOC-I accidents because the pre-2008 coding system has two stall/spin related fi nding codes whereas the post-2008 coding system has only one stall related occurrence code, but no fi nding codes. Figure 4. Comparison of number of LOC-I accidents found using the NTSB codes vs. state defi nitions Figure 5. Comparison of presence of top hazardous states for LOC-I accidents in 1999–2008 and 2009–2017 Further, pilot in a disoriented/lacking awareness state (not shown in Figure 5) was present in 6.42% of 2009– 2017 accidents. Th is state involves situations when a pi- lot loses a reference point especially when fl ying through poor weather (for example, low visibility and instrumental meteorological conditions). Figure 6 shows the top ten triggers in LOC-I acci- dents. Additional fi ndings into LOC-I accidents such as improper infl ight planning/decision-making, improper maintenance, improper prefl ight planning, and improp- er use of procedure or directives could not be identifi ed from a conventional analysis of the NTSB database. In 2009–2017, improper infl ight planning/decision-making has the highest presence (17.34%) in LOC-I accidents. Th is trigger involves scenarios such as reduced/improper judgement or decision-making by pilot, and not recog- nizing or comprehending risks. Improper maintenance and prefl ight planning put the fl ight in a hazardous state Figure 5. Comparison of presence of top hazardous states for LOC-I accidents in 1999–2008 and 2009–2017 N. Majumdar et al. Analysis of General Aviation fi xed-wing aircraft accidents involving infl ight loss of control... 292 (such as an unsafe to fl y aircraft or severe weather condi- tions) even before it starts. Conclusions We extended Rao and Marais’ (2020) state-based approach for rotorcraft accidents to fi xed-wing aircraft accidents by modifying the existing rotorcraft state and trigger defi ni- tions and adding a total of 130 new states, triggers, and ad- ditional information applicable to fi xed-wing aircraft . We created a new category to store additional accident infor- mation called pre-existing condition (PEC) that describes an aircraft ’s environment that remains true throughout a fl ight. We developed a new set of grammar rules to se- quence states and link triggers to states. Th ese grammar rules help to logically infer some of the missing informa- tion and provide additional insights into accidents. We While the NTSB reports terminating occurrences (or end states in this paper) that immediately followed the LOC-I state, the accident codes do not indicate what trig- gers the aircraft to transition from the LOC-I state to the end state. In some cases, the NTSB codes translate to trig- gers that described how an LOC-I state transitioned to an accident such as improper remedial action (which is used in 5.14% of LOC-I accidents) and lack of action (presence of 4.13%). Aviation, 2021, 25(4): 283–294 293 ger information. In related work, we found that the nar- ratives sometimes contain new and detailed information as compared to the NTSB codes. We found this informa- tion by “manually” reading each narrative, which is time consuming, tedious, and prone to subjectivity. An auto- mated text mining approach can alleviate these issues and yield additional information. Augmenting our state-based model by including such a text mining approach can offer an additional source of state and trigger information to code and model accidents more efficiently. Having more data from the narratives would likely reveal new states and triggers. To address this aspect, we consider machine learning as a way to automatically identify potential haz- ardous states, triggers and new grammar rules and thus create a “self-developing coding system” based on the extracted information from the accident narratives. Fur- ther, by investigating potential associations between pre- existing conditions (PECs) and hazardous states, we can create additional rules to gain more insights from accident modeling. For example, the grammar rules may help to find the likelihood of the PEC, wet runway condition, to be associated with the landing to overrun state. Disclosure statement We declare that we have no significant competing finan- cial, professional, or personal interests that might have influenced the performance or presentation of the work described in this manuscript. Conclusions investigated the usefulness of the state‑based approach to model fixed-wing LOC-I accidents and revealed some new findings that were not discernible from the conventional analysis. The state-based approach steps away from the chain of events accident modeling technique by viewing aviation accident as a set of hazardous states and triggers. Our ap- proach also helps to provide a more correct count of the LOC‑I accidents and their causes in the NTSB database by accounting for coding redundancies. By mapping the LOC-I state definition codes, we identified 1,214 addi- tional LOC-I accidents that had not been labelled as such in the NTSB database.h The conventional analysis provides little information about LOC-I accident causation by using tautologies of LOC‑I (such as directional control and aircraft control). These causes provide additional information about the type of LOC‑I (directional or aircraft), but do not mention why loss of control happened. The state-based approach helped to provide a deeper statistical understanding of the LOC-I accidents in the NTSB database. We ranked the top hazardous states and triggers in 5,726 LOC-I accidents in two different timeframes 1999–2008 and 2009–2017 to understand the causal patterns in LOC-I accidents. In addition to the already known causes of LOC-I such as prevailing weather and light conditions and improper airspeed from the conventional analysis, our state-based analysis reveals that hazardous states such as exceeding aircraft performance limits, insufficient qualification/ training, and preflight mechanical issues are prevalent in LOC-I accidents. We also found that triggers such as im- proper inflight planning/decision-making, preflight plan- ning, and improper use of procedures are some of the top causes for LOC-I.hi Funding This research was partially funded by the US Depart- ment of Transportation/Federal Aviation Adminis- tration PEGASAS Center of Excellence under Award No 12-C-GA-PU AM44, 55. The project was managed by Michael Vu. The views expressed in this paper are those of the authors and do not necessarily reflect those of the FAA. The information in this research does not constitute FAA Flight Standards or FAA Aircraft Certification policy. Author contributions The NTSB database sometimes omits important find- ings codes from accidents. This approach helps infer miss- ing codes from reports and construct logical accident se- quences (or stories). By using the grammar rules to model the LOC-I accidents, we inferred that aircraft clipping with object or terrain caused LOC-I in 19.9% of LOC-I accidents in 2009–2017, a finding that was not discernible from the conventional analysis. Additionally, we inferred that 4.84% and 7.46% of the accidents in 1999–2007 and 2009–2017 respectively had missing information about hazardous aircraft state before the start of the flight. These additional insights help to provide a better understanding of loss of control accidents. Further, considering these ad- ditional insights in loss of control prevention and recovery training techniques may help in reducing LOC-I accidents and incidents in the future. NM was responsible for the design and development of the data analysis and wrote the first draft of the article. KM provided oversight of the analysis and reviewed the paper drafts. AR helped develop the data analysis and re- viewed the paper drafts. Aircraft Owners and Pilots Association. (2018). 27th Joseph T. Nall report: General aviation accidents in 2015. AOPA. https:// www.aopa.org/-/media/files/aopa/home/training-and-safety/ References A new approach to modeling aviation accidents [Doctoral dissertation, Purdue University, USA]. https://docs. lib.purdue.edu/open_access_dissertations/993 Ballard, S.-B., Beaty, L. P., & Baker, S. P. (2013). US Commercial air tour crashes 2000–2011: Burden, fatal risk factors and FIA Score Validation. Accident Analysis & Prevention, 57, 49–54. https://doi.org/10.1016/j.aap.2013.03.028 Rao, A. H., & Marais, K. (2015). Identifying high-risk occur- rence chains in helicopter operations from accident data. 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Learning Interdisciplinarity and Systems Approaches in Agroecology: Experience with the Serious Game SEGAE
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Learning interdisciplinarity and systems approaches in agroecology: experience with the serious game SEGAE Julia Jouan, Mireille de Graeuwe, Matthieu Carof, Rim Baccar, Nathalie Bareille, Suzanne Bastian, Delphine Brogna, Giovanni Burgio, Sébastien Couvreur, Michal Cupial, et al. To cite this version: Julia Jouan, Mireille de Graeuwe, Matthieu Carof, Rim Baccar, Nathalie Bareille, et al.. Learning interdisciplinarity and systems approaches in agroecology: experience with the serious game SEGAE. Sustainability, 2020, 12 (11), pp.4351. ￿10.3390/su12114351￿. ￿hal-02650955￿ Distributed under a Creative Commons Attribution 4.0 International License Article Learning Interdisciplinarity and Systems Approaches in Agroecology: Experience with the Serious Game SEGAE Julia Jouan 1, Mireille De Graeuwe 2, Matthieu Carof 1, Rim Baccar 3, Nathalie Bareille 4, Suzanne Bastian 4, Delphine Brogna 2, Giovanni Burgio 5, Sébastien Couvreur 6, Michał Cupiał 7, Benjamin Dumont 2, Anne-Lise Jacquot 8, Serena Magagnoli 5, Joanna Makulska 9, Kevin Maréchal 2, Guénola Pérès 1, Aude Ridier 10, Thibault Salou 1,11, Barbara Tombarkiewicz 9, Fabio Sgolastra 5 and Olivier Godinot 1,* 1 SAS, INRAE, Institut Agro, 35042 Rennes, France; julia.jouan@agrocampus-ouest.fr (J.J.); matthieu.carof@agrocampus-ouest.fr (M.C.); guenola.peres@agrocampus-ouest.fr (G.P.); thibault.salou@supagro.fr (T.S.) 2 ULiège Gembloux Agro-Bio Tech, TERRA research and teaching center, B-5030 Gembloux, Belgium; mdegraeuwe@uliege.be (M.D.G.); delphine.brogna@gmail.com (D.B.); benjamin.dumont@uliege.be (B.D.); k.marechal@uliege.be (K.M.) 3 USC 1432 LEVA, Ecole Supérieure d’Agricultures, INRAE, SFR 4207 QUASAV, 55 rue Rabelais, 49100 Angers, France; r.baccar@groupe-esa.com 4 INRAE, Oniris, BIOEPAR, 44300 Nantes, France; nathalie.bareille@oniris-nantes.fr (N.B.); suzanne.bastian@oniris-nantes.fr (S.B.) 5 DISTAL, Alma Mater Studiorum Università di Bologna, 40126 Bologna, Italy; giovanni.burgio@unibo.it (G.B.); serena.magagnoli4@unibo.it (S.M.); fabio.sgola 5 DISTAL, Alma Mater Studiorum Università di Bologna, 40126 Bologna, Italy; giovanni.burgio@unibo.it (G.B.); serena.magagnoli4@unibo.it (S.M.); fabio.sgolastra2@unibo.it (F.S.) 6 USC 1481 URSE, Ecole Supérieure d’Agricultures, INRAE, 55 rue Rabelais, 49007 Angers, France; s.couvreur@groupe-esa.com 7 University of Agriculture in Krakow, Faculty of Production and Power Engineering, 30-269 Kraków, Poland; michal.cupial@ur.krakow.pl p p 8 PEGASE, INRAE, Institut Agro, 35042 Rennes, France; anne-lise.jacquot@agrocampus-ouest.fr 9 University of Agriculture in Krakow, Faculty of Animal Science, 30-269 Kraków, Poland; rzmakuls@cyf-kr.edu.pl (J.M.); barbara.tombarkiewicz@urk.edu.pl (B.T.) 10 SMART-LERECO, INRAE, Institut Agro, 35042 Rennes, France; aude.ridier@agrocampus-ouest.fr 11 ITAP, Univ Montpellier, INRAE, Institut Agro, 34060 Montpellier, France * Correspondence: olivier.godinot@agrocampus-ouest.fr; Tel.: +33-(0)-2-23-48-55-61 Received: 29 April 2020; Accepted: 21 May 2020; Published: 26 May 2020 Abstract: Agroecology represents a pertinent option to improve the sustainability of agriculture. To promote its application, agroecological concepts should be taught to students and professionals in the agricultural sector. However, most agricultural courses are not adapted to teach these concepts due to little interactivity or interdisciplinarity, and a lack of a systems approach to farm management. Serious games help to fill these gaps by simulating complex models in which players can learn by doing. We thus developed a serious computer game, called SEGAE (SErious Game for AgroEcology learning), which represents a mixed crop–livestock farm and assesses impacts of farming practices on indicators related to environmental, economic, and social sustainability. Sustainability 2020, 12, 4351; doi:10.3390/su12114351 HAL Id: hal-02650955 https://hal.inrae.fr/hal-02650955v1 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 1. Introduction European agriculture is facing many challenges, such as producing food and other ecosystem services in sufficient quantity and quality and providing economic benefits for farmers and food- chain actors, while decreasing negative impacts on the environment [1,2]. Several authors consider agroecology as a pertinent option to reconcile the environmental, economic, and social pillars of agricultural sustainability [3,4]. Agroecology is broadly defined as “the ecology of sustainable food systems” [5]. This article considers a more restrictive definition that focuses on the farm level: “The study of the interactions between plants, animals, humans, and the environment within agricultural systems” [6]. Agroecological practices aim to increase levels of ecosystem services in order to sustain production while decreasing environmental impacts by decreasing anthropogenic inputs [7]. In particular, agroecological practices are based on diversifying the components of the farming system and increasing the interactions among them to close energy and material cycles [8]. Beyond studying agroecological practices and transitions [9], there is an urgent need to build awareness and teach agroecological concepts to future professionals of the agricultural sector (i.e., high-school and university students training to become farmers or extension agents), as well as current professionals [10,11]. However, most agricultural programs in European countries are not completely adapted to teach these concepts, due to a double weakness. First, their courses often fall into a narrow range of specialized disciplines, each of which focuses on its specific subjects. This approach does not train students to address complex agricultural issues, which generally need to be addressed through an interdisciplinary approach to farm management that includes animal and veterinary sciences, agronomy, applied ecology, and soil, environmental, economic, and social sciences [12]. Closely related to the lack of interdisciplinarity, agricultural courses do not develop enough systems approaches: They address most agricultural issues using a narrow range of methods and approaches that do not represent the complex relationships between farming practices, agricultural production, environmental impacts, sociological consequences, and economic results [13]. Indeed, interdisciplinary and systems approaches are now identified as founding principles of educational programs in sustainable agriculture [14,15]. Learning agroecology is not easy, however, since a variety of lock-in mechanisms may appear [16]. In particular, “cognitive lock-in” has been demonstrated in relation to agroecology [17], in which previous knowledge, skills, and personal beliefs may decrease students’ motivation to acquire new knowledge. In addition, current pedagogical methods often lack interactive and experiential dimensions [18,19]. Article Learning Interdisciplinarity and Systems Approaches in Agroecology: Experience with the Serious Game SEGAE Its pedagogical interest was evaluated through two types of surveys given to university students who played the game during a one-week workshop: A knowledge survey on agroecology, and a feedback survey based on flow theory. Results showed that students increased their knowledge of agroecology significantly, particularly those who had had little knowledge of crop production. More than 86% of the students enjoyed the game, appreciating its interaction and feedback. Thus, SEGAE is an interesting tool to help students acquire knowledge of agroecology in a fun way by facilitating interdisciplinary and collaborative learning. www.mdpi.com/journal/sustainability www.mdpi.com/journal/sustainability Sustainability 2020, 12, 4351; doi:10.3390/su12114351 2 of 15 Sustainability 2020, 12, 4351 Keywords: serious game; agroecology; systems thinking; interdisciplinarity; farming system 1. Introduction Theories on learning suggest that it is more effective when it is active, experiential, situated and problem-based, and provides immediate feedback [20]. Serious games based on digital tools can exhibit these features, which may explain why they have grown rapidly in recent years [21]. They are “designed experiences” in which players can learn through doing and being, rather than assimilating information from reading and lectures [22]. In addition, by simulating complex models or providing control over parameters that usually cannot be monitored, they can allow players to improve their understanding of systems approaches [23]. Finally, by offering players the ability to envision themselves in the future and see consequences of actions instantly, serious games develop capacities to design the future through “visioning thinking”, which can ease assimilation of innovative techniques such as agroecological practices [24]. Nonetheless, few serious games are related to agriculture, and even fewer use interdisciplinary and systems approaches to address agroecological issues. For instance, the game Azteca Chess addresses agroecological practices for shade-coffee production, but focuses on ecological complexity, ignoring farming system management [25]. Certain serious games focus on animal production without considering crop production or socio-economic dimensions [26]. In contrast, other serious games do connect crop and livestock management, but are based on board games, which limits 3 of 15 Sustainability 2020, 12, 4351 interactivity and visioning thinking [27–29]. Finally, based on our experience, most existing serious games do not seem to be adaptable to a variety of pedagogical objectives (e.g., learning interdisciplinary approach, systems thinking, multicriteria assessment of agroecological practices, or the evaluation of farming system sustainability). Given the urgent need to develop more sustainable agriculture and increase the attractiveness of active-learning tools, serious computer games on agroecology can be useful learning tools. However, their pedagogical interest should be assessed to ensure their quality and usefulness. They can be evaluated using a variety of theories [30], including flow theory. “Flow” is a state of profound enjoyment and concentration that increases students’ ability to learn [31]. To reach this state of mind, clear goals, immediate feedback, and a good balance between challenges and players’ skills are essential [32]. This theory has been extended to evaluate serious computer games by considering social interactions and measuring knowledge acquisition more precisely [33,34]. 1. Introduction g g q p y The serious game SEGAE (SErious Game for AgroEcology learning) is the main output of the Erasmus+ SEGAE project, a three-year project that associated six European universities from Belgium, France, Italy, and Poland, and was funded with the support of the European Commission and the French Chair of Agroecology. The aim of this study is to present SEGAE and to assess its pedagogical interest for learning agroecology. To do so, a workshop was organized with students from the partner universities. Two types of surveys were performed: (i) A knowledge survey to evaluate students’ individual knowledge of agroecology before and after the workshop, and (ii) a feedback survey to evaluate students’ perception of the workshop and the game, based in particular on the flow theory. We hypothesized that SEGAE would improve university students’ learning about interdisciplinary and systems approaches in sustainable management of mixed crop–livestock farming systems, particularly students who specialize in specific disciplines related to the functioning of farming systems. The main conclusion is that SEGAE is a relevant tool for learning agroecology in a fun way, since students increased their knowledge of agroecology significantly while generally enjoying the game. 2.1. Development of SEGAE 2.1. Development of SEGAE 2.1.1. Overall Description 2.1.1. Overall Description SEGAE is an online farming simulation game that addresses three main pedagogical objectives. First, players should acquire practical knowledge by learning a variety of agroecological practices and understanding their impacts on the farming system. This practical knowledge is by definition interdisciplinary, since the game includes several scientific disciplines (i.e., plant science, animal and veterinary sciences, soil science, ecology, economic and social sciences). Second, players should acquire a systems approach by assessing the combined impacts of multiple practices on the farming system. Third, they should improve their skills in transition management by testing several options to reach given goals with limited resources. An important feature related to these three objectives is the lack of an automatic balance among choices: Players must verify for themselves the consistency of the agroecological practices they choose (e.g., the cropping system must have sufficient grassland area if the livestock feeding system is based on grass). SEGAE represents a mixed crop–livestock farm oriented to dairy production (Figure 1). This type of farm was chosen because of its importance in all partner countries and the number of ways in which it can develop agroecology, especially by varying the degree of integration between crops and livestock [8]. For the workshop and this article, the farm corresponded to a typical dairy farm from western France, but SEGAE can represent dairy farms from each partner country, or elsewhere. SEGAE simulates crop and livestock management realistically at an annual time step, but it cannot be used as a decision aid tool for real farms, since it is not detailed enough to provide context-relevant results or represent all important biophysical and socio-economic processes that occur on a farm. SEGAE’s specific features are to (i) represent a variety of agroecological practices related to crop and 4 of 15 Sustainability 2020, 12, 4351 livestock management and (ii) give players the ability to assess impacts of these practices on the three pillars of sustainability: Environmental, economic, and social. SEGAE is available online at no cost at https://rebrand.ly/SEGAE. p y Figure 1. The graphical interface of SEGAE (SErious Game for AgroEcology) shows strategic dimensions and a sustainability score of a mixed crop and dairy farm. Figure 1. The graphical interface of SEGAE (SErious Game for AgroEcology) shows strategic dimensions and a sustainability score of a mixed crop and dairy farm. 2.1.2. Underlying Model SEGAE is based on an innovative model created for the game that has three components: (i) A matrix, (ii) a calculation engine, and (iii) a graphical interface that represents the farm. The matrix represents impacts of 124 practices on 591 indicators. Most of the practices are agroecological practices that stemmed from two studies [7,35] and were debated, chosen, and adapted by an interdisciplinary group of experts in the project. They are related to (i) crop management (i.e., rotations, tillage, fertilization, cultivar choice, weeding, pest and disease control), (ii) livestock management (i.e., animal health, feeding systems, manure management), (iii) management of the landscape and biodiversity (planned and natural), and (iv) strategic decisions (i.e., distribution of farm profit, conversion to organic farming, herd size, cattle breed). The indicators reflect technical, economic, environmental, and social aspects of the farm. Each practice influences one or more indicators through multiplicative or additive factors. These factors were determined from a literature review that included (i) meta-analysis (e.g., [36]), (ii) targeted analysis (e.g., [37]), (iii) analysis of more divergent case studies (e.g., [38]), and (iv) quantitative and qualitative expert assessment by experts in the project. Table 1 represents an extract of the matrix: It includes examples of practices for crop, livestock, and landscape management, and links them with examples of indicators reflecting the various aspects of the farm. Players do not see all 591 indicators, since some indicators are aggregated to provide more relevant information (e.g., the time spent on crop management is calculated for each crop each month, but only the total time for all crops each month is shown). Due to the model’s complexity, most practices impact several indicators, which helps players understand the many relations among the three pillars of sustainability: Environmental, economic, and social. Finally, scores of the three pillars are aggregated into an overall sustainability score. 5 of 15 5 of 15 Sustainability 2020, 12, 4351 Table 1. Extract from SEGAE’s matrix of impacts of farm practices on farm indicators. The impacts of practices are represented through multiplicative factors that modify the initial value of the related indicators. Table 1. Extract from SEGAE’s matrix of impacts of farm practices on farm indicators. The impacts of practices are represented through multiplicative factors that modify the initial value of the related indicators. p p p practices are represented through multiplicative factors that modify the initial value of the related indicators. 2.1.2. Underlying Model Category Practice Indicators Technical Environmental Social Economic Winter wheat yield Pollinator abundance Animal welfare Machinery cost Tillage management Conventional tillage 1.00 1.00 1.00 Reduced tillage 1.00 1.01 0.90 No tillage 0.92 1.03 0.80 Fungicides Systematic use of chemical molecules 1.00 1.00 1.00 Sound approach of chemical molecules 1.00 1.01 0.90 Biocontrol product 0.85 1.02 0.85 None 0.85 1.03 0.80 Temporary grassland Grassland-grass 1.00 Grassland-grass-legume mixture 1.02 Grassland-complex mixture 1.05 Cropping plana Maize-wheat 1.00 1.00 1.00 Rapeseed-wheat-maize-wheat 1.02 1.02 1.00 Faba bean-wheat-rapeseed-maize 1.03 1.04 1.10 Grassland (3 years)-maize-wheat 1.02 1.03 0.70 Grassland-maize-wheat-faba bean-rapeseed 1.03 1.05 0.80 Green infrastructurea None 0.80 Wildflower strip 1.30 Hedgerows 1.20 Grass margins 1.05 In-field agroforestry 1.10 Cow housing Tie-stalls with straw 1.00 Cubicles with straw 1.50 Cubicles with slatted floor 1.13 Free-stall (deep bedding) 1.65 Free-stall (slatted floor) 1.20 Feeding systema Indoors all year, maize, soybean 1.00 Indoors all year, maize + alfalfa, soybean 1.00 4 months grazing, soybean 1.31 8 months grazing, faba bean 1.63 9 months grazing, without supplement 1.70 Management of the risk of mastitisb Systematic antibiotic treatment 1.00 Selective antibiotic treatment 1.00 Preventive measures of hygiene 0.98 Notes: Green infrastructure, green infrastructure, feeding system: Not all practices related to this category are represented; management of the risk of mastitis: Only during the cow’s dry period. The calculation engine, programmed in JavaScript, has equations for crop, livestock, and economic management that calculate impacts of all practices each year of the simulation. It also compiles the indicators’ scores to calculate the score of each pillar of sustainability. The code of the calculation engine is open source, which allows future users to add new developments or reuse it in other software. The graphical interface represents the farm’s strategic dimensions: Cow management, crop management, feeding system, fertilization, heifer and fattening cattle management, landscape management, land use management, and strategic decisions (Figure 1). By clicking on each strategic dimension, players can choose from a list of related practices. Then, by clicking on the “Next year” button, the game applies the choices, calculates impacts on indicators, and displays a set of steering indicators. Sustainability 2020, 12, 4351 6 of 15 2.2. Evaluation of SEGAE SEGAE workshop was organized during the project with students and teachers from the partner universities. Its objective was to evaluate SEGAE’s interest as a learning tool, in particular its ability to (i) help students learn agroecology and (ii) put them into a state of flow. Two other objectives— feedback from students on the design of attractive training sessions and identification of game bugs and weaknesses—will be used to improve SEGAE, but are not described here. 2.1.3. Pedagogical Activities Pedagogical activities offered with SEGAE should include (i) presentation of the learning objectives and an overview of the game, (ii) playing the game with a scenario adapted to the context, and (iii) discussion of the results, methodology, and limits of the game with the teacher. Due to SEGAE’s high customization capacity, teachers can develop a wide variety of pedagogical scenarios. We imagined four scenarios to reach different pedagogical goals, but others can be proposed: g p y p y p g g We imagined four scenarios to reach different pedagogical goals, but others can be proposed:  “Sandbox”: Players explore the game’s strategic dimensions and related practices, with the easy goal of simply improving sustainability. This scenario helps players begin to understand impacts of practices and relations between animal and crop production.  “Sandbox”: Players explore the game’s strategic dimensions and related practices, with the easy goal of simply improving sustainability. This scenario helps players begin to understand impacts of practices and relations between animal and crop production.  “Systems thinking”: Players must improve overall sustainability by changing practices related to either crop or animal production. Usually, the improvement in overall sustainability is lower in this scenario than in the “sandbox”. This scenario helps players understand more deeply the close interconnections between crop and animal production.  “Indicator oriented”: Players must improve a specific indicator of the farm (e.g., animal welfare) by developing a step-by-step strategy with other players. This scenario allows players to exchange viewpoints and knowledge, so it can be particularly interesting for a group of students with different backgrounds.  “Sustainability oriented”: Players must improve overall sustainability without decreasing the score of each pillar below a certain threshold. It can be played alone or by small groups of students. This scenario helps players more deeply understand potential trade-offs and synergies among the pillars of sustainability. 2.2.1. The Workshop Notes: ESA = Graduate school of Agricultures; Institut Agro = National Institute of Education for Agriculture, Food and Environment; Oniris = National Veterinary, Agrifood and Food School of Nantes-Atlantique; ULiège = University of Liège; UAK = University of Agriculture in Krakow, UNIBO = University of Bologna. 2.2.1. The Workshop The workshop was held at the Department of Agricultural and Food Sciences, of the University of Bologna (UNIBO) in Italy, during one week (3–7 February 2020). It consisted of three complementary sessions. First, a half-day farm visit was organized to understand the use of agroecological practices in the field. It also allowed students to discuss with farmers their economic strategy for developing agroecology and its positive and negative consequences. Second, six hours of lectures were provided on the main aspects of agroecology (i.e., crop production, animal production, agricultural ecology, economics, and sociology), followed by an additional lecture on sustainability assessment. These lectures highlighted connections between theoretical knowledge and the practices simulated in the game. Third, six hours of game sessions were organized that consisted of a variety of activities, including the four pedagogical scenarios. The workshop assembled 52 students and 15 teachers from the partner universities. The sample of students was diverse, ranging from first-year Master’s students to doctoral students, with most students in the second year of a Master’s degree. The disciplines they studied were agroecology, animal science, crop science, economics applied to agriculture, environmental science, mechatronics, and veterinary science (Table 2). Some students were specialized in their field (i.e., those in veterinary science or, from UNIBO, crop science), while the others were generalists who followed an agricultural engineering education that included agriculture-related disciplines. 7 of 15 Sustainability 2020, 12, 4351 7 of 15 Table 2. Curriculum and discipline of specialization of the 52 students in the intensive one-week training session. Table 2. Curriculum and discipline of specialization of the 52 students in the intensive one-week training session. University Country Engineering (multidisciplinary) Veterinary science Crop science Agroecology Animal science Crop science Economics Environmental science Mechatronics ESA France 3 6 Institut Agro France 4 2 3 Oniris France 8 ULiège Belgium 2 4 3 UAK Poland 3 4 1 UNIBO Italy 9 TOTAL 6 6 12 3 3 4 9 9 Notes: ESA = Graduate school of Agricultures; Institut Agro = National Institute of Education for Agriculture, Food and Environment; Oniris = National Veterinary, Agrifood and Food School of Nantes-Atlantique; ULiège = University of Liège; UAK = University of Agriculture in Krakow, UNIBO = University of Bologna. 2.2.2. Design of Surveys One of the positive outcomes of Erasmus+ projects for students, regardless of the topic, is that they meet other European students, discover other ways of thinking and, more generally, open their minds. It was collectively decided to keep the students in a single group to enhance peer-to-peer exchanges and social mixing among different origins, cultures, and disciplines. Two types of surveys were performed during the workshop. First, a knowledge survey was performed to evaluate students’ individual knowledge of agroecology, since they came from different academic levels and disciplines. This evaluation was based on summative assessments that used pre- and post-testing, a common approach to test effects of new teaching methods [39]. Students answered the knowledge survey once at the beginning of the week, before any lectures or game sessions (hereafter, “pre-test”), and a second time, with the same questions, at the end of the week, after all lectures and game sessions (hereafter, “post-test”). The knowledge survey was answered by the all the students, except four French students in agroecology since they had helped to develop it. The knowledge survey contained 21 multiple-choice or open-ended questions: 10 specific to crop production, 4 specific to animal production, and 7 general questions (Table S1). For each multiple- choice question, the student earned 1 point if the answer was correct, 0 points for no answer, and –1 point if the answer was incorrect. For each open-ended question, the student earned from –1 to 3 points depending on the number of correct (or incorrect) answers given compared to the number of expected correct answers. To ease interpretation of results, students’ scores were converted into a percentage of the maximum score, which was 39 points. The second type of survey, the feedback survey, was performed to evaluate students’ perception of the game and the workshop. In particular, it allowed us to characterize the states of flow that they experienced during the game, using the eight factors of EgameFlow of Fu et al. [34]:  Concentration: The game must provide activities that encourage players’ concentration while minimizing stress from learning overload.  Concentration: The game must provide activities that encourage players’ concentration while minimizing stress from learning overload. Clear goal: Tasks should be clearly defined at the beginning of game sessions.  Clear goal: Tasks should be clearly defined at the beginning of game sessions. 2.2.3. Analysis of Survey Results Analysis of survey results consisted mainly of analyzing students’ scores. For the knowledge survey, scores were summed to calculate students’ pre-test and post-test scores. Mean scores were calculated for the group of all students, by curriculum and discipline, and by theme of the questions. Statistical analysis was then performed using R software v.3.6.1 (Foundation for Statistical Computing, Vienna, Austria). [40]. First, descriptive statistics (e.g., mean, median, standard deviation) of scores and student descriptors (e.g., age, agroecology background) were calculated. Next, equivalence tests with paired data were used to calculate the change in scores between the pre- test and post-test. Finally, Pearson correlation coefficients and one-way analysis of variance were calculated to assess the significance of observed differences. For the feedback survey, each flow factor was assessed by averaging the scores of its questions. Thus, each student was associated with eight mean scores, one for each flow factor. These eight scores were then averaged to obtain an overall assessment of the flow factors. Mean scores were calculated for the group of all students and by discipline. Then, to confirm the results, principal component analysis (PCA) of the mean values of the eight flow factors for each student (i.e., 50 × 8 matrix) was performed. It helped us understand students' perception and explanatory variables more precisely, which could explain the perception of flow. 2.2.2. Design of Surveys  Feedback: Regular feedback should be given to allow players to determine the gap Feedback: Regular feedback should be given to allow players to determine the gap between t current stage of knowledge and the target stage. g g g g  Challenge: The game should offer challenges that fit players’ skill levels.  Autonomy: Players should enjoy taking initiatives in game sessions and asserting total control over their choices.  Immersion: The game should lead players into a state of immersion, which is characterized in particular by an altered sense of time. Social interaction: Tasks in the game should favor social interactions between players.  Knowledge acquisition: The game should increase players’ knowledge. 8 of 15 Sustainability 2020, 12, 4351 Students answered the feedback survey once at the end of the workshop, after all lectures and game sessions. Results of the feedback survey were not collected for two students due to technical issues. The feedback survey contained four open-ended questions about the workshop and a Likert- type inventory of 45 statements: 2–5 statements to assess each of the eight flow factors (e.g., “the overall game goals were presented clearly” assesses the factor “clear goal”) and additional statements to assess the game more generally (Table S2). Students evaluated statements on a four-point scale (1 = strongly disagree, 2 = disagree, 3 = agree, and 4 = strongly agree). The feedback survey also contained background questions to describe students (e.g., age, nationality, level of studies) and their experience in agroecology and gaming (Table S3). 3.1. Results of Knowledge Acquisition On the knowledge survey pre-test, students had a mean score of 43% of answers correct. The scores differed significantly among the disciplines: Students in crop science had the highest scores (51%), while those in mechatronics had the lowest (14%). On the post-test, students’ mean score was significantly (p < 0.001) higher: 51% of answers correct. Overall, 33 of 48 students increased their scores, 6 had the same scores, and 9 decreased their scores slightly (Figure 2). In addition, scores of pre- and post-tests were strongly positively correlated (r = 0.69; p < 0.001); thus, students who had the highest pre-test scores also had the highest post-test scores. 9 of 15 Sustainability 2020, 12, 4351 y , , Figure 2. Comparison of students’ mean scores on the pre-test and post-test knowledge surveys, by their curriculum: Crop science (UNIBO), multidisciplinary (all engineering students), and veterinary science (Oniris and UAK). 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Post-test score (% of answers correct) Pre-test score (% of answers correct) Crop science Multidisciplinary Veterinary science 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Post-test score (% of answers correct) Pre-test score (% of answers correct) Figure 2. Comparison of students’ mean scores on the pre-test and post-test knowledge surveys, by their curriculum: Crop science (UNIBO), multidisciplinary (all engineering students), and veterinary science (Oniris and UAK). All student groups increased their scores significantly, but there was no significant difference in the mean increase among groups (Figure 3; p = 0.10). Although not significant, students in specialized curricula (i.e., veterinary science and crop science) increased their scores more than those in other curricula (by a mean of 14 and 11 percentage points, respectively), which allowed them to catch up with the engineering students and obtain a mean score higher than the overall mean (Table A1, Appendix A). Figure 3. Boxplots of the change in students’ scores on the knowledge survey between the pre-test and post-test, by their curriculum: Crop science (UNIBO), multidisciplinary (all engineering students), and veterinary science (Oniris and UAK). Whiskers show 1.5 times the interquartile range. Figure 3. 3.2. Perception of SEGAE and its Evaluation Through Flow Factors On the feedback survey, 86% of students “agreed” or “strongly agreed” with the statements “I enjoyed playing this game” and “I will advise students from my university to play this simulation game”. Thus, students appreciated SEGAE overall. More precisely, students assessed the flow factors positively, with an overall mean score of 2.96 points (i.e., ≈ “agree”) out of 4. The factors “knowledge acquisition”, “interaction”, and “feedback” were rated particularly highly (Figure 4). Two groups of students differed in their assessment of flow factors. Students in economics gave higher scores for most of the factors, especially knowledge acquisition (3.70 points out of 4). In contrast, students in environmental science were more critical, highlighting a lack of concentration during the game and low clarity of its goal (means of 2.17 and 2.33 points, respectively). Figure 4. Mean scores of the eight flow factors by students on the feedback survey, including those of the two groups of students that gave the highest and lowest scores (in economics and environmental science, respectively). Figure 4. Mean scores of the eight flow factors by students on the feedback survey, including those of the two groups of students that gave the highest and lowest scores (in economics and environmental science, respectively). The first dimension of the PCA (inertia of 51%) was correlated significantly with the eight flow factors (p < 0.001), all of which had r > 0.5. Students gave similar scores for most of the flow factors (i.e., most high or most low). Since the second dimension of the PCA had an inertia of only 10%, it was excluded from further analysis. Four qualitative variables were strongly and significantly correlated with the mean flow score. The first, “discipline” (r = 0.51; p < 0.05), showed that economics and veterinary science students reached higher flow states than environmental science students. The second, agreement with the statement “I will advise students from my university to play this simulation game” (r = 0.51; p < 0.05), showed that students who reached higher flow states were more willing to recommend the game. Finally, the two statements “I learned about an integrated vision of agriculture” (r = 0.73; p < 0.001) and “I learned about agroecology as an interactive set of agricultural practices” (r = 0.57; p < 0.001) were significantly correlated with the PCA dimension. 3.1. Results of Knowledge Acquisition Boxplots of the change in students’ scores on the knowledge survey between the pre-test and post-test, by their curriculum: Crop science (UNIBO), multidisciplinary (all engineering students), and veterinary science (Oniris and UAK). Whiskers show 1.5 times the interquartile range. Sustainability 2020, 12, 4351 10 of 15 Students in agroecology and crop science (in engineering or specialized in crop science) had the highest scores. Although mechatronics students increased their mean score by nearly 50% (6 percentage points), it remained the lowest (i.e., 21% of answers correct). By theme of the questions, the mean score increased the most overall for animal-production questions (i.e., by 12 percentage points). 4. Discussion Results showed that students increased their knowledge of agroecology significantly, with a mean increase of nine percentage points in their scores on the knowledge survey. This confirms the relevance of serious games as learning tools to teach complex concepts [41]. Nonetheless, students’ mean score remained low (51% of answers correct). This result may be explained by students having given many vague answers to the open-ended questions, which were not considered correct. The survey may also have been too difficult, since teachers and four second-year Master’s students in agroecology designed it so that it would not be too simple for agroecology students. In addition, the high diversity of the students was not optimal for developing activities that could help all of them increase their knowledge in a variety of disciplines. Students in veterinary science made the most progress, which shows that SEGAE is particularly useful for specialized students who lack a systems approach or knowledge of crop or animal production. Thus, it confirms the hypothesis that SEGAE helps specialized students acquire interdisciplinary knowledge in agroecology. The modest increase in performance of other students (i.e., in mechatronics), however, also shows limits of this learning tool. In this case, some students in mechatronics may have had difficulty getting involved in the game (their mean score for the flow factor “immersion” was 2.40 out of 4 points). They may also have suffered from a lack of knowledge of biology or problems understanding English. Thus, particular attention should be paid to students’ prerequisites and academic curricula to reach the pedagogical goals. In addition, to decrease language barriers, we plan to translate SEGAE into several languages. The knowledge survey had two drawbacks that could limit the scope of these results. First, the sizes of student groups varied greatly, from large (e.g., French students, crop science students) to small (e.g., mechatronics students). Second, there was no control group [42]. Although the lectures were designed to explain only SEGAE’s mechanisms and not to provide new knowledge, the lack of a control raises questions about whether the game helped students learn more than they would have with the lectures alone. To strengthen the validity of our conclusions, it would be necessary to compare the results of students who experienced only lectures to those who experienced both the lectures and the game. Regarding student perceptions, 86% of students enjoyed playing SEGAE, which shows high appreciation of the game. 3.2. Perception of SEGAE and its Evaluation Through Flow Factors Since these statements were included to assess interdisciplinarity, the correlation showed that students who experienced higher flow states also Sustainability 2020, 12, 4351 11 of 15 perceived that they had acquired knowledge of agroecology, in particular its interdisciplinary aspects. The scores on the knowledge survey and of flow factors were not significantly correlated. Thus, some students (e.g., those in economics) may have enjoyed and been involved in the serious game even if they did not increase their scores on the knowledge survey greatly. 4. Discussion They also assessed the eight flow factors positively overall, but some students identified difficulties with concentration, the clarity of goals, or the need for autonomy. These results highlight a slight difficulty in becoming immersed in the game, which may be due to insufficient guidelines on how to play it. Nonetheless, these results are encouraging, since the game was not completely finished by the time of the workshop. Since then, some “gamification” features have been added to SEGAE, including some requests from the workshop, which should improve students’ engagement with this learning tool. SEGAE is an interesting tool to help students learn agroecology in a fun way. By allowing players to apply agroecological practices directly on a virtual farm and then analyze their impacts, this game represents “learning by doing in silico”. It is particularly relevant because it helps overcome the knowledge–action gap, which is identified as a critical skill for learning agroecology [43]. Another interesting feature of SEGAE is that it highlights animal production, especially its reconnection to the land, as a core element of agroecology, which is rarely done in agroecological thinking [4]. However, the sets of practices in the game related to animal production, biodiversity management, and socio- economic decisions are less developed than that related to crop production. Thus, SEGAE could be improved by adding practices related to these aspects. In addition, some students would have appreciated having access to more theoretical information in the game to help them understand Sustainability 2020, 12, 4351 12 of 15 impacts of practices before applying them. We decided to limit theoretical information in the game to improve SEGAE’s playability, simplify its graphical interface, and encourage “learning by doing”. However, this design can make it difficult for undergraduate students to play the game and improve their understanding of relations between practices and impacts. Using SEGAE with undergraduate students requires pedagogical activities that allow the students to play the game step-by-step, including more directive scenarios than those described here. The initial goal of the SEGAE project was to build a serious game that would also be suitable for high-school students and extension agents. Using SEGAE with high-school students would require more preparation of game scenarios and perhaps simplification of the practices available and sustainability indicators, while being careful not to lose the game’s interdisciplinary or systems approaches. 4. Discussion In contrast, extension agents may be frustrated by SEGAE’s low flexibility, in particular the fact that players cannot create crop rotations or feeding rations but must choose them from a short list. Since SEGAE is available online at no cost, individuals can play it independently, but exchanges between students and teachers appear to be crucial to learn effectively and highlight the game’s limits. The wide range of practices available in the game, which highlights interactions among the three pillars of sustainability, shows that SEGAE favors an interdisciplinary approach. The game and its related pedagogical activities could be improved to move closer to a transdisciplinary approach. In particular, more non-academic participants, such as farmers and extension agents, should be included in lectures to enhance participatory approaches and thus transdisciplinarity [44]. Similarly, many students asked for more farm visits, but this could be difficult due to the universities’ economic and logistical constraints. As suggested by [45], however, additional case studies can be developed in the future; adapting the French farm currently in SEGAE to represent farms in each partner country is an initial step that is underway. Like SEGAE, other digital tools can be developed to learn agroecology. In particular, Massive Open Online Courses (MOOC) represent an interesting way to provide educational content similar to that of lectures to as many people as possible. A MOOC on agroecology is already available and complements SEGAE, since it offers more structured and theoretical content on agroecological practices [46]. Another interesting free online tool is the Dictionary of Agroecology [47], which contains simple definitions of terms related to agroecology, which are often poorly understood. It values knowledge in a participative way, allowing collaboration among researchers, students, and actors in the field, who illustrate the definitions with their experiences. Finally, many associations and extension services offer face-to-face training on agroecology for both novices and farmers. These initiatives, whether digital or not, contribute greatly to the agroecological transition of European agriculture and should become more closely interconnected to increase their effectiveness. 5. Conclusions European agriculture is facing many challenges, and agroecology is considered as a pertinent option to reconcile the various pillars of agricultural sustainability. However, most agricultural courses are not adapted to teach agroecological concepts. The objective of this study was to present SEGAE and to assess the pedagogical interest of this serious game for learning agroecology. Results showed that students increased their knowledge of agroecology significantly, with a mean increase of nine percentage points in their scores on the knowledge survey. Students in veterinary science made the most progress, which confirmed the hypothesis that SEGAE helps specialized students acquire interdisciplinary knowledge. However, the modest increase in performance of other students highlighted the need to check students’ prerequisites and academic curricula to reach the pedagogical goals. Regarding student perceptions, 86% of students enjoyed playing SEGAE, which shows high appreciation of the game. They particularly appreciated the available feedback and the possibility of interacting, but some students identified difficulties with concentration. Overall, it can be concluded that SEGAE is a relevant tool for learning agroecology in a fun way. Since this serious game is available online at no cost, it can be used in addition to other trainings on agroecology, whether digital or not, and thus contribute greatly to the agroecological transition of European agriculture. Sustainability 2020, 12, 4351 13 of 15 Supplementary Materials: The following are available online at www.mdpi.com/2071-1050/12/11/4351/s1, Table S1: Knowledge survey, Table S2: Feedback survey; Table S3: Database. Supplementary Materials: The following are available online at www.mdpi.com/2071-1050/12/11/4351/s1, Table S1: Knowledge survey, Table S2: Feedback survey; Table S3: Database. Author Contributions: J.J. and M.D.Gr. are co-first authors who contributed equally to this work. Individual contributions of co-authors are as follows: Conceptualization, O.G., J.J., and M.D.G.; methodology, J.J., M.D.G, M.Ca., O.G., T.S., R.B., N.B., S.B., D.B., G.B., S.C., M.Cu., B.D., A.L.J., S.M., J.M., G.P., A.R., B.T., and F.S.; software development, M.Ca. and O.G.; formal analysis, M.D.G.; data curation, M.D.G. and J.J.; writing—original draft preparation, J.J.; writing—review and editing, all co-authors; supervision, O.G.; project administration, O.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the European Commission through the Erasmus+ program (project no. 2017-1-FR01-KA203-037254) and by the French Chair of Agroecology. The article processing charge was funded by the European Commission. 5. Conclusions This publication is binding only on its authors, and the Commission is not responsible for any use which may be made of the information contained therein. Acknowledgments: The authors thank all students who participated in the workshop, especially the four students who helped develop the knowledge survey. They thank Anthony Tedde for his suggestions about database management and his explanations of some advanced functions of R. They also thank Yves Brostaux and Hélène Soyeurt for their advice on statistical analysis. Finally, they thank Michael Corson for proofreading the manuscript’s English. Mireille De Graeuwe gives personal thanks to Timothée Collin for his unconditional support, especially during writing of the article, which coincided with the Covid-19 pandemic. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Grouping of data (and number of students) Pre-test Post-test Increase All students 42.7 (2.4) 51.4 (2.3) 8.7 *** By curriculum  Veterinary science (9) 40.2 (2.6) 53.9 (3.9) 13.7 *  Crop science (9) 42.5 (3.0) 53.6 (3.7) 11.1 **  Multidisciplinary (30) 43.6 (3.6) 50.0 (3.3) 6.4 ** By discipline of specialization  Veterinary science (9) 40.2 (2.6) 53.9 (3.9) 13.7 *  Agroecology (2) 46.2 (2.6) 57.7 (1.3) 11.5 ns  Animal science (6) 35.9 (10.1) 45.7 (7.9) 9.8 ns  Environmental science (3) 36.8 (3.1) 45.3 (5.2) 8.6 ns  Crop science (21) 50.9 (2.8) 57.8 (2.3) 6.8**  Mechatronics (4) 14.1 (4.0) 20.5 (6.8) 6.4 ns  Economics (3) 48.7 (6.5) 53.9 (7.4) 5.1 ns By theme of the questions  Crop production 44.2 (3.0) 50.4 (2.3) 6.1 **  Animal production 52.9 (3.3) 65.1 (3.1) 12.2 ***  General 35.7 (2.8) 45.1 (3.2) 9.4 ** Notes: The “crop science” discipline includes engineering students in crop science and students specialized in crop science; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant (p > 0.05). 1. Garnett, T.; Appleby, M.C.; Balmford, A.; Bateman, I.J.; Benton, T.G.; Bloomer, P.; Burlingame, B.; Dawkins, M.; Dolan, L.; Fraser, D.; et al. Sustainable intensification in agriculture: Premises and policies. Science 2013, 341, 33–34, doi:10.1126/science.1234485. Appendix A Table A1. Mean (and standard error) of students’ scores (percentage of answers correct) on the knowledge survey during the pre-test and post-test, and the increase between the tests (percentage points). p Grouping of data (and number of students) Pre-test Post-test Increase All students 42.7 (2.4) 51.4 (2.3) 8.7 *** By curriculum  Veterinary science (9) 40.2 (2.6) 53.9 (3.9) 13.7 *  Crop science (9) 42.5 (3.0) 53.6 (3.7) 11.1 **  Multidisciplinary (30) 43.6 (3.6) 50.0 (3.3) 6.4 ** By discipline of specialization  Veterinary science (9) 40.2 (2.6) 53.9 (3.9) 13.7 *  Agroecology (2) 46.2 (2.6) 57.7 (1.3) 11.5 ns  Animal science (6) 35.9 (10.1) 45.7 (7.9) 9.8 ns  Environmental science (3) 36.8 (3.1) 45.3 (5.2) 8.6 ns  Crop science (21) 50.9 (2.8) 57.8 (2.3) 6.8**  Mechatronics (4) 14.1 (4.0) 20.5 (6.8) 6.4 ns  Economics (3) 48.7 (6.5) 53.9 (7.4) 5.1 ns By theme of the questions  Crop production 44.2 (3.0) 50.4 (2.3) 6.1 **  Animal production 52.9 (3.3) 65.1 (3.1) 12.2 ***  General 35.7 (2.8) 45.1 (3.2) 9.4 ** Notes: The “crop science” discipline includes engineering students in crop science and students specialized in crop science; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant (p > 0.05). 2. 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Outcomes following hematopoietic stem cell transplantation in patients treated with standard chemotherapy with or without gemtuzumab ozogamicin for acute myeloid leukemia
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Outcomes following hematopoietic stem cell transplantation in patients treated with standard chemotherapy with or without gemtuzumab ozogamicin for acute myeloid leukemia Cécile Pautas 1 ●Emmanuel Raffoux2 ●Juliette Lambert3 ●Ollivier Legrand4 ●Sylvain Chantepie5 ● Lauris Gastaud6 ●Jean-Pierre Marolleau7 ●Xavier Thomas8 ●Pascal Turlure9 ●Rebecca J. Benner10 ● Erik Vandendries11 ●Karïn Gogat12 ●Hervé Dombret2 ●Sylvie Castaigne13 Cécile Pautas 1 ●Emmanuel Raffoux2 ●Juliette Lambert3 ●Ollivier Legrand4 ●Sylvain Chantepie5 ● Lauris Gastaud6 ●Jean-Pierre Marolleau7 ●Xavier Thomas8 ●Pascal Turlure9 ●Rebecca J. Benner10 ● Erik Vandendries11 ●Karïn Gogat12 ●Hervé Dombret2 ●Sylvie Castaigne13 Received: 30 June 2020 / Revised: 17 November 2020 / Accepted: 15 December 2020 / Published online: 9 February 20 © The Author(s) 2021. This article is published with open access 6 Service hématologie, Centre Lacassagne, Nice, France 7 Service hématologie, CHU Amiens, Amiens, France 8 Service hématologie, Centre Hospitalier Lyon Sud, Lyon, France 9 Service hématologie et de thérapie cellulaire, CHU de Limoges, Limoges, France 10 Global Product Development, Pfizer Inc, Groton, CT, USA 11 Pfizer Inc, Cambridge, MA, USA 12 Global Clinical Development, Pfizer Inc, Paris, France 13 Service d’Hématologie et Oncologie, Centre Hospitalier de Versailles, Université de Versailles Saint Quentin, Le Chesnay, France Abstract The phase 3 ALFA-0701 trial demonstrated improved outcomes with fractionated-dose gemtuzumab ozogamicin (GO) combined with standard chemotherapy vs. standard chemotherapy alone in adults with de novo acute myeloid leukemia (AML). We examined post-transplant outcomes and occurrence of hepatic veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) in patients who received hematopoietic stem cell transplantation (HSCT) as follow-up therapy in ALFA-0701. Patients aged 50–70 years were randomized to standard chemotherapy with or without GO (3 mg/m2 on days 1, 4, and 7 of induction and day 1 on each of two consolidation courses). Allogeneic HSCT was recommended for patients in first complete remission with matched (related or unrelated) donor, except those with core-binding factor AML or normal karyotype and either NPM1+/FLT3-ITDwt or CEBPA+ AML. Eighty-five patients (GO: n = 32; control: n = 53) received HSCT in first complete remission or after relapse/primary induction failure. Three patients (GO: n = 2; control: n = 1 [received GO as follow-up therapy]) developed VOD/SOS after HSCT or conditioning. Post-transplant survival, non-relapse mortality, and relapse were not different between arms. Results indicate fractionated-dose GO as part of induction and consolidation chemotherapy for AML does not induce excess post-transplant VOD/SOS or mortality and thus does not preclude the use of HSCT as consolidation treatment. Gemtuzumab ozogamicin (GO), a humanized anti-CD33 monoclonal antibody linked to calicheamicin, is indicated for Gemtuzumab ozogamicin (GO), a humanized anti-CD33 monoclonal antibody linked to calicheamicin, is indicated for the treatment of adult and pediatric (aged ≥1 month) patients with newly diagnosed CD33-positive (CD33+) acute mye- loid leukemia (AML) and adult and pediatric (aged ≥2 years) patients with relapsed/refractory CD33+AML [1]. The phase 3 ALFA-0701 trial demonstrated significant improvement of event-free and relapse-free survival with fractionated-dose the treatment of adult and pediatric (aged ≥1 month) patients with newly diagnosed CD33-positive (CD33+) acute mye- loid leukemia (AML) and adult and pediatric (aged ≥2 years) patients with relapsed/refractory CD33+AML [1]. The phase 3 ALFA-0701 trial demonstrated significant improvement of event-free and relapse-free survival with fractionated-dose Supplementary information The online version of this article (https:// doi.org/10.1038/s41409-020-01207-4) contains supplementary material which is available to authorized users doi.org/10.1038/s41409-020-01207-4) contains supplementary material, which is available to authorized users. Bone Marrow Transplantation (2021) 56:1474–1477 https://doi.org/10.1038/s41409-020-01207-4 Bone Marrow Transplantation (2021) 56:1474–1477 https://doi.org/10.1038/s41409-020-01207-4 CORRESPONDENCE Abstract 6 Service hématologie, Centre Lacassagne, Nice, France 7 Service hématologie, CHU Amiens, Amiens, France 8 Service hématologie, Centre Hospitalier Lyon Sud, Lyon, France 9 Service hématologie et de thérapie cellulaire, CHU de Limoges, Limoges, France 10 Global Product Development, Pfizer Inc, Groton, CT, USA 11 Pfizer Inc, Cambridge, MA, USA 12 Global Clinical Development, Pfizer Inc, Paris, France 13 Service d’Hématologie et Oncologie, Centre Hospitalier de Versailles, Université de Versailles Saint Quentin, Le Chesnay, France * Cécile Pautas cecile.pautas@aphp.fr * Cécile Pautas cecile.pautas@aphp.fr 1 Service d’Hématologie et de thérapie cellulaire, Hôpital Henri Mondor, Créteil, France 2 Hôpital Saint-Louis (AP-HP), EA 3518, Université de Paris, Paris, France 3 Service d’Hématologie et Oncologie, Centre Hospitalier de Versailles, Le Chesnay, France 4 Service d’hématologie clinique et de thérapie cellulaire, Hôpital Saint-Antoine (AP-HP), Université Paris Pierre et Marie Curie, Paris, France 13 Service d’Hématologie et Oncologie, Centre Hospitalier de Versailles, Université de Versailles Saint Quentin, Le Chesnay, France 5 Institut d’Hématologie de Basse-Normandie, Caen, France Outcomes following hematopoietic stem cell transplantation in patients treated with standard. . . 1475 achieved CR in the GO (n = 29) and control (n = 44) arms, respectively, median time from first CR to HSCT was 6.4 and 7.8 months. GO added to standard chemotherapy vs. standard che- motherapy alone [2, 3]. Veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) is a concern with GO, particularly when administered before hematopoietic stem cell transplantation (HSCT) [4]. However, lower, fractio- nated GO dosing may mitigate this risk [5, 6]. Post-transplant/conditioning VOD/SOS occurred in three patients (GO: n = 2; control: n = 1; Table S2), for a rate of 6.5% (n = 2/31) among GO-treated patients in the GO arm and 7.7% (n = 3/39) among all patients receiving GO before HSCT. Of these, one patient in the GO arm first developed VOD/SOS during induction. GO was permanently discontinued. The patient recovered within ~1.5 months and subsequently received two allogeneic HSCTs with reduced-intensity conditioning. VOD/SOS occurred again after second HSCT from an HLA-matched donor (relatedness unknown) following rejection of first HSCT; symptoms included painful hepatomegaly and moderate cholestatic jaundice. No treatment was given. The patient fully recovered within ~1 month but died ~14.8 months after first HSCT. This retrospective analysis examined VOD/SOS and post-transplant outcomes in patients who received HSCT as follow-up therapy in ALFA-0701. Study design and patient eligibility have been described [3]. Abstract Briefly, 271 patients, aged 50–70 years, with previously untreated de novo AML were randomized to a 3 + 7 induction course of daunorubicin (days 1–3) and cytarabine (days 1–7) with or without GO 3 mg/m2 (maximum dose 5 mg; days 1, 4, and 7). Patients in complete remission (CR) following induction received two consolidation courses of daunor- ubicin and cytarabine with or without GO (3 mg/m2 on day 1) according to their initial randomization. Patients with delayed count recovery were not given GO for con- solidation. Allogeneic HSCT was recommended for patients in first CR with matched (related or unrelated) donor, except patients with core-binding factor AML or with normal karyotype and either NPM1+/FLT3-ITDwt or CEBPA+AML. A 2-month interval between last GO dose and HSCT was recommended. Conditioning type was left to the discretion of the transplant center. The second patient in the GO arm developed VOD/SOS after reduced-intensity conditioning but before allogeneic HSCT from an HLA-matched unrelated donor. Symptoms included weight gain, abdominal distension with frank ascites, peritoneal effusion revealed by ultrasound, and increased liver function tests with cytolysis. Severe sepsis was a concomitant event. Treatment included defibrotide. After progressive normalization of liver function tests within 10 days, the patient experienced a recurrence of cholestatic injury with hepatocellular injury associated with respiratory distress, leading to the patient’s death within a month of HSCT. VOD/SOS was not resolved at the time of death. Data on VOD/SOS were collected as described [2]. Diagnosis and grading were based on investigator judgment. VOD/SOS was classified as pre- or post-transplant relative to first HSCT date. Time-to-event endpoints were post- transplant survival, non-relapse mortality (NRM), relapse, and overall survival (OS; see Supplementary Methods). The third patient, who was in the control arm and received GO as follow-up therapy before HSCT, devel- oped VOD/SOS after autologous HSCT with myeloa- blative conditioning. Symptoms included hepatomegaly with signs of portal hypertension and intraperitoneal effusion with thickening of gallbladder walls revealed by abdomen ultrasound, and increased total bilirubin and liver enzymes. Treatment included furosemide and heparin. The patient fully recovered within 1 month and was still alive ~28.7 months after HSCT. ALFA-0701 was approved by the ethics committee of Saint-Germain en Laye, France, and institutional review board of the French Regulatory Agency, and conducted in compliance with the Declaration of Helsinki. All patients provided written informed consent (EudraCT Number, 2007-002933-36; ClinicalTrials.gov, NCT00927498). Abstract at risk GO arm Control arm 0 Cumulative incidence (%) 0 20 40 60 80 100 HR 1.69 (95% CI: 0.74–3.87) P = 0.2103 GO Control 32 51 n 28.7 (14.1–45.2) 21.6 (11.5–33.8) NRM Rate at End of Month 12, % (95% CI) B 6 12 18 24 30 36 42 48 54 B 31.4% (19.1–44.4). Post-transplant survival did not differ between arms in patients receiving HSCT in first CR or after relapse/induction failure (Fig. S1). OS did not differ between arms in patients who received HSCT (hazard ratio [HR] 0.97; 95% CI: 0.54–1.75; P = 0.9190; median OS: 34.0 vs. 32.5 months), but was longer in the GO vs. control arm in patients who did not receive HSCT (HR 0.68; 95% CI: 0.48–0.97; P = 0.0333; median OS: 23.7 vs. 14.9 months). Our findings indicated that fractionated-dose GO added to standard chemotherapy was associated with a low incidence of VOD/SOS after HSCT in this cohort. The rate of post-transplant/conditioning VOD/SOS in GO-exposed patients (7.7%) was consistent with other 0 32 51 Time (months) Cumulative incidence (%) 0 20 40 60 80 100 HR 1.69 (95% CI: 0.74–3.87) P = 0.2103 GO Control 32 51 n 28.7 (14.1–45.2) 21.6 (11.5–33.8) NRM Rate at End of Month 12, % (95% CI) GO Control 32 51 n 9.4 (2.3–22.6) 31.4 (19.1–44.4) Relapse rate at end of month 12, % (95% CI) 0 32 51 Time (months) Cumulative incidence (%) 0 20 40 60 80 100 HR 0.60 (95% CI: 0.27–1.33) P = 0.2181 B C 6 12 18 24 30 36 42 48 54 25 25 14 14 8 8 4 4 0 31 31 21 21 10 10 4 4 0 6 12 18 24 30 36 42 48 54 25 25 14 14 8 8 4 4 0 31 31 21 21 10 10 4 4 0 No. at risk GO arm Control arm No. at risk GO arm Control arm C GO Control 32 51 n 9.4 (2.3–22.6) 31.4 (19.1–44.4) Relapse rate at end of month 12, % (95% CI) 0 32 51 Time (months) Cumulative incidence (%) 0 20 40 60 80 100 HR 0.60 (95% CI: 0.27–1.33) P = 0.2181 C 6 12 18 24 30 36 42 48 54 25 25 14 14 8 8 4 4 0 31 31 21 21 10 10 4 4 0 No. at risk GO arm Control arm 31.4% (19.1–44.4). Abstract at risk GO arm Control arm 27 43 25 35 22 30 20 28 15 25 15 22 14 21 12 20 11 15 8 10 7 7 6 7 6 7 4 5 3 3 0 1 0 3 2 Time (months) survival did not differ HSCT i fit CR 0.68; 95% CI: 0.48–0.97; P = 0.0333; median OS: 23.7 vs. 14 9 th ) 0 32 51 Time (months) Cumulative incidence (%) 0 20 40 60 80 100 HR 1.69 (95% CI: 0.74–3.87) P = 0.2103 GO Control 32 51 n 28.7 (14.1–45.2) 21.6 (11.5–33.8) NRM Rate at End of Month 12, % (95% CI) GO Control 32 51 n 9.4 (2.3–22.6) 31.4 (19.1–44.4) Relapse rate at end of month 12, % (95% CI) 0 32 51 Time (months) Cumulative incidence (%) 0 20 40 60 80 100 HR 0.60 (95% CI: 0.27–1.33) P = 0.2181 B C 6 12 18 24 30 36 42 48 54 25 25 14 14 8 8 4 4 0 31 31 21 21 10 10 4 4 0 6 12 18 24 30 36 42 48 54 25 25 14 14 8 8 4 4 0 31 31 21 21 10 10 4 4 0 No. at risk GO arm Control arm No. at risk GO arm Control arm survival did not differ g HSCT in first CR or S1) OS did not differ 0.68; 95% CI: 0.48–0.97; P = 0.0333; median OS: 23.7 vs. 14.9 months). Our findings indicated that fractionated dose GO added 0 32 51 Time (months) Cumulative incidence (%) 0 20 40 60 80 100 HR 1.69 (95% CI: 0.74–3.87) P = 0.2103 GO Control 32 51 n 28.7 (14.1–45.2) 21.6 (11.5–33.8) NRM Rate at End of Month 12, % (95% CI) GO Control 32 51 n 9.4 (2.3–22.6) 31.4 (19.1–44.4) Relapse rate at end of month 12, % (95% CI) 0 32 51 Time (months) Cumulative incidence (%) 0 20 40 60 80 100 HR 0.60 (95% CI: 0.27–1.33) P = 0.2181 B C 6 12 18 24 30 36 42 48 54 25 25 14 14 8 8 4 4 0 31 31 21 21 10 10 4 4 0 6 12 18 24 30 36 42 48 54 25 25 14 14 8 8 4 4 0 31 31 21 21 10 10 4 4 0 No. at risk GO arm Control arm No. Abstract Eighty-five patients (GO: n = 32; control: n = 53) received HSCT as follow-up therapy. One patient received autologous HSCT. Eight patients in the control arm received GO as follow-up therapy before HSCT, and one patient in the GO arm received HSCT but not GO, totaling 39 patients (GO: n = 31; control: n = 8) who received GO before HSCT. VOD/SOS occurred before HSCT in three patients (GO: n = 2; control: n = 1; Table S3). Both patients in the GO arm developed VOD/SOS during induction and fully recovered. One of these patients developed VOD/SOS again after second HSCT (described above). The patient in the control arm developed VOD/SOS after follow-up therapy with GO and cytarabine and fully recovered. Baseline and transplant characteristics were generally similar between arms (Table S1). In the GO and controls arms, respectively, transplant was performed during first CR in 53.1 and 41.5%, after induction failure in 6.3 and 17.0%, and after relapse in 40.6 and 41.5% of patients. One patient received HSCT<2 months after last GO dose (as follow-up therapy). Most patients received reduced-intensity con- ditioning (GO: 78.1%; control: 75.5%). In patients who Overall, post-transplant outcomes did not differ between treatment arms (Fig. 1). In the GO vs. control arm, median survival was 21.4 vs. 17.1 months; the 12-month rate (95% confidence interval [CI]) of NRM was 28.7% (14.1–45.2) vs. 21.6% (11.5–33.8) and relapse 9.4% (2.3–22.6) vs. 1476 C. Pautas et al. A 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 54 51 48 Survival probability (%) GO Control 32 53 n 21.4 (9.4–NE) 17.1 (7.3–NE) Median OS, mo (95% CI) 39.4 (21.9–56.5) 44.5 (30.5–57.6) 3-year survival, % (95% CI) 100 90 80 70 60 50 40 30 20 10 0 Time (months) HR 0.97 (95% CI: 0.53–1.75) P = 0.9081 A 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 54 51 48 Survival probability (%) GO Control 32 53 n 21.4 (9.4–NE) 17.1 (7.3–NE) Median OS, mo (95% CI) 39.4 (21.9–56.5) 44.5 (30.5–57.6) 3-year survival, % (95% CI) 100 90 80 70 60 50 40 30 20 10 0 Time (months) HR 0.97 (95% CI: 0.53–1.75) P = 0.9081 A 32 53 No. Abstract Post-transplant survival did not differ between arms in patients receiving HSCT in first CR or after relapse/induction failure (Fig. S1). OS did not differ between arms in patients who received HSCT (hazard ratio [HR] 0.97; 95% CI: 0.54–1.75; P = 0.9190; median OS: 34.0 vs. 32.5 months), but was longer in the GO vs. control arm in patients who did not receive HSCT (HR 0.68; 95% CI: 0.48–0.97; P = 0.0333; median OS: 23.7 vs. 14.9 months). 0.68; 95% CI: 0.48–0.97; P = 0.0333; median OS: 23.7 vs. 14.9 months). Our findings indicated that fractionated-dose GO added to standard chemotherapy was associated with a low incidence of VOD/SOS after HSCT in this cohort. The rate of post-transplant/conditioning VOD/SOS in GO-exposed patients (7.7%) was consistent with other References 1. Pfizer Inc. Mylotarg (gemtuzumab ozogamicin) [prescribing informa- tion]. 2020. Available at: https://www.accessdata.fda.gov/drugsatfda_ docs/label/2020/761060s004lbl.pdf. In conclusion, these findings suggest that fractionated-dose GO as part of induction and consolidation chemotherapy for adult AML does not induce excess post-transplant mortality and VOD/SOS and thus does not preclude the use of HSCT as consolidation treatment following induction or salvage treatment. 2. Lambert J, Pautas C, Terre C, Raffoux E, Turlure P, Caillot D, et al. Gemtuzumab ozogamicin for de novo acute myeloid leukemia: final efficacy and safety updates from the open-label, phase III ALFA-0701 trial. Haematologica. 2019;104:113–9. 3. Castaigne S, Pautas C, Terre C, Raffoux E, Bordessoule D, Bastie JN, et al. Effect of gemtuzumab ozogamicin on survival of adult patients with de-novo acute myeloid leukaemia (ALFA-0701): a randomised, open-label, phase 3 study. Lancet. 2012;379:1508–16. Acknowledgements The authors would like to thank Regis Peffault de Latour for providing his expertise in HSCT procedure and contributing to patient care during the study. This study was sponsored by Pfizer. Medical writing support was provided by Emily Balevich, PhD, of Engage Scientific Solutions and was funded by Pfizer. 4. McKoy JM, Angelotta C, Bennett CL, Tallman MS, Wadleigh M, Evens AM, et al. Gemtuzumab ozogamicin-associated sinusoidal obstructive syndrome (SOS): an overview from the research on adverse drug events and reports (RADAR) project. Leuk Res. 2007;31:599–604. Data sharing statement Upon request, and subject to certain criteria, conditions, and exceptions (see for more information), Pfizer will provide access to individual de-identified participant data from Pfizer- sponsored global interventional clinical studies conducted for medi- cines, vaccines, and medical devices (1) for indications that have been approved in the US and/or EU or (2) in programs that have been terminated (i.e., development for all indications has been dis- continued). Pfizer will also consider requests for the protocol, data dictionary, and statistical analysis plan. Data may be requested from Pfizer trials 24 months after study completion. The de-identified par- ticipant data will be made available to researchers whose proposals meet the research criteria and other conditions, and for which an exception does not apply, via a secure portal. To gain access, data requestors must enter into a data access agreement with Pfizer. 5. Burnett AK, Russell N, Hills RK, Cavenagh J, Kell J, Jones G, et al. Outcomes following hematopoietic stem cell transplantation in patients treated with standard. . . 1477 received consultant fees and honoraria from Pfizer. The other authors have no conflicts of interest to declare. received consultant fees and honoraria from Pfizer. The other authors have no conflicts of interest to declare. recent retrospective reports of GO-exposed patients who received HSCT [7, 8]. Fractionated dosing, the use of reduced-intensity conditioning in most patients, and the 2- month interval between last GO dose and HSCT, followed in all but one patient, may have contributed to our low observed rate. Another small study of patients with high- risk, relapsed/refractory AML who received fractionated- dose GO plus chemotherapy found that no patient who underwent allogeneic HSCT—the majority with reduced-intensity conditioning—developed VOD/SOS [6]. It should be noted that VOD/SOS prophylaxis data were not systematically collected in our study; therefore, we cannot exclude the possibility that some patients may have received prophylactic defibrotide. 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. Post-transplant outcomes were similar between arms, although the small sample size may have precluded the detection of small differences in outcomes. Alternatively, transplant may have eliminated any differences in outcomes between arms, as OS was improved in the GO vs. control arm in patients who did not receive HSCT, but did not differ between arms in patients who received HSCT. References A comparison of single dose gemtuzumab ozogamicin 3 mg/m2 and 6 mg/m2 combined with induction chemotherapy in younger patients with AML: Data from the UK NCRI AML17 trial. Blood. 2014; 124:2308–2308. 6. Paubelle E, Ducastelle-Lepretre S, Labussiere-Wallet H, Nicolini FE, Barraco F, Plesa A, et al. Fractionated gemtuzumab ozogamicin combined with intermediate-dose cytarabine and daunorubicin as salvage therapy in very high-risk AML patients: a bridge to reduced intensity conditioning transplant? Ann Hematol. 2017;96:363–71. 7. Battipaglia G, Labopin M, Candoni A, Fanin R, El Cheikh J, Blaise D, et al. Risk of sinusoidal obstruction syndrome in allogeneic stem cell transplantation after prior gemtuzumab ozogamicin treatment: a retrospective study from the Acute Leukemia Working Party of the EBMT. Bone Marrow Transpl. 2017;52:592–9. Compliance with ethical standards 8. Ho VT, St Martin A, Perez WS, Steinert P, Zhang MJ, Chirnomas D, et al. Prior gemtuzumab ozogamicin exposure in adults with acute myeloid leukemia does not increase hepatic veno-occlusive disease risk after allogeneic hematopoietic cell transplantation: a center for international blood and marrow transplant research analysis. Biol Blood Marrow Transplant. 2020;26:884–92. 8. Ho VT, St Martin A, Perez WS, Steinert P, Zhang MJ, Chirnomas D, et al. Prior gemtuzumab ozogamicin exposure in adults with acute myeloid leukemia does not increase hepatic veno-occlusive disease risk after allogeneic hematopoietic cell transplantation: a center for international blood and marrow transplant research analysis. Biol Blood Marrow Transplant. 2020;26:884–92. Conflict of interest CP has received travel grants from Pfizer and served on advisory boards for Pfizer. RJB, EV, and KG are employees of and own stock in Pfizer. HD has received research funding and personal fees for participating on advisory boards from Pfizer. SCa has
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Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae
Frontiers in plant science
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Keywords: secretome, Sclerotinia, psychrophily, effector candidates, amino acid usage, intrinsic disorder, antifreeze protein, lytic polysaccharide monooxygenase Edited by: Fungal plant pathogens produce secreted proteins adapted to function outside fungal cells to facilitate colonization of their hosts. In many cases such as for fungi from the Sclerotiniaceae family the repertoire and function of secreted proteins remains elusive. In the Sclerotiniaceae, whereas Sclerotinia sclerotiorum and Botrytis cinerea are cosmopolitan broad host-range plant pathogens, Sclerotinia borealis has a psychrophilic lifestyle with a low optimal growth temperature, a narrow host range and geographic distribution. To spread successfully, S. borealis must synthesize proteins adapted to function in its specific environment. The search for signatures of adaptation to S. borealis lifestyle may therefore help revealing proteins critical for colonization of the environment by Sclerotiniaceae fungi. Here, we analyzed amino acids usage and intrinsic protein disorder in alignments of groups of orthologous proteins from the three Sclerotiniaceae species. We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are significantly associated with S. borealis proteins. We designed an index to report bias in these properties and found that high index proteins were enriched among secreted proteins in the three Sclerotiniaceae fungi. High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identified through our pipeline as candidate proteins involved in colonization of the environment. Our findings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases. Reviewed by: Jana Sperschneider, Commonwealth Scientific and Industrial Research Organisation, Australia Kim Marilyn Plummer, La Trobe University, Australia *Correspondence: Sylvain Raffaele, Laboratoire des Interactions Plante Micro-organismes, 24 Chemin de Borde Rouge – Auzeville, 31326 Castanet Tolosan, France sylvain.raffaele@toulouse.inra.fr Specialty section: This article was submitted to Plant Biotic Interactions, a section of the journal Frontiers in Plant Science Received: 23 June 2015 Accepted: 10 September 2015 Published: 24 September 2015 ORIGINAL RESEARCH published: 24 September 2015 doi: 10.3389/fpls.2015.00776 Thomas Badet 1, 2, Rémi Peyraud 1, 2 and Sylvain Raffaele 1, 2* 1 Laboratoire des Interactions Plantes-Microorganismes, Institut National de la Recherche Agronomique, UMR441, Castanet-Tolosan, France, 2 Laboratoire des Interactions Plantes-Microorganismes, Centre National de la Recherche Scientifique, UMR2594, Castanet-Tolosan, France Edited by: Delphine Vincent, Department of Environment and Primary Industries, Australia Introduction Genes involved in host-parasite interactions such as pathogen effectors are often subject to strong balancing or directional selection. For example, oomycete effectors commonly evolve rapidly, and natural selection can maintain many different alleles in a population (Raffaele et al., 2010; Oliva et al., 2015). Therefore, signatures of positive selection are frequent in effector genes and this property has been used to identify novel effector candidates (Wicker et al., 2013; Rech et al., 2014; Sperschneider et al., 2014). However, most of our understanding of the molecular evolution of effector genes and genes involved in colonization of the environment comes from studies of the pairwise co- evolution of a given pathogen with a single host plant. By contrast, fungal pathogens in the Sclerotiniaceae interact with a wide range of hosts in multiple environmental conditions and should therefore be considered as evolving under “diffuse” (or “generalized”) interactions (Juenger and Bergelson, 1998). In the Ascomycete genus Metarhizium, signatures of positive selection were observed less frequently in the genome of fungal pathogens FIGURE 1 | Sclerotinia borealis colonizes different niches than its close relatives S. sclerotiorum and Botrytis cinerea. Number of host plant genera (A) and geographic distribution (B) of the three fungal species according to the USDA Systematic Mycology and Microbiology Laboratory Fungus-Host Database (Farr and Rossman, 2015). under diffuse co-evolution compared to Metarhizium acridum evolving under pairwise co-evolution (Hu et al., 2014). It is thus expected that in the Sclerotiniaceae, some genes important for colonization of environment, including fungal effectors involved in diffuse interactions, may escape detection by positive selection analysis, and additional detection criteria would be useful. Compared to B. cinerea and S. sclerotiorum, the snow mold pathogen Sclerotinia borealis colonizes a reduced range of environments. Indeed, according to the Fungus-Host database of the U.S. Department of Agriculture (Farr and Rossman, 2015), S. borealis has been reported to infect 14 plant genera only, compared to 332 and 469 for S. sclerotiorum and B. cinerea respectively (Figure 1A). S. borealis host plants include notably Agropyron, Agrostis, Elymus, and Festuca species that have not been reported as hosts for S. sclerotiorum or B. cinerea to date. S. borealis has an economic impact in countries with cold climates, where it causes snow mold on winter cereals and grasses (Schneider and Seaman, 1987). Its geographic range is restricted to the Arctic Circle, including North of Japan, North America, Scandinavia, and Russia, whereas B. cinerea and S. Introduction FIGURE 1 | Sclerotinia borealis colonizes different niches than its close relatives S. sclerotiorum and Botrytis cinerea. Number of host plant genera (A) and geographic distribution (B) of the three fungal species according to the USDA Systematic Mycology and Microbiology Laboratory Fungus-Host Database (Farr and Rossman, 2015). Fungi from the Sclerotiniaceae family include several devastating plant pathogens with a broad host range. Among those are Botrytis cinerea, the causal agent of gray rot, and Sclerotinia sclerotiorum, causal agent of white and stem rot, each able to infect several hundreds of plant genera and causing multi- billion dollar losses in agriculture every year (Figure 1A) (Bolton et al., 2006; Dean et al., 2012). The geographic distribution of these two fungi is also remarkably broad since they have been reported across five continents (Figure 1B). Sequencing of the genome of B. cinerea and S. sclerotiorum (Amselem et al., 2011) opened the way to systematic searches for the molecular bases of pathogenicity in these fungi (Guyon et al., 2014; Heard et al., 2015). However, the repertoire of molecules contributing to the ability of plant pathogenic fungi, such as fungi from the Sclerotiniaceae family, to colonize a wide range of hosts and environments remains elusive. Fungal pathogens secrete diverse sets of degrading enzymes and toxins to facilitate colonization of their hosts (Möbius and Hertweck, 2009; Kubicek et al., 2014). In addition, fungal pathogens use molecules designated as effectors to manipulate host cells and achieve successful infection. Their activities include the inactivation of plant defenses, interference with plant hormone balance, or dismantling of the plant cell. However, effectors may also trigger specific plant defense responses, leading to plant resistance, when recognized directly or indirectly by the plant immune system (Jones and Dangl, 2006). Typical effectors are small secreted proteins, but secondary metabolites and small RNAs can also play the role of effectors (Schardl et al., 2013; Weiberg et al., 2013). Although a subset of bacterial and oomycete protein effectors can be identified based on conserved N-terminal targeting signals and other sequence signatures (Schornack et al., 2009; McDermott et al., 2011; Meyer et al., 2013), this is not the case in fungi. Effector detection in fungal pathogens relies largely on specific host responses revealing effector recognition, and bioinformatics approaches based on whole genome sequences and deduced protein repertoires remain challenging (Sperschneider et al., 2015). Citation: Badet T, Peyraud R and Raffaele S (2015) Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae. Front. Plant Sci. 6:776. doi: 10.3389/fpls.2015.00776 September 2015 | Volume 6 | Article 776 Frontiers in Plant Science | www.frontiersin.org 1 Protein signatures in Sclerotiniaceae fungi Badet et al. Frontiers in Plant Science | www.frontiersin.org Introduction sclerotiorum are cosmopolitan fungi found in arctic, temperate and tropical climates (Figure 1B). Consistently, S. borealis is a psychrophile, with an optimal growth temperature about 4–10◦C, whereas optimal growth temperature is ∼23◦C for B. cinerea and S. sclerotiorum (mesophiles) (Wu et al., 2008; Hoshino et al., September 2015 | Volume 6 | Article 776 2 Protein signatures in Sclerotiniaceae fungi Badet et al. 2010; Judet-Correia et al., 2010). To successfully thrive in cold environments, psychrophilic pathogens must synthesize enzymes and effectors that perform effectively at low temperatures. Cold-temperature environments present several challenges, in particular reduced reaction rates, increased viscosity, and phase changes of the surrounding medium. A draft genome sequence of S. borealis strain F-4128 has recently been released (Mardanov et al., 2014a,b) providing an opportunity to better understand its adaptation to its ecological niche and particularly to cold environment. The total size of the assembled genome of S. borealis is 39.3 Mb, with a G+C content of 42%, including 10,171 predicted protein coding sequences (Mardanov et al., 2014a). These characteristics are similar for the genomes of S. sclerotiorum 1980 and B. cinerea B05.10 with total sizes of 38.3 Mb and 42.3 Mb respectively, G+C content of 41.8 and 43.1% respectively, and 14,503 and 16,448 predicted protein coding genes respectively (Amselem et al., 2011). the search for signatures of adaptation to S. borealis lifestyle may help revealing proteins essential for host and environment colonization in the Sclerotiniaceae. In this work, we focused our analysis on adaptations to S. borealis environment that lead to alterations in core functions (genes and proteins) conserved in S. sclerotiorum and B. cinerea. We analyzed a set of 5531 groups of core orthologous proteins for amino acid usage and intrinsic protein disorder patterns specifically associated with S. borealis proteins. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identified through our pipeline as candidate proteins involved in colonization of the environment. Our findings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases and for the bio-conversion of plant biomass. Frontiers in Plant Science | www.frontiersin.org Introduction Cellular adaptations to low temperatures and the underlying molecular mechanisms are not fully understood but include membrane fluidity, the production of cold-acclimation and antifreeze proteins and maintenance of enzyme-catalyzed reactions and protein-protein interactions involved in essential cellular processes (Feller, 2003; Casanueva et al., 2010). Attempts to correlate protein thermal adaptation with sequence and structure derived features have accumulated with the multiplication of genomic sequencing programs. For instance, analysis of the complete predicted proteome of the psychrophilic bacterium Colwellia psychrerythraea supported the view that psychrophilic lifestyle probably involves specific sets of genes in addition to changes in the overall genome content and amino acid composition (Methé et al., 2005). Because microorganisms are at complete thermal equilibrium with their environment, it is indeed conceivable that adaptation to low temperature lead to global alterations of proteomes in psychrophiles. Comparative genomic and metagenomic analyses in prokaryotes demonstrated that the summed frequency of amino acids Ile, Val, Tyr, Trp, Arg, Glu, Leu (IVYWREL) correlates with optimal growth temperature (Zeldovich et al., 2007). In another study on bacteria, Ala, Asp, Ser, and Thr were found preferred in the genome of psychrophiles compared to mesophiles, whereas Glu and Leu are less frequent (Metpally and Reddy, 2009). The analysis of amino acid usage in thermophilic fungi showed that these species indeed have a higher total frequency of IVYWREL amino acids than their mesophilic relatives, but show also significant depletion in Gly and enrichment in Arg and Ala (Van Noort et al., 2013). At the structural level, cold environments seem to release selective pressure for stable proteins, and increase selection for highly active heat-labile enzymes, relying on improved intrinsic disorder to maintain optimal conformation dynamics (Feller, 2003, 2007). Besides these seemingly general principles and given the existence of psychrophiles in lineages across the tree of life, multiple mechanisms contributing to cold adaptation may exist. For a fungal pathogen such as S. borealis, completion of its A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments g p g g Several studies reported specific amino acid usage patterns and intrinsic disorder frequency in proteins from psychrophilic bacteria as compared to related mesophilic bacteria (Methé et al., 2005; Metpally and Reddy, 2009). To test whether S. borealis proteins have a distinctive pattern of amino acid usage and disorder compared to S. sclerotiorum and B. cinerea proteins, we designed a bioinformatics pipeline to process complete proteomes deduced from the whole genome sequences of these three fungal pathogens (Figure 2) (Amselem et al., 2011; Mardanov et al., 2014a). To exclude patterns that may be due to factors unrelated to adaptation in S. borealis proteins, we focused our analysis on core groups of orthologous proteins with one member from each species. A total of 6717 core orthologous groups (COGs) were identified using two pairwise InParanoid proteome comparisons (Ostlund et al., 2010) as explained in material and methods section and presented in Figure 2, covering between ∼42% (B. cinerea) to ∼66% (S. borealis) of complete predicted proteomes. We used multiple alignments of the three proteins in each COG to select S. sclerotiorum protein regions conserved in S. borealis and B. cinerea. To retrieve core protein regions conserved in all three members of COGs, we ran another round of InParanoid pairwise comparisons using conserved regions of S. sclerotiorum proteins as input. Short alignments can artificially cause strong variations in amino acid proportions. To reduce this confounding effect, we excluded alignments producing a consensus sequence shorter than 200 amino acids. We obtained a total of 5531 COG alignments matching these criteria that were processed for amino acid frequency and intrinsic protein disorder analysis. S. borealis Proteins Show Specific Intrinsic Disorder and Amino Acid Usage Patterns Compared to Their Sclerotiniaceae Orthologs To document intrinsic protein disorder and amino acid usage in Sclerotiniaceae COGs, we calculated frequencies of each of the 20 amino acids in the aligned protein regions as well S. borealis Proteins Show Specific Intrinsic Disorder and Amino Acid Usage Patterns Compared to Their Sclerotiniaceae Orthologs For a fungal pathogen such as S. borealis, completion of its life cycle requires successful plant colonization, and a subset of secreted virulence factors is likely involved in essential cellular processes. Results A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments Besides, secreted proteins in both yeasts and mammals were shown to evolve slightly faster than intracellular proteins (Julenius and Pedersen, 2006; Liao et al., 2010), suggesting that p g To document intrinsic protein disorder and amino acid usage in Sclerotiniaceae COGs, we calculated frequencies of each of the 20 amino acids in the aligned protein regions as well September 2015 | Volume 6 | Article 776 3 Badet et al. Protein signatures in Sclerotiniaceae fungi FIGURE 2 | Bioinformatics pipeline for the identification of S. borealis protein sequence signatures in multiple ortholog alignments. Our pipeline uses complete predicted proteomes of S. borealis, S. sclerotiorum, and B. cinerea as inputs. It identifies orthologous protein pairs in S. borealis and S. sclerotiorum; and in S. borealis and B. cinerea using Inparanoid. Using S. sclerotiorum proteins as a reference, it identifies non-redundant core ortholog groups (COG) and overlapping regions (1). A second Inparanoid run is then used to define the longest aligned region in all three genomes (“consensus”) for each COG (2). Next, protein sequence metrics (disorder probability and amino acid frequencies) are calculated for consensus regions of all proteins (3). Finally, Wilcoxon sum rank tests are performed to identify metrics significantly different in S. borealis proteins. FIGURE 2 | Bioinformatics pipeline for the identification of S. borealis protein sequence signatures in multiple ortholog alignments. Our pipeline uses complete predicted proteomes of S. borealis, S. sclerotiorum, and B. cinerea as inputs. It identifies orthologous protein pairs in S. borealis and S. sclerotiorum; and in S. borealis and B. cinerea using Inparanoid. Using S. sclerotiorum proteins as a reference, it identifies non-redundant core ortholog groups (COG) and overlapping regions (1). A second Inparanoid run is then used to define the longest aligned region in all three genomes (“consensus”) for each COG (2). Next, protein sequence metrics (disorder probability and amino acid frequencies) are calculated for consensus regions of all proteins (3). Finally, Wilcoxon sum rank tests are performed to identify metrics significantly different in S. borealis proteins. when Wilcoxon sum rank tests were significant (p < 0.05) for S. borealis—S. sclerotiorum and S. borealis—B. cinerea comparisons but not (p > 0.05) for S. sclerotiorum—B. cinerea comparison. The “hot loops” frequencies measure of intrinsic protein disorder was found significantly associated with S. borealis COG aligned regions, whereas “Coils” and “Remark465” were not (Figure 3A). A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments borealis and the other fungi (p < 0.05) but not between S. sclerotiorum and B. cinerea (p > 0.05). These properties were considered as associated with S. borealis lifestyle. FIGURE 3 | Adaptation to S. borealis lifestyle associates with specific amino acid usage and protein disorder patterns. Distribution of the p-values of Wilcoxon sum rank tests performed to identify intrinsic disorder probabilities (A) and amino acid frequencies (B) that are significantly different in S. borealis core orthologs. For each amino acid frequency and intrinsic disorder probability, three pairwise tests were performed to compare (i) values in B. cinerea and S. sclerotiorum orthologs (p-values shown along the X-axis), (ii) values in S. borealis and B. cinerea orthologs (p-values shown along the Y-axis in green), and (iii) values in S. borealis and S. sclerotiorum orthologs (p-values shown along the Y-axis in red). Amino acid frequencies and intrinsic disorder probabilities that fell in the shaded areas were considered significantly different between S. borealis and the other fungi (p < 0.05) but not between S. sclerotiorum and B. cinerea (p > 0.05). These properties were considered as associated with S. borealis lifestyle. and 5.91% in B. cinerea proteins. Lys and Glu were significantly depleted in S. borealis. Lysine represented 5.26% of amino acids in S. borealis instead of 5.41% in S. sclerotiorum and B. cinerea proteins; Glu represented 6.43% of amino acids in S. borealis instead of 6.54% in S. sclerotiorum and 6.57% in B. cinerea proteins (Figure 3B). These findings are consistent with the view that cold adaptation includes the directional adaptation of pre- existing protein functions (intrinsic protein structure and amino acid composition) in addition to specific sets of genes conferring a psychrophilic lifestyle, such as previously reported in bacteria (Methé et al., 2005). proteomes of S. borealis, S. sclerotiorum, and B. cinerea. First, we compared the distribution of sTEKhot values in COGs by plotting all values in a ternary plot (Figure 4A). This revealed that sTEKhot values are distributed along an axis pointing toward S. borealis angle, clearly showing that sTEKhot values of S. borealis orthologs are major contributors to the structure of the dataset. There was 692 COGs in which S. borealis sTEKhot value accounted for >40% of the total sTEKhot for the COG, but only 388 and 345 in which S. sclerotiorum and B. A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments “Hot loops,” corresponding to protein regions predicted not to adopt helix or strand secondary structure and having a high degree of flexibility, were found significantly depleted in S. borealis COG aligned regions. S. borealis proteins had a median hot loop frequency of 3.43% in COG aligned regions, vs. 3.67% in S. sclerotiorum and 3.71% in B. cinerea proteins. Regarding frequency of amino acids, three were found significantly associated with S. borealis aligned COG regions. Thr frequency was significantly enriched, representing 6.00% of amino acids in S. borealis instead of 5.93% in S. sclerotiorum as their disorder frequencies. Determination of the disorder frequencies were obtained by assigning to each amino acid of the aligned protein regions their disorder probability obtain by submitting the full length protein to disEMBL analyses (Linding et al., 2003). The disEMBL output contained three measures of intrinsic protein disorder designated as “Coils,” “Hot loops,” and “Remark465” corresponding to their probability to be involved in disorder region. To test whether any of these 20 amino acid frequencies plus 3 disorder metrics frequencies showed a significantly different distribution in S. borealis COG aligned regions compared to S. sclerotiorum and B. cinerea, we performed pairwise Wilcoxon sum rank tests to compare distributions of each of the 23 properties in S. borealis and S. sclerotiorum, in S. borealis and B. cinerea, and in S. sclerotiorum and B. cinerea (Table S1). We considered that a protein property was significantly associated with S. borealis COG aligned regions September 2015 | Volume 6 | Article 776 Frontiers in Plant Science | www.frontiersin.org 4 Protein signatures in Sclerotiniaceae fungi Badet et al. FIGURE 3 | Adaptation to S. borealis lifestyle associates with specific amino acid usage and protein disorder patterns. Distribution of the p-values of Wilcoxon sum rank tests performed to identify intrinsic disorder probabilities (A) and amino acid frequencies (B) that are significantly different in S. borealis core orthologs. For each amino acid frequency and intrinsic disorder probability, three pairwise tests were performed to compare (i) values in B. cinerea and S. sclerotiorum orthologs (p-values shown along the X-axis), (ii) values in S. borealis and B. cinerea orthologs (p-values shown along the Y-axis in green), and (iii) values in S. borealis and S. sclerotiorum orthologs (p-values shown along the Y-axis in red). Amino acid frequencies and intrinsic disorder probabilities that fell in the shaded areas were considered significantly different between S. A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments cinerea sTEKhot values respectively accounted for >40% of the total sKTEHhot for the COG (Figure 4A). Consistently, S. borealis has the highest sTEKhot value in 42.7% of COGS (2761), whereas S. sclerotiorum and B. cinerea have the highest sTEKhot value in 28.3% (1845) and 28.8% (1865) of the COGs respectively (Figure 4B). The Distribution of sTEKhot Index Is Biased in S. borealis Orthologous Proteins and Complete Predicted Proteome Therefore, orthologs that have similar sTEKhot values in all three species appear at the center of the plot, while COGs appear near the corner of the species harboring the ortholog with the highest sTEKhot otherwise. The size of bubbles is proportional to the sTEKhot value of S. borealis orthologs. Data points are frequent above the 40% line for S. borealis sTEKhot, and less so for S. sclerotiorum and B. cinerea sTEKhot indicating frequent higher sTEKhot values in S. borealis orthologs. (B) Species distribution of orthologs having the highest sTEKhot value in COGs. (C) Distribution of the sTEKhot index in the whole predicted proteome of the three fungi. FIGURE 4 | The sTEKhot index discriminates S. borealis proteins in core ortholog groups and whole predicted proteomes. (A) Overall distribution of sTEKhot values from the three fungal species within COGs. Each bubble represents a COG positioned according to the contribution of each ortholog (sTEKhot%) to the total sTEKhot of the COG. Therefore, orthologs that have similar sTEKhot values in all three species appear at the center of the plot, while COGs appear near the corner of the species harboring the ortholog with the highest sTEKhot otherwise. The size of bubbles is proportional to the sTEKhot value of S. borealis orthologs. Data points are frequent above the 40% line for S. borealis sTEKhot, and less so for S. sclerotiorum and B. cinerea sTEKhot indicating frequent higher sTEKhot values in S. borealis orthologs. (B) Species distribution of orthologs having the highest sTEKhot value in COGs. (C) Distribution of the sTEKhot index in the whole predicted proteome of the three fungi. simulations indicate that sTEKhot clearly departs from random in describing specific intrinsic protein disorder and amino acid usage patterns in S. borealis proteins. measures to discriminate the proteome of S. borealis from that of S. sclerotiorum and B. cinerea, we randomly shuffled the 23 measures for intrinsic protein disorder and amino acid usage in equation (1) and calculated the proteome median value for shuffled indices in S. borealis, S. sclerotiorum, and B. cinerea (Table S3). In 300 shuffling iterations, the p-value for difference between the distribution of shuffled index in S. borealis and S. sclerotiorum or B. cinerea was < 5.1.e−104 (highest observed p- value) in only 6 instances. The median shuffled index value for S. borealis proteome was higher than the observed sTEKhot median in only 2 instances over 300 (0.6%). The Distribution of sTEKhot Index Is Biased in S. borealis Orthologous Proteins and Complete Predicted Proteome At the whole proteome level, sTEKhot median was 0.366 in S. borealis, but only 0.314 in S. sclerotiorum and 0.313 in B. cinerea (Figure 4C, Table S2). The overall sTEKhot distributions were significantly different when comparing S. borealis to the two other species (p < 5.1.e−104) but not when comparing S. sclerotiorum to B. cinerea (p = 0.84). However, a subset of S. sclerotiorum and B. cinerea proteins appeared to have high sTEKhot values. Indeed, as mentioned previously, S. sclerotiorum and B. cinerea each account for the highest sTEKhot in ∼30% of the COGs. Furthermore, the proportion of proteins with a sTEKhot > 1 was 6.2% in S. borealis, 4.6% in S. sclerotiorum and 5.0% in B. cinerea. This suggests that the general pattern of intrinsic protein disorder and amino acid usage observed in S. borealis protein may be shared by a subset of S. sclerotiorum and B. cinerea predicted proteome. Several studies reported biases in amino acid usage in the proteome of extremophiles and proposed indices able to discriminate proteins from extremophilic and related mesophilic organisms (Suhre and Claverie, 2003; Zeldovich et al., 2007; Wang and Lercher, 2010). To analyze the degree to which intrinsic protein disorder and amino acid usage of individual proteins matches with specific patterns identified in S. borealis predicted proteome, we designed the S. borealis T (Thr), E (Glu), K (Lys), hot (hot loops) index as follows: sTEKhot = T E + K + hot (1) (1) where “T,” “E,” and “K” are the normalized frequencies of Thr, Glu and Lys respectively in a given protein sequence, and “hot” is the normalized frequency of hot loops in this sequence. We computed the sTEKhot index for each protein in the predicted To verify that the sTEKhot index was an optimized combination of intrinsic protein disorder and amino acid usage September 2015 | Volume 6 | Article 776 Frontiers in Plant Science | www.frontiersin.org Badet et al. Protein signatures in Sclerotiniaceae fungi FIGURE 4 | The sTEKhot index discriminates S. borealis proteins in core ortholog groups and whole predicted proteomes. (A) Overall distribution of sTEKhot values from the three fungal species within COGs. Each bubble represents a COG positioned according to the contribution of each ortholog (sTEKhot%) to the total sTEKhot of the COG. The Distribution of sTEKhot Index Is Biased in S. borealis Orthologous Proteins and Complete Predicted Proteome Wilcoxon ranking tests comparing random medians distribution to real sTEKhot median showed p < 4.72e−33 in the three species. The result of these Secreted Enzymes are Enriched among S. borealis Proteins with High sTEKhot To identify protein functions important for adaptation to S. borealis environment, we analyzed annotations of proteins with a sTEKhot value higher than 1 in S. borealis proteome. Overall, 4794 (47%) S. borealis proteins had no Gene Ontology (GO) annotation assigned. There were 635 proteins with sTEKhot > 1, among which 349 (55%) had no GO annotation. We looked for GO term enrichment in the 635 S. borealis with sTEKhot > 1 September 2015 | Volume 6 | Article 776 Frontiers in Plant Science | www.frontiersin.org 6 Protein signatures in Sclerotiniaceae fungi Badet et al. and cell wall compartments. Consistently, enriched “biological processes” and “molecular functions” related to secreted enzymes involved in cell wall modification (glycosyl hydrolases and carboxylic ester hydrolases, among which are pectinesterases and cutinases) and binding to cellulose. Cellulose is a major compared to all annotated proteins. Forty two GO terms appeared significantly enriched (p < 0.05) among proteins with sTEKhot > 1, including 16 leaves (GO with no child term) of the GO network (Figure 5). GO terms for “cellular component” enriched in proteins with sTEKhot > 1 included extracellular FIGURE 5 | Network representation of gene ontologies (GOs) of proteins with sTEKhot >1 in S. borealis proteome. Nodes correspond to GOs are sized according to the number of proteins with sTEKhot >1. They are colored from yellow to orange according to the p-value of a hypergeometric test for enrichment in proteins with sTEKhot >1 compared to whole proteomes. White nodes are GOs not significantly enriched among proteins with sTEKhot > 1 (p > 0.05). GOs labeled in bold font correspond to functions possibly associated with host interaction. FIGURE 5 | Network representation of gene ontologies (GOs) of proteins with sTEKhot >1 in S. borealis proteome. Nodes correspond to GOs are sized according to the number of proteins with sTEKhot >1. They are colored from yellow to orange according to the p-value of a hypergeometric test for enrichment in proteins with sTEKhot >1 compared to whole proteomes. White nodes are GOs not significantly enriched among proteins with sTEKhot > 1 (p > 0.05). GOs labeled in bold font correspond to functions possibly associated with host interaction. September 2015 | Volume 6 | Article 776 Frontiers in Plant Science | www.frontiersin.org 7 Protein signatures in Sclerotiniaceae fungi Badet et al. component of plant cell walls that fungal pathogens are able to detect and bind. Secreted Proteins Have Higher sTEKhot Than Non-secreted Proteins in the Three Sclerotiniaceae Species The enrichment of extracellular proteins among proteins with sTEKhot > 1 prompted us to compare the distribution of sTEKhot for secreted and non-secreted protein in the Sclerotiniaceae. We considered as predicted secreted proteins those identified as secreted with SignalP 4.0 no-TM network and as extracellular by WoLF PSORT. This produced lists of 667, 661, and 748 predicted secreted proteins (secretome) for S. borealis, S. sclerotiorum, and B. cinerea respectively. In all three fungal species, secreted proteins had significantly higher sTEKhot values than non-secreted proteins, with median sTEKhot values for secreted proteins of 1.13 in S. borealis, 1.06 in S. sclerotiorum and 1.08 in B. cinerea (Figure 6A). The distribution of sTEKhot in secreted proteins was found significantly higher than its distribution in non-secreted proteins with p-value of 8.8e−239 in S. borealis, 9.1e−265 in S. sclerotiorum and 4.1e−275 in B. cinerea respectively. To evaluate the likelihood of obtaining such distributions with other intrinsic protein disorder and amino acid usage parameters, we randomly shuffled the 23 measures for intrinsic protein disorder and amino acid usage in Equation (1), and calculated shuffled indices for each protein in the predicted secretome in the three species. In 300 rounds of shuffling, the median secretome index was found higher than the observed median secretome sTEKhot in 3, 1 and 1 instance for S. borealis, S. sclerotiorum and B. cinerea respectively (Table S3). FIGURE 6 | Predicted secreted proteins have high sTEKhot values. (A) Distribution of sTEKhot values in the proteome and the secretome of (Continued) FIGURE 6 | Predicted secreted proteins have high sTEKhot values. (A) Distribution of sTEKhot values in the proteome and the secretome of (Continued) Remarkably, although secreted proteins account for 6.5% of total proteome in S. borealis, 4.5% in S. sclerotiorum and 4.5% in B. cinerea, the proportion of secreted proteins among those with sTEKhot > 1.5 raised to 76.9% (206 out of 268) in S. borealis, 68.2% (182 out of 267) in S. sclerotiorum and 65.0% (206 out of 317) in B. cinerea, representing ∼13.6 fold enrichment in secreted proteins (Figure 6B). These results suggest that intrinsic protein disorder and amino acid usage patterns associated with S. borealis lifestyle and secretion are largely overlapping in the Sclerotiniaceae. Secreted Enzymes are Enriched among S. borealis Proteins with High sTEKhot Also plants aerial parts are protected by a cuticle composed by cutin. Fungal pathogens are able to hydrolyze cutin through cutinases, thus facilitating host colonization. In addition, proteins involved in carbohydrate metabolism were enriched among proteins with sTEKhot > 1. These functions are associated with colonization of the environment, especially plant- associated environment. Similar enrichments where observed when looking at GO annotations for S. sclerotiorum and B. cinerea proteins harboring a sTEKhot > 1 (Figures S1, S2). In addition, copper ion binding GO was found to be enriched in S. sclerotiorum and B. cinerea. FIGURE 6 | Predicted secreted proteins have high sTEKhot values. (A) Distribution of sTEKhot values in the proteome and the secretome of (Continued) Frontiers in Plant Science | www.frontiersin.org FIGURE 6 | Continued S. borealis, S. sclerotiorum and B. cinerea. (B) Proportion of predicted secreted proteins according to sTEKhot cutoff values. In complete proteomes (sTEKhot ≥0), the proportion of secreted proteins is ∼5% in all three fungal proteomes, whereas among proteins with sTEKhot ≥1.5 (dotted line) it reaches an average ∼70%. (C) Proportion of whole proteomes and proteins with sTEKhot > 1.5 that are secreted, contain GPI-anchors, are N-glycosylated or contain transmembrane (TM) domains. Enrich., enrichment fold among sTEKhot > 1.5 as compared to whole proteomes. To test whether proteins with high sTEKhot could be enriched in other types of motifs, we predicted glycosylphosphatidylinositol (GPI) anchors, transmembrane (TM) domains and N-glycosylation sites in the proteome of S. borealis, S. sclerotiorum and B. cinerea. We found an average of 5.0% of proteins with GPI-anchors, 9.9% proteins with TM domains and 3.8% of proteins with >10 predicted N-glycosylation sites in the Sclerotiniaceae species (Table S6, Figure 6C). As compared to whole proteomes, the list of proteins with sTEKhot >1.5 showed an average 7.1-fold enrichment in proteins with GPI-anchors, 2.1-fold enrichment in proteins with >10 predicted N-glycosylation sites and no enrichment in proteins with TM domains (Figure 6C). Secreted proteins showed the strongest enrichment among proteins with sTEKhot >1.5. Overall these analyses suggest that a significant overlap exists between the constraints imposed on protein sequence by adaptation to S. borealis lifestyle and to secretion in the Sclerotiniaceae. non-secreted proteins) could be rejected with p < 0.05 for all three fungal species. Among the 23 measures for protein disorder and amino acid usage, 21 could be significantly associated with fungal secretomes, supporting the view that function outside the cell imposes specific constraints on amino acids usage in secreted proteins, such as evolution toward reduced synthetic cost of proteins (Smith and Chapman, 2010). Similar to patterns associated with S. borealis lifestyle, we found that enrichment in Thr, depletion in Glu and reduced frequency of hot loops disorder are among the properties most significantly associated with secretion (p-values ranging from 7.62e−3 to 2.67e−194) (Table S4). We considered several hypotheses to explain the observed common signatures for S. borealis lifestyle and secretion. First, we envisaged that prevalence of secreted proteins in COGs may have biased signatures of S. borealis lifestyle toward properties associated with secretion. However, ratios of secreted proteins in COG sets were similar to those observed for total proteomes (7% in S. borealis, 6.7% in S. Secreted Proteins Have Higher sTEKhot Than Non-secreted Proteins in the Three Sclerotiniaceae Species To independently validate this observation, we compared the distribution of all amino acid frequencies and the distribution of the three intrinsic protein disorder measures used previously in secreted and non-secreted proteins from the three fungal species. We considered that a protein property is associated with secretion when the null hypothesis of the Wilcoxon sum-rank test (distribution of property no different between secreted and FIGURE 6 | Predicted secreted proteins have high sTEKhot values. (A) Distribution of sTEKhot values in the proteome and the secretome of (Continued) September 2015 | Volume 6 | Article 776 8 Protein signatures in Sclerotiniaceae fungi Badet et al. with their closest homolog in S. sclerotiorum, this average score was 521.6 for S. borealis secreted proteins (Figure S4). This indicates that globally, S. borealis secretome is more divergent from S. sclerotiorum proteome than S. borealis non-secreted proteins. A similar tendency was observed when comparing S. borealis and B. cinerea proteomes. The high sTEKhot average observed in Sclerotiniaceae secretomes is therefore not due to higher similarity in secretomes compared to non-secreted proteins. FIGURE 6 | Continued sclerotiorum and 6.4% in B. cinerea proteins from the set of 5531 COGs). Furthermore, we excluded COGs that comprised secreted proteins and tested whether amino acid usage patterns associated with S. borealis proteins as previously. Amino acids enriched in S. borealis proteins included Thr and amino acids depleted in S. borealis included Glu and Lys (p < 0.05), similar to what we found in our initial analysis taking all COGs into account. In addition, we also found His enriched in S. borealis sequences and Asn depleted (p < 0.05). We conclude that the detection of a bias in the usage of these amino acids in S. borealis proteins was not due to the abundance of secreted proteins in COGs (Table S5). Second, we hypothesized that intrinsic protein disorder and amino acid usage in secreted proteins might be due to signal peptide regions. To test this, we analyzed protein properties associated with mature secreted proteins (signal peptide region removed). We found that mature secreted proteins had significantly higher sTEKhot than the rest of the proteome (p < 2.4.e−232), similar to what we found with full length secreted proteins (Figure S3). Therefore high sTEKhot in secretomes is not due to signal peptide sequence. Third, we considered that high sTEKhot in secretomes could arise if secretomes were be less divergent than the rest of the proteomes, leading to S. borealis signature being more conserved in secreted proteins of S. sclerotiorum and B. cinerea. To test this, we analyzed the distribution of similarity between S. borealis proteins and their closest homologs in S. sclerotiorum and B. cinerea. Whereas the average BLASTP score was 630.9 for S. borealis non-secreted proteins aligned S. Sclerotiorum Genes Encoding Proteins with High sTEKhot are Enriched in Genes Induced in planta Frontiers in Plant Science | www.frontiersin.org S. Sclerotiorum Genes Encoding Proteins with High sTEKhot are Enriched in Genes Induced in planta p To further support the association between high sTEKhot index and colonization of the environment, and particularly host plants, we analyzed the distribution of sTEKhot values in S. sclerotiorum genes differentially regulated in planta. For this, we took advantage of S. sclerotiorum microarray gene expression data generated by Amselem et al. from infected sunflower cotyledons (Amselem et al., 2011). In this dataset, out of 14 503 predicted protein coding genes, 615 were induced at least two-fold during infection of sunflower (4.31%) and 458 genes down-regulated at least two-fold (3.21%). The proportion of genes induced in planta reached 27.1% of S. sclerotiorum genes encoding proteins with sTEKhot ≥2, representing ∼6.3-fold enrichment (Figure 7). The proportion of genes down-regulated in planta reached 12.1% of S. sclerotiorum genes encoding proteins with sTEKhot ≥2, representing ∼3.8-fold enrichment. S. sclerotiorum proteins with sTEKhot > 1 include six proteins with CFEM domain, a Cys-rich domain with proposed role in fungal pathogenesis, two proteins with a cerato-platanin domain, one of which being the ortholog of B. cinerea pathogen associated molecular pattern BcSpl1 (Frías et al., 2011), 27 proteins with a pectin lyase fold found in Aspergillus virulence factors (Mayans et al., 1997), and 29 out of 78 effector candidates proposed by Guyon et al. (2014). These findings are consistent with important role in the colonization of the host plant for some proteins with high sTEKhot values. September 2015 | Volume 6 | Article 776 Frontiers in Plant Science | www.frontiersin.org 9 Protein signatures in Sclerotiniaceae fungi Badet et al. may stabilize folding like, although these residues were not predicted to form disulfide bonds by Disulfind (Ceroni et al., 2006). Antifreeze proteins have been reporting that rely on disulfide bonds for folding (Basu et al., 2015) whereas others do not (Kondo et al., 2012; Sun et al., 2014). Like other known fungal antifreeze proteins (Kondo et al., 2012), but unlike Maxi, SBOR_9046 and its orthologs are predicted to be secreted. A unique feature of Maxi among antifreeze proteins is the presence of ice-binding residues buried within the four-helix bundle instead of exposed on their surface (Sun et al., 2014). A prediction of SS1G_10836 dimer structure supports the existence of rather hydrophilic pockets buried within the four-helix bundle, suggesting that the mechanism of ice binding of Maxi could be conserved in SS1G_10836 and its orthologs (Figure 8C). S. Sclerotiorum Genes Encoding Proteins with High sTEKhot are Enriched in Genes Induced in planta To get insights into SS1G_10836 function, we analyzed the expression of the corresponding gene in mycelium grown in Potato Dextrose Broth (PDB), during the colonization of Arabidopsis plants and in sclerotia by quantitative RT-PCR. This revealed a 3.3-fold induction (log2 = 1.7) specific to sclerotia (Figure 8F). Since sclerotia overwinter in the soil, putative antifreeze proteins may contribute to survival of these structures both in arctic and temperate climates. FIGURE 7 | Proportion of S. sclerotiorum proteins encoded by genes differentially expressed in planta according to sTEKhot cutoff values. In S. sclerotiorum complete genome (sTEKhot ≥0), the proportion of genes induced ≥2-fold in planta is ∼4.31%, whereas it reaches ∼27.1%.among proteins with sTEKhot ≥2 (dotted line). FIGURE 7 | Proportion of S. sclerotiorum proteins encoded by genes differentially expressed in planta according to sTEKhot cutoff values. In S. sclerotiorum complete genome (sTEKhot ≥0), the proportion of genes induced ≥2-fold in planta is ∼4.31%, whereas it reaches ∼27.1%.among proteins with sTEKhot ≥2 (dotted line). p The COG including SS1G_03146, BC1G_07573, and SBOR_1255 is remarkable for including three proteins with high (>1) but with very variable sTEKhot, ranging from 1.58 (SS1G_03146) to 7.07 (BC1G_07573). No interproscan domain or GO terms were associated with these proteins of 223 amino acids in average, but all three were predicted to include a N-terminal signal peptide for secretion. To get insights into their putative function, we performed protein structure modeling and fold recognition using the I-TASSER server (Zhang, 2008). The closest structural analog was Aspergillus oryzae AA11 (AoAA11) Lytic Polysaccharide Monooxygenase (LPMO) (Hemsworth et al., 2014). Sequence similarity with AoAA11 was limited (from 9.6% identity for SBOR_1255 to 10.9% identity for SS1G_03146), superimposition of SS1G_03146 predicted structure with AoAA11 structure showed a Root Mean Square Deviation < 3.1Å and a TM-score of 0.677, indicating structural similarity deviating significantly from random (Figures 8D,E). Similar to the Sclerotiniaceae proteins, full length AoAA11 (accession number XM_001822611) harbors a N-terminal signal peptide. AoAA11, SBOR_1255, and BC1G_07573 feature two conserved predicted disulfide bonds, SS1G_03146 is predicted to contain only one (Figure 8D). The catalytic triad of AoAA11 appears nicely conserved in the Sclerotiniaceae proteins, with the exception of the catalytic Tyr replaced by a Ser in SS1G_03146 (Figure 8D). LPMOs are enzymes oxidizing recalcitrant polysaccharides such as cellulose, starch and chitin. They present excellent potential for use in biomass conversion and the production of biofuels. S. Sclerotiorum Genes Encoding Proteins with High sTEKhot are Enriched in Genes Induced in planta Aspergillus oryzae AA11 represents a new class of LPMOs that include a putative chitin-binding domain (Hemsworth et al., 2014). We analyzed the expression of the SS1G_03146 gene in mycelium grown in PDB, during the colonization of Arabidopsis plants and in sclerotia by quantitative RT-PCR. This revealed up to 9.5-fold induction (log2 = 3.25) during plant infection (Figure 8F). This suggests that SS1G_03146 may be involved in colonization of the Frontiers in Plant Science | www.frontiersin.org High sTEKhot Index and Secretion Signal Reveal Candidate Proteins Associated with Colonization of the Environment To illustrate the value of the sTEKhot index for the exploration of the proteome of fungi from the Sclerotiniaceae, we analyzed in detail the sequence of two proteins with high sTEKhot but with no assigned function. Over the three proteomes analyzed, S. borealis SBOR_9046 had the highest sTEKhot (10.01). In S. sclerotiorum, its ortholog is SS1G_10836 which ranked as the 5th highest sTEKhot in S. sclerotiorum (7.34). In B. cinerea, its ortholog is BC1G_03854 which ranked as the 23rd highest sTEKhot in B. cinerea (4.29). No interproscan domain or GO terms were associated with these proteins of 171 amino acids (except SS1G_10836 which is 173 amino acids long). To get insights into their putative function, we performed protein structure modeling and fold recognition using the I-TASSER server (Zhang, 2008). The closest structural analog was the antifreeze protein Maxi from winter flounder (Pseudopleuronectes americanus) (Sun et al., 2014). Although sequence similarity with Maxi was limited (from 15.2% identity for SBOR_9046 to 16.2% identity for SS1G_10836), superimposition of SS1G_10836 predicted structure with Maxi structure showed a Root Mean Square Deviation < 2.3Å and a TM-score of 0.875, indicating structural similarity deviating significantly from random (Figures 8A,B). Analysis of SBOR_9046, SS1G_10836 and BC1G_03854 sequence by TargetFreeze (He et al., 2015) supported the prediction as antifreeze proteins. The Sclerotiniaceae proteins contain four Cys residues located in the kink of predicted structures that Frontiers in Plant Science | www.frontiersin.org September 2015 | Volume 6 | Article 776 10 Badet et al. Protein signatures in Sclerotiniaceae fungi FIGURE 8 | Candidate proteins associated with colonization of the environment identified based on high sTEKhot values. (A) Multiple protein sequence alignment of B. cinerea BC1G_03854 (sTEKhot = 4.29), S. borealis SBOR_9046 (sTEKhot = 10.01), S. sclerotiorum SS1G_10836 (sTEKhot = 7.34) and the hyperactive Type I antifreeze protein “Maxi” from Pseudopleuronectes americanus (4KE2_A). (B) Superimposition of Maxi antifreeze protein structure (tan) and SS1G_10836 model structure (rainbow). (C) Surface hydrophobicity of SS1G_10836 model dimer. Dotted line corresponds to the position of the section shown on the right, illustrating the characteristic hydrophilic inner core of the dimer. (D) Multiple protein sequence alignment of B. cinerea BC1G_07573 (sTEKhot = 7.07), S. borealis SBOR_1255 (sTEKhot = 3.79), S. sclerotiorum SS1G_03146 (sTEKhot = 1.58) and the AA11 Lytic Polysaccharide Monooxygenase from Aspergillus oryzae (4MAH_A). (E) Superimposition of A. oryzae AA11 structure (tan) and SS1G_03146 model structure (rainbow). High sTEKhot Index and Secretion Signal Reveal Candidate Proteins Associated with Colonization of the Environment (F) SS1G_10836 and SS1G_03146 gene expression in vitro (PDB, Potato Dextrose Broth), during colonization of Arabidopsis thaliana (lesion periphery and lesion center) and in sclerotia. Error bars show standard error of the mean from two independent biological replicates. FIGURE 8 | Candidate proteins associated with colonization of the environment identified based on high sTEKhot values. (A) Multiple protein sequence alignment of B. cinerea BC1G_03854 (sTEKhot = 4.29), S. borealis SBOR_9046 (sTEKhot = 10.01), S. sclerotiorum SS1G_10836 (sTEKhot = 7.34) and the hyperactive Type I antifreeze protein “Maxi” from Pseudopleuronectes americanus (4KE2_A). (B) Superimposition of Maxi antifreeze protein structure (tan) and SS1G_10836 model structure (rainbow). (C) Surface hydrophobicity of SS1G_10836 model dimer. Dotted line corresponds to the position of the section shown on the right, illustrating the characteristic hydrophilic inner core of the dimer. (D) Multiple protein sequence alignment of B. cinerea BC1G_07573 (sTEKhot = 7.07), S. borealis SBOR_1255 (sTEKhot = 3.79), S. sclerotiorum SS1G_03146 (sTEKhot = 1.58) and the AA11 Lytic Polysaccharide Monooxygenase from Aspergillus oryzae (4MAH_A). (E) Superimposition of A. oryzae AA11 structure (tan) and SS1G_03146 model structure (rainbow). (F) SS1G_10836 and SS1G_03146 gene expression in vitro (PDB, Potato Dextrose Broth), during colonization of Arabidopsis thaliana (lesion periphery and lesion center) and in sclerotia. Error bars show standard error of the mean from two independent biological replicates. FIGURE 8 | Candidate proteins associated with colonization of the environment identified based on high sTEKhot values. (A) Multiple protein sequence alignment of B. cinerea BC1G_03854 (sTEKhot = 4.29), S. borealis SBOR_9046 (sTEKhot = 10.01), S. sclerotiorum SS1G_10836 (sTEKhot = 7.34) and the hyperactive Type I antifreeze protein “Maxi” from Pseudopleuronectes americanus (4KE2_A). (B) Superimposition of Maxi antifreeze protein structure (tan) and SS1G_10836 model structure (rainbow). (C) Surface hydrophobicity of SS1G_10836 model dimer. Dotted line corresponds to the position of the section shown on the right, illustrating the characteristic hydrophilic inner core of the dimer. (D) Multiple protein sequence alignment of B. cinerea BC1G_07573 (sTEKhot = 7.07), S. borealis SBOR_1255 (sTEKhot = 3.79), S. sclerotiorum SS1G_03146 (sTEKhot = 1.58) and the AA11 Lytic Polysaccharide Monooxygenase from Aspergillus oryzae (4MAH_A). (E) Superimposition of A. oryzae AA11 structure (tan) and SS1G_03146 model structure (rainbow). (F) SS1G_10836 and SS1G_03146 gene expression in vitro (PDB, Potato Dextrose Broth), during colonization of Arabidopsis thaliana (lesion periphery and lesion center) and in sclerotia. Error bars show standard error of the mean from two independent biological replicates. Discussion Understanding how fungal plant pathogens colonize their environment, including their host plants, is critical for food security and the sustainable management of ecosystems (Roux et al., 2014). In particular B. cinerea and S. sclerotiorum are threatening hundreds of plant species and important crop species in the majority of regions of the globe. Fungi also represent a remarkable reservoir of enzymes with very diverse catalytic abilities that are employed in industrial processes. We have conducted a comparative analysis of the proteome and secretome of fungal species from the Sclerotiniaceae revealing common principles of sequence optimization for secreted proteins. g y Enrichment analyses revealed that signatures associated with S. borealis lifestyle are frequent in plant cell wall degrading enzymes, carbohydrate binding domain containing proteins and ion binding proteins. More generally, secreted proteins showed high sTEKhot values in S. borealis, S. sclerotiorum and B. cinerea. The proportion of predicted secreted proteins reaches over 75% of S. borealis proteins with sTEKhot > 1.5 and the proportion of proteins encoded by in-planta induced genes reaches over 27% of S. sclerotiorum proteins with sTEKhot > 2, suggesting that sTEKhot may be a useful criterion to identify proteins associated with environmental adaptation or potential virulence factors. More specifically, there were 117 proteins predicted to be secreted and harboring a sTEKhot > 1.5 with no annotation in S. sclerotiorum that could include uncharacterized virulence factors. Although some classes of protein effectors from bacteria and oomycete pathogens can be identified relatively easily thanks to conserved N-terminal sequence signals, this strategy has proven limited for fungal pathogens. Alternative bioinformatics approaches have been developed exploiting known effector properties for searching effector candidates in the secretome of fungal pathogens (Saunders et al., 2012; Guyon et al., 2014). Typical effector properties include the presence of a N-terminal secretion signal, small protein size, high Cys content, the absence of characterized protein domains, high rate of non-synonymous over synonymous substitutions (Hacquard et al., 2012; Saunders et al., 2012; Persoons et al., 2014; Sperschneider et al., 2014). However, validated virulence factors do not all comply with these properties, such as Verticillium dahlia isochorismatase VdIsc1 harboring an isochorismatase domain but no conventional secretion signal (Liu et al., 2014) or Melampsora lini AvrM that lacks any Cys (Catanzariti et al., 2006). High sTEKhot Index and Secretion Signal Reveal Candidate Proteins Associated with Colonization of the Environment September 2015 | Volume 6 | Article 776 Frontiers in Plant Science | www.frontiersin.org 11 Protein signatures in Sclerotiniaceae fungi Badet et al. plant, but functional analysis will be required to determine its actual role. are therefore consistent with some previous observations made for psychrophilic proteins. Nevertheless, our approach does not allow dissociating psychrophily and other specific life traits of S. borealis (specific host range, geographic habitat) as drivers of the observed protein signatures. We observed a reduction in the frequency of intrinsic disorder in hot loops in S. borealis proteins. By contrast, cold adapted enzymes were often reported to harbor low conformational stability to maintain high reaction rates at low temperature (Feller, 2007; Casanueva et al., 2010) and intrinsically disordered proteins were shown to be more resistant to cold than globular proteins (Tantos et al., 2009). A global study of intrinsic protein disorder in 332 prokaryotes showed however that psychrophilic bacteria have a lower level of intrinsic disorder than mesophiles, although this was proposed to be due to the loss of cellular functions relying on intrinsically disordered proteins (Burra et al., 2010). This analysis also supports the view that adaptations to S. borealis lifestyle include directional changes in the sequence of conserved proteins, in addition to possible gene gains and losses that have not been analyzed in this work. Based on these predicted functions, we propose that SS1G_10836 and SS1G_03146 have important functions in the colonization of the environment, the identification of which was facilitated by the implementation of the sTEKhot index. Functional studies will be required to test predicted functions of these proteins. Furthermore, these two proteins have predicted properties that may be exploited for biotechnology purposes. Frontiers in Plant Science | www.frontiersin.org Discussion In Pseudomonas syringae, amino acid biases and patterns at the N-terminus were used to identify type III September 2015 | Volume 6 | Article 776 12 Protein signatures in Sclerotiniaceae fungi Badet et al. effector candidates. Enrichment in Thr and depletion in Leu is a characteristic of bacterial type III proteins secreted into animal and plant cells, although high sequence variability and high tolerance of mutations make these properties difficult to generalize (Arnold et al., 2009; McDermott et al., 2011; Schechter et al., 2012). To identify novel effectors in Fusarium sp., Stagonospora nodorum, and Puccinia graminis f.sp. tritici fungi, Sperschneider et al. performed unsupervised clustering based on 35 sequence-derived features, including amino acid composition (Sperschneider et al., 2013, 2014). Several clusters were characterized by strong biases in amino acid usage, such as the cluster including the three S. nodorum effectors SnToxA, SnTox1 and SnTox3 enriched in small and non-polar amino acids and the cluster including F. oxysporum f. sp. lycopersici SIX3 featuring high average positive protein charge and a significantly higher percentage of Pro, Ser and Thr (Sperschneider et al., 2013). Similarly, secreted effectors of fungi from the Sclerotiniaceae family could be enriched in Thr and depleted in Glu and Lys compared to the rest of the proteome. This suggests that amino acid usage bias is a property that may be shared by sets of secreted proteins with unrelated function and from distant pathogen lineages. Consistent with Glu and Lys being disorder-promoting amino acids, we found that secreted proteins of Sclerotiniaceae species show lower disorder frequency in hot loops that the rest of the proteome. Effectors of bacterial pathogens were shown to be highly enriched in long disordered regions, presumably to facilitate effector translocation into the host cell, host function mimicry and evasion of the host immune system (Marín et al., 2013). Intrinsic protein disorder was shown to promote high specificity and low affinity protein-ligand interactions (Zhou, 2012; Chu and Wang, 2014). While these properties could be advantageous for host-specific effectors of biotrophic pathogens, for which avoiding detection by the host is critical, opposite requirements may shape the evolution of effectors from broad- range necrotrophic pathogens. Indeed, a relatively low specificity may allow effectors to function during colonization of diverse host species. It is also believed that detection by the host would not be detrimental, and could even be beneficial, to some necrotrophic plant pathogens (Govrin and Levine, 2000). Genome Sources We retrieved three predicted proteomes (Sclerotinia sclerotiorum v1.0, Botrytis cinerea v1.0 and Sclerotinia borealis F-4157) from the Joint Genome Institute (http://jgi.doe.gov/) and NCBI (http://www.ncbi.nlm.nih.gov/) in fasta format. As a cautionary note: the proteome sequences that form the basis of our analyses had originally been predicted by various techniques and may thus be of varying quality and completeness. S. sclerotiorum gene expression data was obtained from http://urgi.versailles.inra.fr/ Data/. Discussion Consistent with Glu and Lys being disorder-promoting amino acids, we found that secreted proteins of Sclerotiniaceae species show lower disorder frequency in hot loops that the rest of the proteome. Effectors of bacterial pathogens were shown to be highly enriched in long disordered regions, presumably to facilitate effector translocation into the host cell, host function mimicry and evasion of the host immune system (Marín et al., 2013). Intrinsic protein disorder was shown to promote high specificity and low affinity protein-ligand interactions (Zhou, 2012; Chu and Wang, 2014). While these properties could be advantageous for host-specific effectors of biotrophic pathogens, for which avoiding detection by the host is critical, opposite requirements may shape the evolution of effectors from broad- range necrotrophic pathogens. Indeed, a relatively low specificity may allow effectors to function during colonization of diverse host species. It is also believed that detection by the host would not be detrimental, and could even be beneficial, to some necrotrophic plant pathogens (Govrin and Levine, 2000). In that case, effectors with high affinity for their targets would not be counter-selected by the host immune system, and would instead favor Sclerotiniaceae fungi in the competition with other microbes for plant-derived resources. Discussion In the present study we designed a bioinformatics pipeline aiming at identifying species-specific patterns of amino acid usage and intrinsic protein disorder in the proteome of closely related species. We applied this pipeline to agriculturally important fungal pathogens from Sclerotiniaceae family to reveal specific signatures associated with S. borealis lifestyle. Compared to S. sclerotiorum and B. cinerea orthologs, we observed in S. borealis proteins a significant increase in Thr usage and a significant decrease in Glu and Lys usage. To minimize the impact of phylogenetic distance on the definition of S. borealis sequence signature, we have restricted our analysis to species from the Sclerotiniaceae family and we discarded any sequence signature differing significantly between S. sclerotiorum and B. cinerea. It is also worth noting that S. borealis, S. sclerotiorum and B. cinerea have a very similar G+C content, so that G+C bias is not expected to have an impact on the differential usage of amino acids. Specific trends in amino acid composition have been reported to associate with protein stability at extreme temperatures. Given the diversity of ecological groups including psychrophiles, it has been challenging to identify universal trends in amino acids usage associated with cold adaptation (Casanueva et al., 2010). Enrichment in Thr has been reported in solvent-accessible areas of proteins from two cold-adapted Archaea (Goodchild et al., 2004) and in proteins from several psychrophilic bacteria (Metpally and Reddy, 2009). This was proposed to reduce surface charge while minimizing risk of aggregation (Goodchild et al., 2004). Frequent substitutions of Glutamate were observed in exposed sites of selected psychrophilic enzymes (Gianese et al., 2001) and more generally in the proteome of the psychrophilic Archea Halorubrum lacusprofundi (Dassarma et al., 2013). Glu is also part of a set of amino acids shown to correlate significantly with optimal growth temperature of prokaryotes (Zeldovich et al., 2007). Specific signatures of amino acid usage we found in S. borealis Amino acid composition is a feature used to predict candidate bacterial effectors. Positive charge, richness in alkaline (H, R, K) amino acids and Glu in the 30 C-terminal amino acids is for instance a property often found in type IV secreted effectors (Meyer et al., 2013; Zou et al., 2013; Wang et al., 2014). Gene Ontology Annotation and Enrichment Analysis The Gene Ontology was collected from the Gene Ontology Consortium website (http://geneontology.org/) in obo format. Assignment of the Gene Ontology annotation of the three sets of protein sequences was performed using InterProScan (Jones et al., 2014). GO enrichments analysis was performed using the Biological Networks Gene Ontology plug-in (Maere et al., 2005) in Cytoscape 3.2.1 with the following parameters: a hypergeometric test for statistical analysis with a Bonferroni Family-Wise Error rate correction and a significance level of 0.05. Cross species comparative analysis has been successfully applied to the identification of novel and specialized virulence mechanisms on the one hand, and to the identification of optimization principles governing the evolution of proteins under given constraints on the other hand. In nature, S. borealis proteins have undergone optimization under specific environmental constraints, including cold, over an irreproducible time at the scale of human life. Comparative genomics approaches therefore have the potential to reveal protein specialization and optimization principles that are not easily accessible through experimental evolution experiments. Indeed, selecting optimized enzyme variants, especially for thermostability, through random mutagenesis often requires exploring a large library of mutants or experimental setups Frontiers in Plant Science | www.frontiersin.org Discussion In that case, effectors with high affinity for their targets would not be counter-selected by the host immune system, and would instead favor Sclerotiniaceae fungi in the competition with other microbes for plant-derived resources. maintaining an appropriate pressure of selection to collect the optimized variants (Kuchner and Arnold, 1997; Lebbink et al., 2000). Comparative genomics can accelerate discoveries usually relying on time consuming screens (Xiao et al., 2008). The biochemical properties of cold-active proteins make them attractive in biochemical, bioremediation, and industrial processes for food, biofuels and pharmaceutical production notably (Cavicchioli et al., 2011). Plant pathogenic fungi in particular present a vast reservoir of biopolymer degrading enzymes adapted to a wide range of temperatures and environments. Functional analyses will be required to test whether the activity of candidates highlighted in this work have applied potential. In the long term, the analysis of optimization principles governing the evolution of secreted proteins from important fungal pathogens may prove useful in improving plant health with the design of crops resistant to broad host range pathogens and to cold stress, and to develop novel strategies for the production of renewable energy relying on the bio-conversion of plant biomass. effector candidates. Enrichment in Thr and depletion in Leu is a characteristic of bacterial type III proteins secreted into animal and plant cells, although high sequence variability and high tolerance of mutations make these properties difficult to generalize (Arnold et al., 2009; McDermott et al., 2011; Schechter et al., 2012). To identify novel effectors in Fusarium sp., Stagonospora nodorum, and Puccinia graminis f.sp. tritici fungi, Sperschneider et al. performed unsupervised clustering based on 35 sequence-derived features, including amino acid composition (Sperschneider et al., 2013, 2014). Several clusters were characterized by strong biases in amino acid usage, such as the cluster including the three S. nodorum effectors SnToxA, SnTox1 and SnTox3 enriched in small and non-polar amino acids and the cluster including F. oxysporum f. sp. lycopersici SIX3 featuring high average positive protein charge and a significantly higher percentage of Pro, Ser and Thr (Sperschneider et al., 2013). Similarly, secreted effectors of fungi from the Sclerotiniaceae family could be enriched in Thr and depleted in Glu and Lys compared to the rest of the proteome. This suggests that amino acid usage bias is a property that may be shared by sets of secreted proteins with unrelated function and from distant pathogen lineages. Ortholog Prediction Ortholog prediction was performed with standalone InParanoid 4.0 (Ostlund et al., 2010) using all vs. all Basic Local Alignment Search Tool (BLAST) algorithms and the following parameters: the BLOSUM62 matrix, a score cut-offof 50 bits and a minimal sequence overlap area of 0.5 (Altschul et al., 1990; Remm et al., 2001). Two pairwise InParanoid comparisons (S. borealis vs. S. sclerotiorum and S. borealis vs. B. cinerea) were ran first on complete proteomes, leading to the identification 6717 COGs, then using only conserved regions of S. sclerotiorum proteins (“overlapping regions”) as input (Figure 2). Finally alignments September 2015 | Volume 6 | Article 776 13 Protein signatures in Sclerotiniaceae fungi Badet et al. producing a consensus sequence shorter than 200 amino acids were excluded leading to 5531 COGs. producing a consensus sequence shorter than 200 amino acids were excluded leading to 5531 COGs. enriched or depleted amino acids and disorder frequency in S. borealis common set of core ortholog groups’ alignments compared to S. sclerotiorum and B. cinerea core ortholog groups alignments, but found to be not significantly different between S. sclerotiorum and B. cinerea, were further used for computing the environmental condition adaptation index (sTEKhot). Thr frequency (Tf) found to be over represented in S. borealis were added to the numerator of the index, whereas Lys (Kf), Glu (Ef) and hot loops (HotLOOPf) frequencies found to be under represented were added to the denominator. Each metrics were normalized by their own median (Xmf, where X is the considered metric) through the all set of proteome used in the analysis (S. borealis plus S. sclerotiorum plus B. cinerea). This normalization assures similar contribution of each metrics to the index. Gene Expression Analysis One-centimeter long leaves were collected and grinded twice for 30 s at maximum frequency in a Retsch MM40 mixer. Total RNA extraction was performed with Macherey-Nagel Nucleospin RNA extraction kit following the manufacturer’s instructions. One µg of total RNA was used for cDNA synthesis in a 20-µL reaction according to Roche Transcriptor Reverse Transcriptase protocol, using 0.5 µL of SuperScript II reverse transcriptase Random Shuffling of sTEKhot Protein amino acid usage was assessed by calculating the frequency of each of 20 amino acids in protein sequences. Prediction of disorder probability of protein amino acid was performed with DisEMBL vs. 1.4 computational tool (Linding et al., 2003) on the full length proteins. In case of analysis of a protein sequence subset, like for the core ortholog groups alignments (see previous section), the disorder probability of each amino acid in the subset were taken from the disorder probability of this amino acid in the full length protein. This was done to avoid miss attribution of disorder probability in a subset of a sequence since surrounding of amino acid in the sequence are of importance to calculate its own disorder probability. Random sTEKhot indexes were calculated by shuffling amino acid and hotloop frequencies in Equation (2) with any of the observed amino acid and hotloop frequencies for a given organism. The random index is therefore defined by Equation (3) in which W, X, Y, and Z are randomly selected observed frequencies. RANDOMindex = Xf Xmf Yf Ymf + Zf Zmf + Wf Wmf (3) (3) Indexes were calculated separately for the three proteomes and secretomes. Random sTEKhot medians and Wilcoxon ranking test p-values were extracted from 300 independent runs. Protein Structure Modeling and Analysis g y Protein structure modeling was performed with the I-TASSER server (Zhang, 2008) using SS1G_10836 and SS1G_03146 full length sequences as queries. SS1G_10836 best model C-score was -3.22; best TM score was 0.875 (RMSD 2.27Å) with model 4KE2. SS1G_03146 best model C-score was -2.28; best TM score was 0.677 (RMSD 3.07Å) with model 4MAH. Pipeline for Collecting Multiple Ortholog Alignments g First, ortholog predictions were performed as described in previous section between one organism, called reference organism in the following (S. sclerotiorum), and each other organism included in the analysis (B. cinerea and S. borealis). Only core groups of orthologous proteins harboring one member from each species were retained. Then, the common overlapping sequences in the reference organism to the others organisms were selected according to BLAST begin and end alignment positions. The maximal begin and the minimal end were used to defined the overlapping sequences. Overlapping sequences with lower than 200 amino acids length were excluded. The obtained overlapping sequences in the reference organism were used to run a new round of ortholog prediction with each other organisms. The consensus sequences, or core ortholog groups alignments, in each organisms were selected accordingly to BLAST begin and end alignment positions using the minimal begin and the maximal end obtained through the all orthologs predicted. The consensus sequences with lower than 200 amino acids length were excluded. First, ortholog predictions were performed as described in previous section between one organism, called reference organism in the following (S. sclerotiorum), and each other organism included in the analysis (B. cinerea and S. borealis). Only core groups of orthologous proteins harboring one member from each species were retained. Then, the common overlapping sequences in the reference organism to the others organisms were selected according to BLAST begin and end alignment positions. h l b d h l d d d fi d h sTEKhot = Tf Tmf Kf Kmf + Ef Emf + HotLOOPf HotLOOPmf (2) (2) sTEKhot value was calculated for every protein of the three proteomes according to (2). The list of proteins with the top 635 sTEKhot (>1) corresponded exactly to proteins with the top Tf-(Ef+Kf+HotLOOPf) values supporting the robustness of the arithmetic design of the sTEKhot index in this dataset. Secretome Prediction and Protein Motif Annotation Analysis by SignalP4.1 was performed at http://www.cbs.dtu.dk using default parameters. Protein localization was predicted with PSORT II software using the WoLF PSORT extension (Horton et al., 2007) for organism type “fungi.” Proteins were defined as part of the secretome when containing both signal peptide and extracellular predicted localization and were excluded if they possess a trans-membrane region predicted by TMHMM (Sonnhammer et al., 1998). Glycosylphosphatidylinositol anchored proteins were identified using Fraganchor (Poisson et al., 2007); N-glycosylation sites were predicted using GlycoEP (Chauhan et al., 2013). References Chauhan, J. S., Rao, A., and Raghava, G. P. S. (2013). In silico platform for prediction of N-, O- and C-glycosites in eukaryotic protein sequences. PLoS ONE 8:e67008. doi: 10.1371/journal.pone.0067008 Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410. doi: 10.1016/S0022- 2836(05)80360-2 Chu, X., and Wang, J. (2014). Specificity and affinity quantification of flexible recognition from underlying energy landscape topography. PLoS Comput. Biol. 10:e1003782. doi: 10.1371/journal.pcbi.1003782 Amselem, J., Cuomo, C. A., van Kan, J. A. L., Viaud, M., Benito, E. P., Couloux, A., et al. (2011). Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet. 7:e1002230. doi: 10.1371/journal.pgen.1002230 Dassarma, S., Capes, M. D., Karan, R., and Dassarma, P. (2013). Amino acid substitutions in cold-adapted proteins from halorubrum lacusprofundi, an extremely halophilic microbe from antarctica. PLoS ONE 8:e58587. doi: 10.1371/journal.pone.0058587 Arnold, R., Brandmaier, S., Kleine, F., Tischler, P., Heinz, E., Behrens, S., et al. (2009). Sequence-based prediction of type III secreted proteins. PLoS Pathog. 5:e1000376. doi: 10.1371/journal.ppat.1000376 Dean, R., Van Kan, J. A. L., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro, A., Spanu, P. D., et al. (2012). The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 13, 414–430. doi: 10.1111/j.1364- 3703.2011.00783.x Basu, K., Graham, L. A., Campbell, R. L., and Davies, P. L. (2015). Flies expand the repertoire of protein structures that bind ice. Proc. Natl. Acad. Sci. U.S.A. 112, 737–742. doi: 10.1073/pnas.1422272112 Farr, D. F., and Rossman, A. Y. (2015). Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Available online at: http://nt.ars- grin.gov/fungaldatabases/ (Retrieved March 31, 2015). Bolton, M. D., Thomma, B. P. H. J., and Nelson, B. D. (2006). Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. Mol. Plant Pathol. 7, 1–16. doi: 10.1111/j.1364-3703.2005.00316.x grin.gov/fungaldatabases/ (Retrieved March 31, 2015). Feller, G. (2003). Molecular adaptations to cold in psychrophilic enzymes. Cell. Mol. Life Sci. 60, 648–662. doi: 10.1007/s00018-003-2155-3 Burra, P. V., Kalmar, L., and Tompa, P. (2010). Reduction in structural disorder and functional complexity in the thermal adaptation of prokaryotes. PLoS ONE 5:e12069. doi: 10.1371/journal.pone.0012069 Feller, G. (2007). Life at low temperatures: is disorder the driving force? Extremophiles 11, 211–216. doi: 10.1007/s00792-006-0050-1 Casanueva, A., Tuffin, M., Cary, C., and Cowan, D. A. (2010). Molecular adaptations to psychrophily: the impact of “omic” technologies. Trends Microbiol. 18, 374–381. Author Contributions TB, RP, and SR designed and performed analyses. SR conceived the study. TB, RP, and SR wrote the manuscript. Figure S3 | Distribution of sTEKhot values for non-secreted proteins and mature secreted proteins (signal peptide removed) in S. borealis, S. sclerotiorum and B. cinerea. Statistical Analysis and sTEKhot Index Determination All statistical tests were computed with R.Studio software. Wilcoxon test was used for significance analysis. Difference was considered significant for p-values inferior to 0.05. Significantly September 2015 | Volume 6 | Article 776 Frontiers in Plant Science | www.frontiersin.org 14 Protein signatures in Sclerotiniaceae fungi Badet et al. BBRIC computational facilities for providing bioinformatics tools. BBRIC computational facilities for providing bioinformatics tools. (Invitrogen), 1 µg of oligo(dT), and 10 nmol of dNTP. cDNAs (diluted 1:10) were used as templates in the quantitative RT- PCR analysis. Quantitative RT-PCR was performed using gene- specific primers (Table S6) with LightCycler 480 apparatus (Roche Diagnostics). Quantitative PCR reaction was performed using the SYBR GREEN I protocol (5 pmol of each primer and 5 µL of RT reaction product in a 7 µL final reaction volume). The PCR conditions were 9 min at 95◦C, followed by 45 cycles of 5 s at 95◦C, 10 s at 65◦C, and 20 s at 72◦C. Expression values of SS1G_10836 and SS1G_03146 were normalized based on expression of SS1G_04652 and SS1G_12196 housekeeping genes. Values from two biological replicates are shown, error bars show standard error of the mean. (Invitrogen), 1 µg of oligo(dT), and 10 nmol of dNTP. cDNAs (diluted 1:10) were used as templates in the quantitative RT- PCR analysis. Quantitative RT-PCR was performed using gene- specific primers (Table S6) with LightCycler 480 apparatus (Roche Diagnostics). Quantitative PCR reaction was performed using the SYBR GREEN I protocol (5 pmol of each primer and 5 µL of RT reaction product in a 7 µL final reaction volume). The PCR conditions were 9 min at 95◦C, followed by 45 cycles of 5 s at 95◦C, 10 s at 65◦C, and 20 s at 72◦C. Expression values of SS1G_10836 and SS1G_03146 were normalized based on expression of SS1G_04652 and SS1G_12196 housekeeping genes. Values from two biological replicates are shown, error bars show standard error of the mean. Supplementary Material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015. 00776 Figure S1 | Network representation of gene ontologies (GOs) of proteins with sTEKhot >1 in S. sclerotiorum proteome. Nodes correspond to GOs are sized according to the number of proteins with sTEKhot >1. They are colored from yellow to orange according to the p-value of a hypergeometric test for enrichment in proteins with sTEKhot >1 compared to whole proteomes. Figure S2 | Network representation of gene ontologies (GOs) of proteins with sTEKhot >1 in B. cinerea proteome. Nodes correspond to GOs are sized according to the number of proteins with sTEKhot >1. They are colored from yellow to orange according to the p-value of a hypergeometric test for enrichment in proteins with sTEKhot >1 compared to whole proteomes. Acknowledgments Figure S4 | Distribution of best BlastP bit scores (log-scaled scores) using S. borealis non-secreted proteins and secreted proteins as queries against S. sclerotiorum or B. cinerea proteomes. Lower scores for searches using S. borealis secretome as query indicate that S. borealis secreted proteins are less conserved than non-secreted proteins. P-values of a Student t-test for differences between non-secreted and secreted proteins are indicated. Figure S4 | Distribution of best BlastP bit scores (log-scaled scores) using S. borealis non-secreted proteins and secreted proteins as queries This work was supported by a Starting Grant of the European Research Council (ERC-StG 336808 project VariWhim) and a Marie Curie grant (MC-CIG 334036 project SEPAraTE) to SR and the French Laboratory of Excellence project TULIP (ANR-10-LABX-41; ANR-11-IDEX-0002-02). We thank the against S. sclerotiorum or B. cinerea proteomes. 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Science 343, 795–798. doi: 10.1126/science.1247407 Copyright © 2015 Badet, Peyraud and Raffaele. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Tantos, A., Friedrich, P., and Tompa, P. (2009). Cold stability of intrinsically disordered proteins. FEBS Lett. 583, 465–469. doi: 10.1016/j.febslet.2008.12.054 Van Noort, V., Bradatsch, B., Arumugam, M., Amlacher, S., Bange, G., Creevey, C., et al. (2013). Consistent mutational paths predict eukaryotic thermostability. BMC Evol. Biol. 13:7. doi: 10.1186/1471-2148-13-7 September 2015 | Volume 6 | Article 776 Frontiers in Plant Science | www.frontiersin.org 17
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Artificial intelligence and cyber-physical mechanical processing systems in digital industry
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Assessment in people with PIMD: Pilot study into the usability and content validity of the Inventory of the personal Profile and Support
Cogent psychology
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University of Groningen Assessment in people with PIMD Wessels, Marleen D.; van der Putten, Annette A.J. Published in: Cogent Psychology DOI: 10.1080/23311908.2017.1340082 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2017 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Wessels, M. D., & van der Putten, A. A. J. (2017). Assessment in people with PIMD: Pilot study into the usability and content validity of the Inventory of the personal Profile and Support. Cogent Psychology, 4(1), 1-10. https://doi.org/10.1080/23311908.2017.1340082 University of Groningen Assessment in people with PIMD Wessels, Marleen D.; van der Putten, Annette A.J. 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 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 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Wessels, M. D., & van der Putten, A. A. J. (2017). Assessment in people with PIMD: Pilot study into the usability and content validity of the Inventory of the personal Profile and Support. Cogent Psychology, 4(1), 1-10. https://doi.org/10.1080/23311908.2017.1340082 Citation for published version (APA): Wessels, M. D., & van der Putten, A. A. J. (2017). Assessment in people with PIMD: Pilot study into the usability and content validity of the Inventory of the personal Profile and Support. Cogent Psychology, 4(1), 1-10. https://doi.org/10.1080/23311908.2017.1340082 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). | Assessment in people with PIMD: Pilot study into the usability and content validity of the Inventory of the personal Profile and Support Assessment in people with PIMD: Pilot study into the usability and content validity of the Inventory of the personal Profile and Support *Corresponding author: Marleen D. Wessels, Faculty of Behavioural and Social Sciences, Centre for Special Needs Education & Youth Care, University of Groningen, Grote Rozenstraat 38, 9712 TJ Groningen, The Netherlands E-mail: m.d.wessels@rug.nl Reviewing editor: Linda Visser, DIPF, Germany Additional information is available at the end of the article Received: 29 November 2016 Accepted: 06 June 2017 Marleen D. Wessels1* and Annette A.J. van der Putten1 Marleen D. Wessels1* and Annette A.J. van der Putten1 Received: 29 November 2016 Accepted: 06 June 2017 Abstract: Providing appropriate support for people with profound intellectual and multiple disabilities (PIMD) is challenging, since valid and reliable instruments that can be used for assessment in persons with PIMD are scarce. Therefore, this study analyzes the usability and validity of an instrument developed for persons with PIMD, the Inventory of the personal Profile and Support (IPS). This instrument is part of a person-centered, goal-oriented, interdisciplinary intervention for persons with PIMD, which is called the “support program.” A first step in the support program is to draw up a personal profile, on which a long term goal for the person with PIMD can be based. When the IPS is used in combination with another instrument, the Behavior Assessment Scales (BAS), a support profile can be written. However, the IPS has not been studied on psychometric qualities yet. To look into the usability and content validity of the IPS, two questionnaires that were developed for this study were used for eight direct support persons of three care facilities. Although filling in the IPS is time-consuming and is therefore sometimes considered to be a burden, in general usability and content validity were rated as good. This study underlines the importance of using assessment instruments during different stages of support in a consistent, systematic way, using the input of all involved persons in support of the person with PIMD. Reviewing editor: Linda Visser, DIPF, Germany Additional information is available at the end of the article Copyright strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of th or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/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. down policy believe that this document breaches copyright please contact us providing details, and we will remove access to the wo vestigate 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 Wessels & van der Putten, Cogent Psychology (2017), 4: 1340082 https://doi.org/10.1080/23311908.2017.1340082 © 2017 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. 1. Introduction 1. Introduction In order to give direction to support or intervention for people with intellectual disabilities, assess- ment is of great importance (Hogg & Langa, 2005). Moreover, according to Mansell and Beadle- Brown (2004), assessment should be tailored to the circumstances and individual needs. However, for people with profound intellectual and multiple disabilities (PIMD), this is particularly challenging. The group persons with PIMD consists of people with a profound intellectual disability in combina- tion with a severe or profound motor disability (Nakken & Vlaskamp, 2007). Moreover, they have a number of additional sensory impairments such as visual impairments (Woodhouse, Griffiths, & Gedling, 2000) and general health problems, such as gastroesophageal reflux disease (Bohmer et al., 1999) and epilepsy (Hogg, 1992; Van Timmeren et al., 2016). People with PIMD experience extensive problems in communication. Language is limited or non-existent and communication may include very subtle cues including physiological signals such as a rise in body temperature or change in level of alertness (Vlaskamp, 2005). Persons with PIMD are dependent on support professionals to be sufficiently knowledgeable about their needs, possibilities, and preferences to be able to provide them with appropriate support (Carnaby, 2007; Forster & Iacono, 2014; Lyons, Walla, & Arthur-Kelly, 2013; Vlaskamp, Hiemstra, & Wiersma, 2007). However, providing appropriate support for persons with PIMD is difficult, since valid and reliable instruments that can be used for the assessment in persons with PIMD are scarce (Carnaby, 2007; Vlaskamp, Van der Meulen, & Zijlstra, 2002). Due to the physical disabilities of people with PIMD, standardized tests that are commonly used such as the Bayley Scales (Bayley, 1967) or the Vineland Adaptive Behavior Scales (Sparrow, Cicchetti, & Balla, 2005) do not lead to a valid esti- mation of a developmental level because they rely heavily on motor functioning (Carnaby, 2007; Visser, Ruiter, van der Meulen, Ruijssenaars, & Timmerman, 2014). In order to develop a sound personal profile about the wishes, needs, functional (dis)abilities, and preferences of the individual with PIMD, a multidimensional framework is necessary (Carnaby, 2007; Vlaskamp, 2005). Therefore, several instruments were developed, including the Inventory of the per- sonal Profile and Support (IPS) and the Behavior Assessment Scales (BAS) (Vlaskamp, Van Wijck, & Poppes, 2015). Using the IPS, information can be gathered about the person with PIMD and about the person in relation to persons that are important for him or her. Subjects: Social Sciences; Behavioral Sciences; Development Studies Subjects: Social Sciences; Behavioral Sciences; Development Studies Keywords: assessment; people with profound intellectual and multiple disabilities (PIMD); support profile; developmental assessment instruments PUBLIC INTEREST STATEMENT The research group is part of the centre for special needs education and youth care, which has a main research focus on assessment and interventions for vulnerable groups of adults and children, including the development of assessment instruments and improving support of people with profound intellectual and multiple disabilities (PIMD). The focus of the current article is on assessment procedures in the support of persons with PIMD, aiming to tune the support to the preferences and needs of people with PIMD. For people with profound intellectual and multiple disabilities (PIMD), sharing information is of crucial importance in order to provide appropriate support. This group consists of people with a profound intellectual disability and a severe or profound motor disability. Often, they have several additional impairments and general health problems. They are dependent on others in all areas of life and experience extensive communication problems. For this group, providing appropriate support is challenging, because reliable and valid assessment instruments that can be used in the support of people with PIMD are scarce. Therefore, the current study focuses on the usability and content validity of an assessment instrument that was developed for people with PIMD, the Inventory of the personal Profile and Support (IPS). In general, direct support persons of people with PIMD-rated usability and content validity of the IPS as good. © 2017 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. © 2017 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. Page 1 of 10 Wessels & van der Putten, Cogent Psychology (2017), 4: 1340082 https://doi.org/10.1080/23311908.2017.1340082 1. Introduction Furthermore, the BAS can be used to measure the functional abilities of a person with PIMD in five developmental domains (Vlaskamp, Van der Meulen, & Smrkovsky, 1999). The instrument can be used in practice to make clear in what devel- opmental domains the person is functioning better in comparison to other developmental domains. This way, strengths and weaknesses of the person with PIMD can be described (Vlaskamp et al., 1999). This approach is in accordance to a model such as the ICF (World Health Organization, 2001), which has a focus on establishing profiles of strengths and weaknesses as well (Buntinx & Schalock, 2010). The BAS and IPS supplement each other by collecting different information from different sources. This corresponds to the approach as described in Lyons, De Bortoli, and Arthur-Kelly (2016), who use the triangulated proxy reporting of data: a technique to improve communication between people with PIMD and their support persons. The IPS and BAS can be used in combination to collect as much information about the person with PIMD as possible, to be able to write a support profile, which contains all available information about the wishes, needs, preferences, and functional abilities of a person with PIMD. The BAS and IPS are both part of a person-centered, goal-oriented, interdisci- plinary program for persons with PIMD, called the “support programme,” which was evaluated as ef- fective by the admission committee of support of persons with a disability (Vlaskamp, Poppes, & Van der Putten, 2015; Vlaskamp & van der Putten, 2009). Vlaskamp and van der Putten (2009) found that implementing the program resulted in more individualized support that is tuned to the wishes and needs of the individual and an increased knowledge of staff about the individual with PIMD. Page 2 of 10 Wessels & van der Putten, Cogent Psychology (2017), 4: 1340082 https://doi.org/10.1080/23311908.2017.1340082 The BAS has adequate psychometric properties (Vlaskamp et al., 2002). However, the IPS has not been studied on psychometric qualities yet. Moreover, assessment with the IPS should be done by interviewing health care professionals and parents, which is time consuming and partly inapplicable in practice. As a result, health care professionals often fill in the IPS without an interview which could have consequences for the accuracy of the information gained. 1. Introduction Therefore, a new version of the IPS was developed, which has a different way of gathering information and can be filled in without an interview (Vlaskamp, Van Wijck, et al., 2015). This version of the IPS has not been studied for psychometric qualities or usability yet. Therefore, this study will focus on the usability and content validity of the IPS in combination with the BAS, by looking at the support profile based on the combination of the instruments. This is important, be- cause without reliable and valid instruments, offering people with PIMD appropriate support is chal- lenging. When the content validity of the support profiles based on the BAS and IPS is high, it is possible to better adjust the support of direct support professionals to the needs and possibilities of persons with PIMD, which could lead to knowledge and strategies that increase their quality of life (Lyons et al., 2016; Maes, Lambrechts, Hostyn, & Petry, 2007). 2.1. Participants A convenience sample was used consisting of eight persons with PIMD and their eight direct support persons from three residential care facilities. Moreover, two health care psychologists of two of the persons with PIMD were involved as well. Inclusion criteria for the persons with PIMD were (Nakken & Vlaskamp, 2007): •  An estimated developmental age of 24 months or lower. •  Severe or profound motor impairments. •  Written informed consent from family or legal representatives to participate in the current study. For the persons with PIMD, exclusion criteria were: For the persons with PIMD, exclusion criteria were: •  Having a severe progressive illness or disorder. •  Having a severe progressive illness or disorder. •  Having recently experienced a fundamental change in their environment (such as a movement to another care institution or surgery). In Table 1, the demographics and additional impairments of the participants can be found. Table 1. Additional impairments of the participants Person Residential home Gender Age Impairment Visual Auditory Epilepsy Eating and drinking problems Sleeping problems Contractures 1 1 Male 26 Yes No Yes Yes Yes No 2 1 Male 22 Yes No Yes Yes Yes Yes 3 1 Male 66 Yes Yes No No No Yes 4 1 Male 68 Yes Yes No Yes No Yes 5 1 Female 45 Yes No Yes Yes Yes Yes 6 2 Female 63 Yes Yes Yes Yes No Yes 7 2 Male Missing No No Yes Yes Yes Yes 8 3 Female 33 Yes No Yes Yes Yes Yes Table 1. Additional impairments of the participants Wessels & van der Putten, Cogent Psychology (2017), 4: 1340082 https://doi.org/10.1080/23311908.2017.1340082 For informed consent, all legal guardians of the involved participants received an information let- ter, which contained information about: •  the BAS, IPS, and the aim of the study; •  processing all personal information anonymously; •  how information about the participant from the assessment instruments would be used by the researcher for the aim of the study, but that it would also be available to the direct support per- sons and the legal guardian; •  the training the researcher who would do the assessment had; •  how participation was completely voluntary and could be stopped at any time; •  contact information of the researcher. Ethical approval for this study was granted by the Ethics Committee of Pedagogical and Educational Sciences of the University of Groningen. 2.2.1. IPS 2.2.1. IPS The IPS focuses on the support that the person with PIMD receives and the context of support. It measures the possibilities of the person with PIMD and in relation to important persons for the per- son with PIMD (Vlaskamp & Van Wijck, 1997). A new version of this instrument was developed which can be filled in by a person who is involved in the support of the person with PIMD (Vlaskamp, Van Wijck, et al., 2015). There are different versions for the different disciplines and persons involved in the support of a person with PIMD, including a version for parents, direct support person, health care psychologist, occupational therapist, speech therapist, physical therapist, and physician. The content consists of three parts: the development history of the person, characteristics of the person, and future goals for the person. In the development, the focus is on motor, cognitive and communicative skills, and in- dependency in daily life of the person. Moreover, the test and observation results and diagnoses are included in this part. The characteristics of the person concerns questions about emotions, needs, relations with others, basic mood, and changes in the mood of a person. The future goals part con- siders questions about the needs, wishes, and goals that the involved person has for the person with PIMD (Vlaskamp, Van Wijck, et al., 2015). 2.1. Participants Moreover, two of the involved residential care facilities dis- cussed the study in an internal meeting and approved the study during that meeting. The other in- volved residential care facility had an ethical board who approved the study. All participants with PIMD experienced severe problems in communication. Language was limited or non-existent and communication included reflex responses, sounds, facial expressions, and bod- ily movements (Vlaskamp, 2005). Of the eight direct support persons and the two health care psy- chologists who filled in the questionnaires about the IPS, there were eight women and three men. They were all involved as direct support persons in the living facility and all working with the person with PIMD for at least a year. 2.3. Procedure Various facilities were asked by phone and e-mail to participate in the study. Health care psycholo- gists were asked to select participants and their direct support persons. 2.2.2. BAS The BAS measures the functional abilities of a person with PIMD on five different domains: emotional and communicative behavior (3 items), receptive language skills (9 items), general communicative behavior (15 items), visual behavior (36 items), and explorative behavior (37 items) (Vlaskamp et al., 1999). Each domain consists of dichotomous items: the answers consist of a yes/no construction, thus indicating that the higher the total score per domain, the more often the person scores a yes on that domain (Vlaskamp et al., 2002). Part of the BAS, 28 items, is based on knowledge of a proxy of the person with PIMD and is scored by an interview of that proxy. These items are, for example, Page 4 of 10 Wessels & van der Putten, Cogent Psychology (2017), 4: 1340082 https://doi.org/10.1080/23311908.2017.1340082 about whether the person with PIMD recognizes other familiar persons and how this can be seen. The other items are scored by observation of the person with PIMD. Using different objects and ma- terials it is tried to provoke behavior of the person. For instance, these items are about reaction to auditory and visual stimuli. The BAS is designed to give the person with PIMD maximal opportunities to show his or her functional possibilities. It has no age boundaries or cut-off scores and contains small steps between different items, making it possible to detect small differences in development. There is no time limit for the person with PIMD to show the behavior, nor a strict order of the items (Vlaskamp et al., 1999). Raw scores are converted to quartile scores on the five domains which re- sults in a personal profile about the higher and lower possibilities of a person. The BAS was evaluated as being practical useful by health care psychologists and support staff working with persons with PIMD (Visser, Oldenburger, & Van der Meulen, 2014). Factor analysis showed that the internal consistency of the BAS was very high (Vlaskamp et al., 1999). Moreover, the inter-observer reliability was good and the instrument was evaluated as reliable. Content validity was evaluated as good (Vlaskamp et al., 2002). 2.4. Analysis 2.4.1. Usability of IPS All eight direct support persons were asked to fill in the IPS and they were asked about the usability of the IPS. The health care psychologists were not involved in this part due to time constraints. For measuring the usability of the IPS, a questionnaire was constructed. Usability was defined by four quality components: time to fill in the IPS, time filling in the IPS compared to the previous version, comprehensibility of the IPS and information covered by the questions of the IPS. The questions about the usability of the IPS were: •  How much time did it take to fill in the IPS? •  Did you work with the previous version of the IPS? If yes, did filling in the new version take more or less time than the previous version? •  How many questions or concepts were difficult to understand? Which ones were difficult to understand? •  Is there any information about the person with PIMD that is not included by filling in the IPS? If yes, what information did you miss? yes, what information did you miss? Personal information of the participants with PIMD and their direct support persons was coded to guarantee anonymity. Personal information of the participants with PIMD and their direct support persons was coded to guarantee anonymity. Descriptives concerning gender, age, and additional impairments of the participants were com- puted. The analysis was descriptive, by describing for every direct support person if he or she worked with the previous version of the IPS, how much time filling in the IPS was indicated by the direct support persons and whether there was enough space for filling in the questions. The analysis was qualitative, using descriptive qualitative analysis (Sandelowski, 2000) to analyze what items where difficult to understand and why and to analyze extra comments that were made. 2.4.2. Content validity IPS For the eight participants, the BAS was also filled in, to analyze content validity according to Tadema, Vlaskamp, and Ruijssenaars (2007). Part of the BAS was filled in by observation of the person with PIMD and part of the BAS was filled in by interviewing the direct support persons. All eight direct sup- port persons filled in the IPS. Moreover, in order to write a support profile, for some persons with Page 5 of 10 Page 5 of 10 Wessels & van der Putten, Cogent Psychology (2017), 4: 1340082 https://doi.org/10.1080/23311908.2017.1340082 PIMD, parents, physicians, physiotherapists, and health care psychologists were also asked to fill in the IPS. Based on the information of both the IPS and BAS, a support profile was written. A second questionnaire was used consisting of five questions about the support profile. The questions that were asked were: PIMD, parents, physicians, physiotherapists, and health care psychologists were also asked to fill in the IPS. Based on the information of both the IPS and BAS, a support profile was written. A second questionnaire was used consisting of five questions about the support profile. The questions that were asked were: •  To what degree is the description of the person in this support profile consistent with your own impression of who this person is? •  What impression does the support profile give of the functional abilities of this person with PIMD? •  What impression does the support profile give you of the wishes and needs of this person? •  What impression does the support profile give of the difficulties in the support of this person? •  Does the support profile contain enough information to formulate goals in the support of the person? The questions were scored on a five point Likert Scale. The first question ranged from very inconsist- ent to very consistent (1 = very inconsistent, 2 = inconsistent, 3 = neither inconsistent nor consist- ent, 4 = consistent, 5 = very consistent). 2.4.2. Content validity IPS Question two till four ranged from very bad impression to very good impression (1 = very bad impression, 2 = bad impression, 3 = neither good nor bad impres- sion, 4 = good impression and 5 = very good impression), and the last question ranged from “very bad possible” to “very good possible” (1 = very bad possible, 2 = bad possible, 3 = neither good nor bad possible, 4 = good possible, 5 = very good possible). After every question, a question for clarifica- tion was asked, i.e. “Can you explain why you think this is a good/bad impression of the functional abilities?” Using a Likert Scale, direct support persons could score the quality of the support profile and using the open questions it was possible to further explain why they would give a specific score. The analy- sis was partly quantitative, by computing mean score, standard deviation, minimum and maximum score on the five-point Likert Scale for the five questions computed. Moreover, the answers on the open questions were analyzed using descriptive qualitative analysis (Sandelowski, 2000). Due to the sudden death of one of the participants and time constraints of two other direct support persons, the questionnaire about the support profile was filled in by five persons. Four were the direct support persons who filled in the IPS and one was the involved health care psychologist of one of the persons with PIMD. Moreover, the other involved health care psychologist gave feedback on the support pro- file, together with the direct support person, by filling in one content validity questionnaire together. All questionnaires and instruments that were used in this study were in Dutch. Notes: Question 1 = To what degree is the description of the person in this support profile consistent with your own impression of who this person is?; Question 2 = What impression does the support profile give of the functional abilities of the person with PIMD?; Question 3 = What impression does the support profile give you of the wishes and needs of this person?; Question 4 = What impression does the support profile give you of the difficulties in the support of this person?; Question 5 = Does the support profile contain enough information to formulate goals in the support of the person? 3.1. Usability of the IPS Of the eight direct support persons, five filled in the time spent on filling in the IPS and three values were missing. According to five direct support persons, filling in the IPS took 30 min–120 min (M = 78, sd = 32.52). Six of the eight direct support persons had already worked with the previous version of the IPS, and two did not. Of these six direct support persons, one said that filling in the new version took more time than the previous one, two said that it took about the same time, one direct support person said it took less time and one value was missing. One direct support persons mentioned con- cerns regarding the list for parents, indicating that not all parents may understand all the questions. For part one of the list, the development part, all questions were understood, but two direct support persons indicated that some questions could better be filled in by another expert, such as a physician and a physical therapist for motor development. One direct support person indicated that there was not enough space for answering the questions, due to problems with the digital form of the IPS. Page 6 of 10 Page 6 of 10 Wessels & van der Putten, Cogent Psychology (2017), 4: 1340082 https://doi.org/10.1080/23311908.2017.1340082 For part 2 of the IPS, the personal characteristics, all the questions were understood by the partici- pants. Again, one direct support person indicated that there was not enough space for answering the questions, due to problems in the digital version. Moreover, one direct support person suggested that it would be better to fill in the questions about personal characteristics by two persons instead of one. For the last part of the list, about the future goals for the person with PIMD, all the questions were understood and there was enough space to fill in the questionnaires. All the direct support persons (N = 8) felt that they were the right person and had the necessary knowledge to answer the questions. 3.2. Content validity of the IPS In Table 2, the mean scores and standard deviation on the five-point Likert Scale of the question- naire of the support profile can be found. In this table it is shown that the mean score on the five- point Likert Scale is high in general and the range is small. In general, all direct support persons indicated that they were content with the support profiles and said that the support profiles were well written. One of the involved health care psychologists noted that it was remarkable that a support profile that is solely based on a IPS and a BAS and was written by someone who barely knew the person with PIMD, could describe the characteristics, needs, and wishes of a person with PIMD so accurately. On question one to four, there were, how- ever, some comments made. At the first question, one person indicated that information regarding what the person likes and what goals were reached in the past year was missing in the support profile. However, this was in- cluded in the answers on the IPS. Moreover, information about the motor activation, the way the person structures his or her world and what support the person needs was missing according to one person who filled in the questionnaire. Whereas the question of what support a person needs is in- cluded in the IPS, specific questions about motor activation and the way a person structures his or her world are not. The direct support person who filled in the IPS did not indicate it at other ques- tions. Moreover, one direct support person mentioned that there was no information about the diet the person with PIMD had because of diabetics and this information was also not part of the ques- tions in the IPS. At the question about functional possibilities, one direct support person mentioned that there was too little information about the auditory impairments and the motor impairments of the person with PIMD. Also, the health care psychologist of one of the persons indicated that the information that was mentioned regarding functional disabilities, was limited. Table 2. 5. Discussion Although in general usability and content validity of the instrument were rated as good, some com- ments should be made. First of all, a concern of one direct support person was about the usability of the IPS for parents; a direct support person indicated that probably some parents may experience the list as a burden and may have difficulty with understanding the terms and questions. Since par- ents can be considered to be experts in the care of persons with PIMD and are essential in formulat- ing their wishes and needs (Jansen, Van der Putten, & Vlaskamp, 2013), it is of great importance that they are able to fill in the questionnaire as well. By filling in the instruments, all the available information about the person with PIMD is gathered and support can be specifically adapted to the highly complex support needs of the person with PIMD (Vlaskamp & van der Putten, 2009). This can increase quality of support, which is strongly re- lated to the quality of life of people with PIMD (Lyons et al., 2016). According to the majority of the participants, the support profile was consistent with their own impression of who the person is. It gave enough information about the difficulties in the support of the person with PIMD and it con- tained enough information to formulate goals in the support of the person with PIMD. Some methodological limitations of this study should be taken into account. The qualitative study of the IPS concerned a small sample, and one usability questionnaire was not filled in, but feedback was sent by the direct support professional without using the format. One of the questions about us- ability concerned the time it takes to fill in the IPS, which is not an entirely objective indicator of usabil- ity as some direct support persons are willing to invest more time in assessment than others. However, the question still yields valuable information, since there is a limit as to how much time direct support persons can invest in filling in instruments. Moreover, the time-related question of the IPS is especially interesting in combination with the questionnaire about content validity: investing time in filling in the IPS will be valued more if filling in the instrument yields the information that is needed in support of the person with PIMD. 3.2. Content validity of the IPS Mean score, standard deviation, minimum, and maximum score on the support profile questionnaire Question Mean Standard deviation Minimum Maximum 1 4.20 0.45 4 5 2 3.90 0.60 2 4 3 3.60 0.55 3 4 4 4 0 4 4 5 4 0 4 4 Notes: Question 1 = To what degree is the description of the person in this support profile consistent with your own impression of who this person is?; Question 2 = What impression does the support profile give of the functional abilities of the person with PIMD?; Question 3 = What impression does the support profile give you of the wishes and needs of this person?; Question 4 = What impression does the support profile give you of the difficulties in the support of this person?; Question 5 = Does the support profile contain enough information to formulate goals in the support of the person? Page 7 of 10 Page 7 of 10 Wessels & van der Putten, Cogent Psychology (2017), 4: 1340082 https://doi.org/10.1080/23311908.2017.1340082 At the third question, one direct support person noted that determining the wishes and needs was difficult for all involved persons, so answering these questions was difficult in the IPS and thus diffi- cult to find in the support profile. Another direct support person mentioned there was not enough information about the need the person with PIMD had to always have a familiar support person close to him. A health care psychologist mentioned that it was important to fill in this part with sev- eral involved persons instead of one. At the question about difficulties in support of a person with PIMD, one direct support person men- tioned that there was not enough information about the alertness of the person with PIMD. One di- rect support person said that there was behavior of the person with PIMD mentioned in the support profile that the person rarely showed and which did not have a large influence in daily life anymore. 4. Conclusion This study focused on the usability and content validity of the IPS. In general, usability and content validity were good according to the majority of the direct support professionals. Concerning usabili- ty, all questions were clear and understood and there was enough space to fill it in. This was accord- ing to expectations, since the new version of the IPS was developed to be filled in by the involved persons without an interview with a health care psychologist (Vlaskamp, Van Wijck, et al., 2015). Scores on the questionnaire about the support profile, that were an indication for content validity, ranged from 3.60–4.20 (on a scale of 1–5), indicating a range from a neither good nor bad impression of the support profile to a (very) good impression of the support profile. In all, the majority of the scores indicated a good impression of the support profile. Citation information Cite this article as: Assessment in people with PIMD: Pilot study into the usability and content validity of the Inventory of the personal Profile and Support, Marleen D. Wessels & Annette A.J. van der Putten, Cogent Psychology (2017), 4: 1340082. Cite this article as: Assessment in people with PIMD: Pilot study into the usability and content validity of the Inventory of the personal Profile and Support, Marleen D. Wessels & Annette A.J. van der Putten, Cogent Psychology (2017), 4: 1340082. Lyons, G. S., Walla, P., & Arthur-Kelly, M. (2013). Towards improved ways of knowing children with profound multiple disabilities: Introducing startle reflex modulation. Developmental Neurorehabilitation, 16, 340–344. https://doi.org/10.3109/17518423.2012.737039 Psychology (2017), 4: 1340082. Author details Author details Marleen D. Wessels1 E-mail: m.d.wessels@rug.nl Annette A.J. van der Putten1 E-mail: a.a.j.van.der.putten@rug.nl 1 Faculty of Behavioural and Social Sciences, Centre for Special Needs Education & Youth Care, University of Groningen, Grote Rozenstraat 38, 9712 TJ Groningen, The Netherlands. Jansen, S. L. G., Van der Putten, A. A. J., & Vlaskamp, C. (2013). What parents find important in the support of a child with profound intellectual and multipledisabilities. Child: Care, Health and Development, 39, 432–441. Jansen, S. L. G., Van der Putten, A. A. J., & Vlaskamp, C. (2013). What parents find important in the support of a child with profound intellectual and multipledisabilities. Child: Care, Health and Development, 39, 432–441. Lyons, G., De Bortoli, T., & Arthur-Kelly, M. (2016). Triangulated proxy reporting: A technique for improving how communication partners come to know people with severe cognitive impairment. Disability and Rehabilitation, 1–7. doi: https://doi org/10 1080/09638288 2016 1211759 Lyons, G., De Bortoli, T., & Arthur-Kelly, M. (2016). Triangulated proxy reporting: A technique for improving how communication partners come to know people with severe cognitive impairment. Disability and Rehabilitation, 1–7. doi: https://doi org/10 1080/09638288 2016 1211759 Funding Hogg, J. (1992). The administration of psychotropic and anticonvulsant drugs to children with profound intellectual disability and multiple impairments. Journal of Intellectual Disability Research, 36, 473–488. Hogg, J. (1992). The administration of psychotropic and anticonvulsant drugs to children with profound intellectual disability and multiple impairments. Journal of Intellectual Disability Research, 36, 473–488. Funding The authors received no direct funding for this research. Competing Interests The authors declare no competing interest. Hogg, J., & Langa, A. (2005). Introduction: Assessment in perspective. In J. Hogg & A. Langa (Eds.), Assessing adults with intellectual disabilities: A service providers’ guide (pp. 1–5). Oxford: The British Psychological Society and Blackwell Publishing Ltd. https://doi.org/10.1002/9780470773697 5. Discussion Furthermore, support profiles were written by a researcher and not someone who knew the person with PIMD well. Moreover, for one of the persons with PIMD, the BAS was filled in by someone else than the researcher, meaning that the researcher who wrote the support profile, never saw the person with PIMD, which may have caused a lower rate of the profile because there was Page 8 of 10 Page 8 of 10 Wessels & van der Putten, Cogent Psychology (2017), 4: 1340082 https://doi.org/10.1080/23311908.2017.1340082 no other information about the person with PIMD available but the information of the IPS and BAS. This was also the person who rated the support profile lowest. In addition, due to time constraints, for seven of the eight profiles, only the BAS and the version of the IPS filled in by the direct support person was used to write a support profile and not the versions of other involved persons. Precisely this is of great importance for writing a support profile. A support profile is more than just a summary about whom the person with PIMD is, it is a complete profile containing all the information of all the involved persons and discrepancies between different persons are of special interest (Vlaskamp, Poppes, & Zijlstra, 2005). This could have influenced the results, since, for example, the health care psychologist who filled in the questionnaire about the support profile did not fill in the IPS. If this version was inte- grated, this opinion may also have been better reflected and the profile would contain more informa- tion. Therefore, the comments that were made about the content validity of the IPS could have been explained by these limitations. In conclusion, in further studies regarding the IPS a recommendation is basing the support profiles on the IPS filled in by different persons who know the person with PIMD well. Moreover, it is important to look at the usability of the list for parents. Recommendations regard- ing practice concern the importance of using good assessment instruments during different stages of support in a consistent, systematic way, using the input of all involved persons in support of the per- son with PIMD. Assessment is crucial to learn about the wishes, needs, and preferences of persons with PIMD in order to deliver individualized support (Lyons et al., 2013). References Bayley, N. (1967). Manual for the Bayley Scales of Infant Development. New York, NY: Psychological Corporation. Maes, B., Lambrechts, G., Hostyn, I., & Petry, K. (2007). Quality- enhancing interventions for people with profound intellectual and multiple disabilities: A review of the empirical research literature. Journal of Intellectual and Developmental Disability, 32, 163–178. https://doi org/10 1080/13668250701549427 Bohmer, C. J. M., Niezen-de Boer, M. C., Klinkenberg-Knol, E. C., Deville, W. L. J. M., Nadorp, J. H. S. M., & Meuwissen, S. G. M. (1999). 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International classification of functioning, Disability and health. Geneva: Author. References World Health Organization. (2001). International classification of functioning, Disability and health. Geneva: Author. © 2017 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. You are free to: Share — copy and redistribute the material in any medium or format Adapt — remix, transform, and build upon the material for any purpose, even commercially. The licensor cannot revoke these freedoms as long as you follow the license terms. Under the following terms: Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. 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&lt;p&gt;Retrospective Analysis of Factors Associated with Long-Stay Hospitalizations in an Acute Psychiatric Ward&lt;/p&gt;
Risk management and healthcare policy
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Dovepress open access to scientific and medical research Dovepress open access to scientific and medical research Risk Management and Healthcare Policy Rosaria Di Lorenzo 1 Giulia Montardi 2 Leda Panza3 Cinzia Del Giovane4 Serena Saraceni2 Sergio Rovesti 5 Paola Ferri 6 Purpose: To evaluate the longest hospitalizations in an acute psychiatric ward [Service of Psychiatric Diagnosis and Treatment (SPDT)] and the related demographic, clinical and organizational variables to understand the factors that contribute to long-stay (LOS) phe- nomenon. The term “long stay” indicates clinical, social and organizational problems responsible for delayed discharges. In psychiatry, clinical severity, social dysfunction and/ or health-care system organization appear relevant factors in prolonging stays. Patients and Methods: We divided all the SPDT hospitalizations from 1 January 2010 to 31 December 2015 into two groups based on the 97.5th percentile of duration: ≤36 day (n=3254) and >36 day (n=81) stays, in order to compare the two groups for the selected variables. Comparisons were made using Pearson’s chi-square for categorical data and t-test for continuous variables, the correlation between the LOS, as a dependent variable, and the selected variables was analyzed in stepwise multiple linear regression and in multiple logistic regression models. 1Psychiatric Intensive Treatment Facility, Department of Mental Health and Drug Abuse, Az-USL Modena, Modena 41122, Italy; 2School of Specialization in Psychiatry, University of Modena and Reggio Emilia, Modena 41124, Italy; 3School of Nursing, University of Modena and Reggio Emilia, Modena 41124, Italy; 4Head of Statistics and Methodology, Institute of Primary Health Care (BIHAM), Bern, Switzerland; 5General and Applied Hygiene, Department of Biomedical, Metabolic and Neural Sciences, Modena 41125, Italy; 6Nursing, Department of Biomedical, Metabolic and Neural Sciences, Modena 41125, Italy Results: The longest hospitalizations were significantly related to the diagnosis of “schizo- phrenia and other psychosis” (Pearson Chi2=17.24; p=0.045), the presence of moderate and severe aggressiveness (Pearson chi2=29; p=0.000), compulsory treatment (Pearson Chi2=8.05; p=0.005), parenteral or other route administration of psycho-pharmacotherapy (Pearson Chi2=12.91; p=0.007), poli-therapy (Pearson Chi2=6.40; p=0.041), complex psychiatric activ- ities (Pearson Chi2=12.26; p=0.002) and rehabilitative programs (Pearson Chi2=37.05; p=0.000) during the hospitalization and at discharge (Pearson Chi2=29.89; p=0.000). Many demographic and clinical variables were statistically significantly correlated to the LOS at our multiple linear and logistic regression model. Conclusion: In our sample, clinical illness severity and need for complex therapeutic and rehabilitative treatments were associated with prolonged psychiatric hospitalizations. Understanding this phenomenon can have not only economic but also clinical, ethical and social relevance. Keywords: psychiatric long-stay, acute psychiatric ward, predictors of long-stay, illness severity Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. This article was published in the following Dove Press journal: Risk Management and Healthcare Policy This article was published in the following Dove Press journal: Risk Management and Healthcare Policy Rosaria Di Lorenzo 1 Giulia Montardi 2 Leda Panza3 Cinzia Del Giovane4 Serena Saraceni2 Sergio Rovesti 5 Paola Ferri 6 Rosaria Di Lorenzo 1 Giulia Montardi 2 Leda Panza3 Cinzia Del Giovane4 Serena Saraceni2 Sergio Rovesti 5 Paola Ferri 6 Risk Management and Healthcare Policy 2020:13 433–442 433 © 2020 Di Lorenzo et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/ terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Di Lorenzo et al demographic and clinical features. Di Lorenzo et al Interestingly, they found different interactions based on the type of ward (acute/emergency or general) and the seniority of psychia- trists: longer practice of the psychiatrist in acute/emergency ward led to longer duration of patient hospitalization, sug- gesting that length of stay depends on the service system rather than individual patient characteristics.6 Another Japanese study highlighted that patients living in regions with fewer home visits for psychiatric nursing care had significantly longer psychiatric stays.21 Also, in Canada, researchers observed that patients with a long wait for psychiatric consultation referral reported the longest dura- tion of psychiatric hospitalizations, suggesting that the quality of health care organization can impact the length of stay.22 In this regard, other studies evidenced that alter- natives to full-time hospitalizations, such as ambulatory care, part-time hospitalizations as well as full-time care integrated in the community, outside of inpatient settings (ie, hospitalizations at home, stays in therapeutic apart- ments, stays in specially trained families, crisis centers and rehabilitation centers), can represent benefits for redu- cing the length of full-time hospitalizations, in accordance with international recommendations for mental health care.23 One of the first articles highlighted that the phenom- enon of “delayed discharges” accounted for 35% of all discharges and was strictly related to social issues since delayed discharge patients represented the poorest section of the population.7 Other historical studies highlighted that, in psychiatry, “delayed discharges” ranged between 27% and 58%, mainly due to patients’ refusal of an assis- tance program and by patients’ loneliness and social maladjustment.8 Successively, another Canadian study highlighted that lack of residential care and long waiting lists to get into a protected facility constituted the organi- zational reasons, whereas schizophrenia represented a clinical cause for delayed discharges in psychiatry.9 In a British study, poor living conditions, such as homeless- ness, significantly increased long-stays in psychiatric hospitals.10 Other more recent research on this phenom- enon has highlighted that clinical severity and the lack of outpatient service programs were the most frequent rea- sons for “delayed discharges”.11–13 Organic comorbidity and old age represented further risk factors for long psy- chiatric hospitalizations, according to other authors.14 In a recent European study, medical comorbidity was asso- ciated with increased length of stay in hospitalized psy- chiatric patients, after adjustment for several potential confounders.15 Other authors highlighted that hostility and manic excitement16 as well as aggressiveness during hospitalization represented risk factors for prolonged hospitalization.17 In a systematic review, Tulloch et al. Introduction In western countries, over the last three decades, the length of psychiatric hospitalizations has been drastically reduced in favor of community health care,1,2 but groups of patients have required frequent re-hospitalizations (“revolving door” phenomenon)3 or long stays in hospital (“new long-stay patients”).4 Due to the significant economic implications of this issue many efforts have been made not only to find which variables could predict the length of stay, but also how they mutually interact to impact on this phenomenon.5,6 Correspondence: Rosaria Di Lorenzo Psychiatrist, Psychiatric Intensive Treatment Facility, Az-USL Modena, Modena 41122, Italy Email r.dilorenzo@ausl.mo.it 433 submit your manuscript | www.dovepress.com http://doi.org/10.2147/RMHP.S238741 DovePress Di Lorenzo et al Dovepress DovePress Di Lorenzo et al (2011)18 found a positive association between LOS and female gender, psychotic disorders and large hospital size. Regarding gender, a recent study hypothesized that shorter length of stay among male patients could be related to their more frequent substance abuse, which could favor to early discharge due to the difficulty in long therapeutic engagement.19 p y submit your manuscript | www.dovepress.com DovePress 434 submit your manuscript | www.dovepress.com Aims In order to better understand the factors that contribute to long-stay phenomenon, we evaluated the correlation between the longest hospitalizations in an acute psychia- tric ward and selected demographic, clinical and organiza- tional variables, which, in accordance with the aforementioned literature can condition the length of stay. 3. Organizational variables [inpatient care activities/ problems: aggressive behavior (need for physical restraints and/or intervention of hospital security guards), rehabilitation programs, activation of com- munity service network (Mental Health Service, Social Service, Drug Addiction Service, etc.), the discharge modalities]. Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. We collected the following: 1. Demographic variables (gender, nationality, age, place of residence). 2. Clinical variables [psychiatric and organic diagnosis according to local diagnostic system, International Classification of Diseases-9th revision-Clinical Modification (ICD-9-CM),29 mono- and poly- pharmacotherapy, drug administration route, compul- sory and voluntary state of admission, extra-psychiatric medical activities (non-psychiatric consultations, clin- ical tests and therapy)]. Risk Management and Healthcare Policy 2020:13 Dovepress Di Lorenzo et al In Italy, after asylum closure, the length of stay in both public and private psychiatric wards has been considerably reduced due to the hypothesis that the institution itself can represent a pathogenic factor, which can drastically wor- sen the course of psychiatric disorders. In accordance with the spirit of Italian Law 180 of 23 May 1978, which mandated the closure of psychiatric asylums, long psychia- tric hospitalizations are considered the conditions which can induce regressive behavior, potentially worsening the course of psychiatric illnesses. In order to observe an Italian psychiatric context, many years after the aforemen- tioned Law was implemented, we decided to analyze long stays in a psychiatric acute ward to deepen the potential critical issues related to this phenomenon. We selected some demographic, clinical and organiza- tional variables, which, in accordance with the aforementioned literature, could influence the duration of hospitalizations. Due to the retrospective design of this study, the choice of the variable was conditioned by their availability in the ward electronic database. Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.18 For personal use only. Up to now, differences in the length of psychiatric stay among countries have been reported and attributed to the range of treatment options available in the community as well as to cultural aspects regarding style of psychiatric care delivery.24 Studies on this topic are few and difficult to compare due to the variability of health-care organiza- tion systems, not only among different countries, but even within a country’s local areas due to differences in clinical governance procedures and health organization policies and bed pressure.20 Nevertheless, a more recent study that evaluated the long-stay predictors in many European countries high- lighted that “clinical severity and social dysfunction” appeared relevant factors despite different organizations and policies of the health-care system.25 Regarding treat- ment policies, a recent review did not highlight any con- sistent result regarding the possibility that community compulsory treatment can reduce readmission or length of inpatient stay.26 Other studies focused on the differences between types of care organization: Bird et al.,20 in a large sample of 2709 patients admitted to 80 adult psychiatry wards, did not find any difference in term of stay length when com- paring critical organizational factors: patients in care trea- ted by the same psychiatrist across both inpatient and outpatient settings stayed on average 7 fewer days than those treated by different psychiatrists, "suggesting factors aside from the organization of care and patient character- istics have an impact on length of stay".20 Regarding this international debate, other authors have outlined that after asylum closure, “virtual asylums” have been created in other places for new long-stay patients, who are defined in the UK national audit as those with admissions lasting between 3 and 6 months.27 In Japan, where the length of hospitalizations often exceeds a period of one year,6 investigated the interaction between hospital organization variables and patients’ Risk Management and Healthcare Policy 2020:13 submit your manuscript | www.dovepress.com 434 DovePress Dovepress Patients and Methods This study was conducted in a 15-bed Italian public psy- chiatric ward, the so-called Service of Psychiatric Diagnosis and Treatment (SPDT), located in a general hos- pital of a town in the Italian Region of Emilia-Romagna, which received voluntary and involuntary patients affected by acute psychiatric diseases from a local population of 260,132 inhabitants up to June 2013 and subsequently from a local population of 511,782 inhabitants. Patients from other regions or towns can be admitted to this local ward but are subsequently transferred to the competent hospitals for specific geographic zone. After having labelled each variable with a number, we built an excel database in order to statistically analyze the data. We used standard statistics for descriptive analyses. Comparisons were made using Pearson’s chi-square for categorical data and t-test for continuous variables, the correlation between the duration of hospitalizations, as a dependent variable, and the selected variables were ana- lyzed in stepwise multiple linear regression, through back- ward selection estimation. We retrospectively collected all hospitalizations recorded in the ward electronic database from 1 January 2010 to 31 December 2015, after having de-identified all patient data. More than one hospitalization per patient in the obser- vation period was eligible for inclusion in the sample since the driver of analysis was all the hospitalizations and not the patients. For a further evaluation of the potential determinants of the longest hospitalizations were analyzed by means of the correlation between the selected variables and the longest hospitalizations as dependent variable (≤36 day hospitali- zations=0, >36 day hospitalizations=1) in multivariate stepwise logistic regression model, through backward selection estimation.30 A probability (p-value) <0.05 was considered statistically significant. The statistical analysis was conducted by means of STATA-12 program (2011). In accordance with literature, which defined “long stays” as the hospitalizations with a duration superior to 95th percentile.28 In order to evaluate the longest ones, we divided all the SPDT hospitalizations in the observation period into two groups based on the 97.5th percentile of duration: ≤36 day (n=3254) and >36 day (n=81) stays. This study was conducted in accordance with the prin- ciples of the Declaration of Helsinki (World Medical Association Declaration of Helsinki, 1964) and good clin- ical practice. Risk Management and Healthcare Policy 2020:13 Patients and Methods All information was collected after approval 435 Risk Management and Healthcare Policy 2020:13 submit your manuscript | www.dovepress.com DovePress Dovepress Di Lorenzo et al “personality disorders”, “anxiety disorders”, “alcohol, drugs and substance abuse” and other disorders, with a statistically significant difference between the two groups (Pearson Chi2=17.24; p=0.045). In the >36 day group, the diagnosis “schizophrenia and other psychoses” was more frequent, whereas “anxiety disorders”, “dementia and organic psycho- sis” and “acute stress reaction” were more common in the ≤36 day hospitalizations (Table 3). The most frequent desti- nation at discharge was “outpatient services”, followed by “other psychiatric wards”. The variable statistically which significantly differed between the two groups (Pearson Chi2=13.02, p=0.023) was represented by “transfer to non- psychiatric wards and/or protected facilities”, more frequent discharge modality after >36 day hospitalizations, whereas “transfer to other psychiatric wards” was more frequent in the other group (Table 3). of the study by the Ethics Committee of Area Vasta Emilia Nord (Italy) (3577 Protocol, 262/17 Practice, 26-9-2017) and the local Mental Health Department and Drug Abuse (1917 Protocol, 20-10-2017). of the study by the Ethics Committee of Area Vasta Emilia Nord (Italy) (3577 Protocol, 262/17 Practice, 26-9-2017) and the local Mental Health Department and Drug Abuse (1917 Protocol, 20-10-2017). Re We 333 tion a m col pat rep gra zati nat gro ≤36 and rou adm two the and Tab D Va Ag Ye Ge Ma Fe Na Ita No Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. We recorded, from 1 January 2010 to 31 December 2015, 3335 hospitalizations for 2074 patients with a mean dura- tion of 10.38±11.13 standard deviation (SD) days and a median of 7 days. During the observation period, we collected our long-stay sample of 81 hospitalizations by 63 patients, with a duration above the 97.5th percentile which represented 2.4% of all hospitalizations. In Table 1, we report the comparison between the demo- graphic variables related to the ≤36 and >36 day hospitali- zations. All demographic variables selected (age, gender, nationality and place of residence) were similar for both groups, without any statistically significant difference. Many demographic and clinical variables were statis- tically significantly correlated to the LOSs at our step- wise multiple regression linear model, as indicated in Table 4: The comparison between the clinical variables of the ≤36 and >36 day hospitalizations showed that psychiatric and rehabilitation activities, psycho-pharmacotherapy and routes of drug administration, aggressiveness and state of admission statistically significantly differed between the two groups (Table 2). ●age (coeff. 0.06), compulsory admission (coeff. 2.29), clinical interview with patient + caregivers (coeff. 4.32) or other professionals (coeff. 2.52), rehabilita- tion programs with staff of ward (coeff. 7.08) or other services (coeff. 2.11) during the hospitalization, mod- erate (coeff. 1.08) and severe (coeff. Re We 333 tion a m col pat rep gra zati nat gro ≤36 and rou adm two the and Tab D Va Ag Ye Ge Ma Fe Na Ita No Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. 4) aggressiveness Regarding the clinical variables at discharge (Table 3), the most common psychiatric diagnosis was “schizophrenia and other psychoses”, followed by “bipolar disorder”, Table 1 Demographic Variables Related to Hospitalizations in SPDT from 1/1/2010 to 31/12/2015, Divided by the Duration Demographic Variables ≤36 Days Hospitalizations N=3,254 (97.57%) >36 Days Hospitalizations N=81 (2.43%) Total Hospitalizations N=3,335 (100%) Statistical Test Probability Age (m± SD) Years 42.28±15.68 41.02±16.34 43.51±16.38 t=0.71 p=0.476 Gender, n (%) Males 1,845 (57%) 40 (49%) 1,885 (55%) Pearson Chi2=1.72 p=0.189 Females 1,409 (43%) 41 (51%) 1,450 (45%) Nationality, n (%) Italian 2,681 (82%) 64 (79%) 2,745 (82%) Pearson Chi2=0.62 p=0.431 Non-Italian 573 (18%) 17 (21%) 590 (18%) Place of Residence, n (%) Catchment area 2,764 (85%) 69 (85%) 2,833 (82%) Pearson Chi2=6.39 p=0.094 Italy (outside the catchment area) 284 (10%) 3 (4%) 287 (12%) Non-Italian residence 134 (4%) 7 (10%) 141 (5%) Homeless 24 (1%) 1 (1%) 25 (1%) s Related to Hospitalizations in SPDT from 1/1/2010 to 31/12/2015, Divided by the Duration 436 DovePress Di Lorenzo et al Dovepress Table 2 Clinical Variables Related to Hospitalizations in SPDT from 1/1/2010 to 31/12/2015, Divided into Two Groups by the Duration Clinical Variables ≤36 Days Hospitalizations N=3,254 (97.57%) >36 Days Hospitalizations N=81 (2.43%) Total Hospitalizations N=3,335 (100%) Statistical Test Probability Psychiatric Activities, n (%) Clinical interview with patient alone 1,433 (45%) 20 (25%) 1,453 (44%) Pearson Chi2=12.26 p=0.002 Clinical interview with patient +caregivers 1,424 (45%) 51 (63%) 1,475 (46%) Clinical interview with patient +caregivers + other professionals 337 (10%) 10 (12%) 347 (10%) Organic Comorbidity, n (%) Present 669 (21%) 14 (17%) 683 (20%) Pearson Chi2=0.52 p=0.471 Absent 2,585 (79%) 67 (83%) 2,652 (80%) Rehabilitation Programs, n (%) Absent 2,006 (61%) 32 (40%) 2,038 (61%) Pearson Chi2=37.05 p=0.000 In the ward 125 (4%) 13 (16%) 138 (4%) With other services 1,123 (35%) 36 (44%) 1,159 (35%) Extra-Psychiatric Clinical Activities, n (%) Present 1,481 (46%) 35 (43%) 1,516 (45%) Pearson Chi2=0.17 p=0.681 Absent 1,773 (54%) 46 (57%) 1,819 (55%) Psycho-Pharmacotherapy, n (%)* No psycho-pharmacotherapy 27 (1%) 0 (0%) 27 (1%) Pearson Chi2=6.40 p=0.041 Monotherapy 561 (17%) 6 (7%) 567 (17%) Politherapy 2,564 (79%) 73 (90%) 2,637 (79%) Routes of Drug Administration, n (%)* Oral 2,362 (73%) 46 (57%) 2,408 (72%) Pearson Chi2=12.91 p=0.000 Parenteral/more than one route 806 (25%) 35 (43%) 841 (25%) State of Admission, n (%) Voluntary 2,389 (73%) 48 (59%) 2,437 (73%) Pearson Chi2=8.05 p=0.005 Compulsory 865 (27%) 33 (41%) 898 (27%) Aggressiveness, n (%)** Absent 2,300 (71%) 39 (48%) 2,339 (70%) Pearson Chi2=29 p=0.000 Moderate 653 (20%) 24 (30%) 677 (20%) Severe (with need for restraints and/or interventions of hospital security guards) 254 (8%) 18 (22%) 272 (8%) Notes: *3% data not available; **2% data not available. Re We 333 tion a m col pat rep gra zati nat gro ≤36 and rou adm two the and Tab D Va Ag Ye Ge Ma Fe Na Ita No Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. elated to Hospitalizations in SPDT from 1/1/2010 to 31/12/2015, Divided into Two Groups by the Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. Notes: *3% data not available; **2% data not available. related to the longest stays (>36 day hospitalizations) as potential factors in prolonging stays, confirming the results of multiple linear regression model: manifested during hospitalization, poly-therapies (coeff. 2.4) administered by parenteral or more than one route (coeff. 2.41) and complex programs at dis- charge with one (coeff. 2.28) or more (coeff. 4.81) than one community outpatient service, with positive correlation; -“rehabilitation programs during the hospitalization” with ward staff (coeff.1.81); -“aggressiveness during hospitalization”, either moder- ate (coeff. 2.33) or severe (coeff. 3.44); ●transfer to other psychiatric wards with negative cor- relation (coeff. −2.99). -“routes of drug administration”, parenteral/more than one route” (coeff. 1.95); At our multiple stepwise logistic regression model (Table 5), only four variables were statistically significantly -“therapeutic and rehabilitative programs at discharge” with more than one outpatient service (coeff. 5.3). Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. 437 submit your manuscript | www.dovepress.com Risk Management and Healthcare Policy 2020:13 DovePress Dovepress Di Lorenzo et al Table 3 Clinical Variables Related to the Discharges from SPDT from 1/1/2010 to 31/12/2015, Divided by the Duration Clinical Variables ≤36 Days Hospitalizations N=3,254 (97.57%) >36 Days Hospitalizations N=81 (2.43%) Total Hospitalizations N=3,335 (100%) Statistical Test Probability Psychiatric Diagnosis (ICD-9-CM) at Discharge, n (%)* Schizophrenia and other psychotic disorders 1,139 (35%) 42 (52%) 1,181 (35%) Pearson Chi2=17.24 p=0.045 Bipolar disorder 570 (18%) 14 (17%) 584 (18%) Anxiety disorders 249 (8%) 1 (1%) 250 (7%) Mental retardation 120 (4%) 3 (4%) 123 (4%) Dementia and organic psychosis 160 (5%) 2 (2%) 162 (5%) Personality disorders 438 (13%) 11 (14%) 449 (13%) Substance, drugs and alcohol abuse 240 (7%) 4 (5%) 244 (7%) Adjustment disorders 142 (4%) 0 (0%) 142 (4%) Other disorders 94 (3%) 4 (5%) 126 (4%) No psychiatric diagnosis 28 (1%) 0 (0%) 28 (1%) Destination at Discharge, n (%)** General practitioner 230 (7%) 8 (10%) 238 (7%) Pearson Chi2=13.02 p=0.023 Community outpatient service 1,572 (48%) 40 (49%) 1,612 (48%) Transfer to other psychiatric wards 827 (25%) 10 (12%) 837 (25%) Transfer to non-psychiatric wards 42 (1%) 2 (2%) 44 (1%) Protected facilities 213 (7%) 11 (14%) 224 (7%) More than one outpatient community service 275 (8%) 7 (9%) 282 (8%) Therapeutic and Rehabilitative Programs at Discharge, n (%) With practitioner 910 (28%) 7 (9%) 917 (27%) Pearson Chi2=29.89 p=0.000 With one community outpatient service 1,450 (45%) 31 (38%) 1,481 (44%) With more than one community outpatient service 894 (27%) 43 (53%) 937 (28%) Notes: *2% data not available; **4% data not available. Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. reserved for acute crisis and for short periods, whereas rehabilitation programs are planned by outpatient commu- nity services.33 In other countries, efforts are still being made to reinforce the cooperation between in- and out- patients services, with the aim to limit the LOSs in order to reduce inpatient resource utilization.19 Nevertheless, a recent Cochrane review which compared stays of less than vs more than 28 days in patients with severe mental illness concluded that there were no benefits from longer hospital stays in terms of readmission and other outcomes, and that short stays were associated with better social functioning.4 submit your manuscript | www.dovepress.com DovePress 438 Risk Management and Healthcare Policy 2020:13 Discussion Our research was focused on the characteristics of the longest hospitalizations in an acute psychiatric ward, a phenomenon that is still only partially analyzed, although it represents a critical issue. This phenomenon is increasingly being studied in many Italian hospitals,31 but the available data on prolonged psychiatric hospitali- zations are difficult to compare due to the differences among the health organizations of various regions or coun- tries. As pointed out in other studies, the extrapolation and generalization of previous findings should take into account the specific characteristics of the regional/national mental health policies and cultures.6 Our results highlighted that patients hospitalized for the longest periods did not differ from other patients for demographic characteristics (mean age, gender, national- ity and place of residence). In our research, foreign and/or homeless patients were most often in the group with the longest hospitalizations, but without any statistically sig- nificant difference, suggesting that difficult environmental and social conditions might be one of the causes of In Italy, after Law 180,32 subsequently included in Law 833 of 23/12/1978, which represented a dramatic change in psychiatric care, especially due to the complete aboli- tion of psychiatric hospitals, the number of hospital beds for patients with psychiatric illnesses was reduced in order to avoid the risk of a chronic dependence on institutions. Currently in our country, hospitalizations in psychiatry are Risk Management and Healthcare Policy 2020:13 438 DovePress Di Lorenzo et al Table 4 Variables Statistically Significantly Related to Dependent Variable, Length of Hospitalization (Stepwise Multiple Linear Regression) Variables Coeff. Discussion Standard Error p-value Confidence Interval 95% Age 0.06 0.013 0.000 0.03; 0.08 Modality of Admission (Voluntary)* Compulsory 2.29 0.42 0.000 1.46; 3.13 Psychiatric Activities (Clinical Interview with Patient Alone)* Clinical interview with patient+caregivers 4.32 0.40 0.000 3.52; 5.11 Clinical interview with patient+ caregivers +other professionals 2.52 0.75 0.001 0.88; 3.79 Rehabilitation Programs During the Hospitalization (Absent)* With staff of ward 7.08 0.91 0.000 5.29; 8.86 With staff of other services 2.11 0.42 0.000 1.29; 2.94 Aggressiveness During the Hospitalization (Absent)* Moderate 1.08 0.51 0.035 0.07; 2.09 Severe (with need for restraints and/or interventions of hospital security guards) 4 0.7 0.000 2.63; 5.37 Psycho-Pharmacotherapy (Monotherapy)* Polytherapy 2.4 0.49 0.000 1.44; 3.36 Routes of Drug Administration (Oral)* Parenteral/more than one route 2.41 0.44 0.000 1.55; 3.27 Destination at Discharge (General Practitioner)* Transfer to other psychiatric wards −2.99 0.77 0.000 −4.51; −1.46 Therapeutic and Rehabilitative Programs at Discharge (With Practitioner)* With one community outpatient service 2.28 0.49 0.000 1.32; 3.23 With more than one community outpatient service 4.81 0.55 0.000 3.72; 5.89 Note: * Reference category. Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. The factors which conditioned the LOS were consti- tuted by complex psychiatric activities during hospitaliza- tion and at discharge involving of family members or caregivers, pharmacological therapies administered by multiple routes, intensive rehabilitation programs provided by ward staff and/or other services, and both mild and severe aggressive behavior. Altogether, these clinical and organizational variables indicate that patients who remain hospitalized for a very long time are characterized by multiple care needs closely related to the severity of psy- chiatric pathologies and not to organic comorbidities or social disadvantage. As suggested by other Authors, in investigating long-stay phenomenon, special attention should be given to indicators of illness severity, repre- sented by functioning scale scores, length and number of previous hospitalizations, employment, marital and hous- ings status.18 The variables significantly related to the length of psy- chiatric hospitalizations indicate that the severity of symp- toms, which often leads to difficulties in socio-relational functioning and adherence to care, can also lead to the risk of extended hospitalizations, requiring the involvement of a wide range of therapeutic, care and rehabilitation programs. Therefore, we hypothesize that, for preventing the long-stay phenomenon, the same Intensive Case Management pro- vided by mental health service for satisfying the unique combination of health and social care needs of people with severe mental illness, as recently highlighted by a Cochrane review,42 could be effective. In accordance with other authors, our results show that length of stay is “multifactorially” determined. More stu- dies evaluating factors that lengthen hospital stay are needed to implement more appropriate and tailored treat- ments in psychiatric services.24,41 Understanding this phe- nomenon has not only organizational and economic but also clinical, ethical and social relevance, as evidenced by most authors.43 The identification of factors associated with long hospitalization makes it possible to organize more appropriate therapeutic programs, reducing the eco- nomic burden on health care and, at the same time, the risk of psychological and physical complications for patients. Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. In particular, among the longest hospitalizations, the presence of aggressiveness, either moderate or severe, and the need for complex therapeutic treatment during the hospitalizations were the statistically significant condition- ing factors of hospitalization prolongation, in line with the results of other authors who highlighted that violence during the hospitalization as well as high number of med- ical conditions requiring medication could prolong the period of stays.35 Aggressiveness, which was particularly frequent among the patients with the longest hospitalizations in our and other studies,14,18 could represent a severe and acute symp- tom of many psychiatric disorders, often responsible for hospital admission, frequent readmissions3 and, according to other research,36 for the frequent aggressions towards staff, in particular nurses,37 in a psychiatric setting. The observation of serious and potentially dangerous aggressive behavior among patients with long-term hospitalizations corresponded with the data of the higher frequency of compulsory treatment in this group, which suggests the lack of illness awareness and behavior control among these patients. This result indirectly confirms that compul- sory treatment does not reduce length of inpatient stay,26 but it can represent an indicator of illness severity in psychiatry and a potential predictor of the long stay, as some Italian Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. Table 5 Variables Statistically Significantly Related to the Longest Hospitalizations (Stepwise Multiple Logistic Regression) Variables Coeff. Standard Error p-value Confidence Interval 95% Rehabilitation Programs During the Hospitalization (Absent)* With ward staff 1.81 0.45 0.016 1.12; 2.95 Aggressiveness During the Hospitalization (Absent)* Moderate 2.33 0.58 0.005 1.25; 3.63 Severe (with need of restraints and/or interventions of hospital security guards) 3.44 1.05 0.000 1.88; 6.26 Routes of Drug Administration (Oral)* Parenteral/more than one route 1.95 0.46 0.005 1.22; 3.11 Therapeutic and Rehabilitative Programs at Discharge (With Practitioner)* With more than one community outpatient service 5.3 2.61 0.001 2.01; 13.93 Note: * Reference category. Note: * Reference category. Note: * Reference category. demographic variables, apart from the above-reported data, only increased age showed a significant, positive but weak, correlation with the LOSs, confirming only in part the results of other authors.14 All our other results psychiatric-delayed discharges, as the first studies on this phenomenon highlighted.7,10,13 Nevertheless, we have to put in evidence that, in our study, this variable did not show any significant correlation with the LOSs. Among psychiatric-delayed discharges, as the first studies on this phenomenon highlighted.7,10,13 Nevertheless, we have to 439 submit your manuscript | www.dovepress.com Risk Management and Healthcare Policy 2020:13 DovePress Dovepress Di Lorenzo et al indicate that risk factors for long stays were mainly repre- sented by clinical and organizational variables. The pathologies in the group of patients with the longest hos- pitalizations were serious and potentially disabling, such as schizophrenia and other psychotic spectrum disorders, in accordance with data reported in the literature.9,34 authors highlighted.38,39 We can hypothesize that aggres- siveness itself could justify the difficulty in discharging patients, especially when it starts a sort of vicious cycle of aggressive escalation, that can trigger pathological depen- dence of patients on the institution. Regarding the discharge modalities, as some authors have already highlighted,3,40,41 our patients with the long- est hospitalizations needed to be sent to protected facilities for implementing complex programs carried out by more than one community service, probably due to the severity of clinical conditions and functioning abilities. Disclosure 17. Gigantesco A, de Girolamo G, Santone G, Miglio R, Picardi A, for PROGRES-Acute Group. Long-stay in short-stay inpatient facilities: risk factors and barriers to discharge. BMC Public Health. 2009;22 (9):306. doi:10.1186/1471-2458-9-306 The authors report no actual or potential conflicts of interest. 18. Tulloch AD, Fearon P, David AS. Length of stay of general psychia- tric inpatients in the United States: systematic review. Adm Policy Ment Health. 2011;38(3):155–168. doi:10.1007/s10488-010-0310-3 Conclusions We conclude by suggesting that the most prolonged hos- pitalizations in an acute psychiatric ward can be related to clinical features of illness severity as well as maladjust- ment or social drift condition, which, in turn, could be induced by serious and chronic mental illnesses. 11. Tulloch AD, Fearon P, David AS. Timing, prevalence, determinants and outcomes of homelessness among patients admitted to acute psychiatric wards. Soc Psychiatry Psychiatr Epidemiol. 2012;47 (7):1181–1191. doi:10.1007/s00127-011-0414-4 More tailored health community programs aimed at reducing this new chronicity could permit us to improve the quality of life of these patients, reducing, at the same time, the economic and social consequences of still unre- solved pathologies. 12. Tulloch AD, Khondoker MR, Fearon P, David AS. Associations of homelessness and residential mobility with length of stay after acute psychiatric admission. BMC Psychiatry. 2012;12:121. doi:10.1186/ 1471-244X-12-121 13. Russolillo A, Moniruzzaman A, Parpouchi M, Currie LB, Somers JM. A 10-year retrospective analysis of hospital admissions and length of stay among a cohort of homeless adults in Vancouver, Canada. BMC Health Serv Res. 2016;16:60. doi:10.1186/s12913-016- 1316-7 Finally, we can conclude by indicating that health treatments and policies, in order to provide a real eco- nomic return and quality of life in the long term, should be mainly addressed to the needs of each patient. We hope to have deepened the understanding of the determinants of long psychiatric stay, contributing to the appropriateness of treatments and care in psychiatry. 14. Lewis R, Glasby J. Delayed discharge from mental health hospitals: results of an English postal survey. Health Soc Care Community. 2006;14(3):225–230. doi:10.1111/j.1365-2524.2006.00614.x 15. Rodrigues-Silva N, Ribeiro L. Impact of medical comorbidity in psychiatric inpatient length of stay. J Ment Health. 2017;1–5. doi:10.1080/09638237.2017.1340605 16. Warnke I, Rössler W, Herwig U. Does psychopathology at admission predict the length of inpatient stay in psychiatry? Implications for financing psychiatric services. BMC Psychiatry. 2011;11:120. doi:10.1186/1471-244X-11-120 Risk Management and Healthcare Policy downloaded from https://www.dovepress.com/ by 155.185.26.79 on 19-May-2020 For personal use only. 8. Zeldow PB, Taub HA. Evaluating psychiatric discharge and aftercare in a VA medical center. Hosp Community Psychiatry. 1981;32 (1):57–58. doi:10.1176/ps.32.1.57 Further prospective studies, which takes into consid- eration the correlations highlighted by this research, could deepen our knowledge of the long-stay phenomenon. 9. Kelly A, Watson D, Raboud J, Bilsker D. Factors in delays in discharge from acute-care psychiatry. Can J Psychiatry. 1998;43 (5):496–501. doi:10.1177/070674379804300508 10. Koffman J, Fulop NJ. Homelessness and the use of acute psychiatric beds: findings from a one-day survey of adult acute and low-level secure psychiatric patients in North and South Thames regions. Health Soc Care Community. 1999;7(2):140–147. doi:10.1046/ j.1365-2524.1999.00156.x Limitations and Strengths g This study has a number of limitations. The retrospective methodology did not allow us to infer any causality and needs prospective research to be confirmed. The selection of variables was conditioned by their database availability, due to the retrospective design, and were not chosen according to a risk model based on a prospective research design. Future research on this topic, based on the results of this and other studies, will be able to build a reliable theoretical model, that will drive the search. Moreover, its results cannot be extrapolated to other psychiatric services. In particular, the generalizability of their findings is Risk Management and Healthcare Policy 2020:13 submit your manuscript | www.dovepress.com 440 DovePress Dovepress Di Lorenzo et al limited due to the important role that context plays in determining length-of-stay in psychiatric wards in Italy. 5. Johnstone P, Zolese G. Length of hospitalisation for people with severe mental illness. Cochrane Database Syst Rev. 2000;2: CD000384. Nevertheless, this study analyzed many variables in a large sample, for a sufficiently long observation period, providing data from Italian psychiatric services to add to the international body of literature, suggesting risk factors that could potentially be addressed with the right kind of outpatient programs. 6. Badriah F, Abe T, Nabeshima Y, Ikeda K, Kuroda K, Hagihara A. Predicting the length of hospital stay of psychiatry patients using signal detection analysis. Psychiatry Res. 2013;210(3):1211–1218. doi:10.1016/j.psychres.2013.09.019 7. Barrette PA. 1,005 delayed days: a study of adult psychiatric discharge. Hosp Community Psychiatry. 1981;32(4):266–268. doi:10.1176/ps.32.4.266 References 1. Furlan PM, Zuffranieri M, Stanga F, Ostacoli L, Patta J, Picci RL. Four-year follow-up of long-stay patients settled in the community after closure of Italy’s psychiatric hospitals. Psychiatr Serv. 2009;60 (9):1198–1202. doi:10.1176/ps.2009.60.9.1198 19. Wullschleger A, Wosniok W, Timm J, Heinze M. Challenges and perspectives in bridging in- and outpatient sectors: the implementation of two alternative models of care and their effect on the average length of stay. Front Psychiatry. 2017;8:196. doi:10.3389/fpsyt.2017.00196 2. Preti A, Rucci P, Gigantesco A, for PROGRES-Acute Group, et al. Patterns of care in patients discharged from acute psychiatric inpatient facilities: a national survey in Italy. Soc Psychiatry Psychiatr Epidemiol. 2009;44(9):767–776. doi:10.1007/s00127-009-0498-2. 20. Bird VJ, Giacco D, Nicaise P, et al. In-patient treatment in functional and sectorised care: patient satisfaction and length of stay. Br J Psychiatry. 2018;212(2):81–87. doi:10.1192/bjp.2017.20 21. Niimura J, Nakanishi M, Yamasaki S, Nishida A. Regional supply of outreach service and length of stay in psychiatric hospital among patients with schizophrenia: national case mix data analysis in Japan. Psychiatry Res. 2017;258:295–298. doi:10.1016/j.psychres.2017.08.067 3. Di Lorenzo R, Sagona M, Landi G, Martire L, Piemonte C, Del Giovane C. The revolving door phenomenon in an italian acute psy- chiatric ward: a 5-year retrospective analysis of the potential risk factors. J Nerv Ment Dis. 2016;204(9):686–692. doi:10.1097/ NMD.0000000000000540 22. Sockalingam S, Alzahrani A, Meaney C, et al. Time to consultation-liaison psychiatry service referral as a predictor of length of stay. Psychosomatics. 2016;57(3):264–272. doi:10.1016/j. psym.2016.01.005 4. Babalola O, Gormez V, Alwan NA, Johnstone P, Sampson S. Length of hospitalisation for people with severe mental illness. Cochrane Database Syst Rev. 2014;1:CD000384. 441 submit your manuscript | www.dovepress.com Risk Management and Healthcare Policy 2020:13 DovePress Di Lorenzo et al Dovepress 23. Gandré C, Gervaix J, Thillard J, Macé JM, Roelandt JL, Chevreul K. The development of psychiatric services providing an alternative to full-time hospitalization is associated with shorter length of stay in French public psychiatry. Int J Environ Res Public Health. 2017;14:3. doi:10.3390/ijerph14030325 34. Di Lorenzo R, Formicola V, Carra E, Piemonte C, Ferri P. Risk factors for long-stay in an Italian acute psychiatric ward: a 7-year retrospective analysis. J Nurs Educ Pract. 2014;4:68–80. 35. Cheng JE, Shumway M, Leary M, Mangurian CV. Patient factors associated with extended length of stay in the psychiatric inpatient units of a large urban county hospital. Community Ment Health J. 2016;52(6):658–661. doi:10.1007/s10597-015-9912-2 24. Baeza FL, da Rocha NS, Fleck MP. References Predictors of length of stay in an acute psychiatric inpatient facility in a general hospital: a prospective study. Braz J Psychiatry. 2018;40(1):89–96. doi:10.1590/1516-4446- 2016-2155 36. Ferri P, Silvestri M, Artoni C, Di Lorenzo R. Workplace violence in different settings and among various health professionals in an Italian general hospital: a cross-sectional study. Psychol Res Behav Manag. 2016;9:263–275. doi:10.2147/PRBM.S114870 25. Dimitri G, Giacco D, Bauer M, et al. Predictors of length of stay in psychiatric inpatient units: does their effect vary across countries? Eur Psychiatry. 2018;48:6–12. doi:10.1016/j.eurpsy.2017.11.001 37. Ferri P, Reggiani F, Di Lorenzo R. Aggressive behavior toward nursing staff in three different health care settings. [corrected]. Prof Inferm. 2011;64(3):143–150. 26. Barnett P, Matthews H, Lloyd-Evans B, Mackay E, Pilling S, Johnson S. Compulsory community treatment to reduce readmission to hospital and increase engagement with community care in people with mental illness: a systematic review and meta-analysis. Lancet Psychiatry. 2018;5 (12):1013–1022. doi:10.1016/S2215-0366(18)30382-1 38. Ielmini M, Poloni N, Caselli I, Gasparini A, Pagani R, Callegari C. Compulsory versus voluntary admission in psychiatry: an observa- tional study. Minerva Psichiatr. 2018;59(3):124–134. 27. Sharma A, Dunn W, O’Toole C, Kennedy HG. The virtual institution: cross-sectional length of stay in general adult and forensic psychiatry beds. Int J Ment Health Syst. 2015;9:25. doi:10.1186/s13033-015- 0017-7 39. Di Lorenzo R, Vecchi L, Artoni C, Mongelli F, Ferri P. Demographic and clinical characteristics of patients involuntarily hospitalized in an italian psychiatric ward: a 1-year retrospective analysis. Acta Biomedica. 2018;89(6–8):17–28. doi:10.23750/abm. v89i6-S.7392 28. Tukey JW. Explorating Data Analysis. Boston: Addison-Wesley; 1977. 40. Glasby J, Lester H. Delayed hospital discharge and mental health: the policy implication of recent research. Soc Policy Admin. 2004;38 (7):744–757. 29. Ministry of Labour, Health and Social Policy. International Classification of Diseases-9th Revision-Clinical Modification. Roma, Italy: Istituto Poligrafico e Zecca dello Stato; 2008. 41. Newman L, Harris V, Evans LJ, Beck A. Factors associated with length of stay in psychiatric inpatient services in London, UK. Psychiatr Q. 2018;89(1):33–43. 30. Berk R. Regression Analysis: A Constructive Critique. Thousand Oaks, CA: Sage Publications; 2004. 31. Mongardi M, Di Ruscio E, Vizioli M, et al. Il fenomeno delle dimissioni ospedaliere ritardate nella regione Emilia-Romagna. Mecosan. 2013;86:65–78. 42. Dieterich M, Irving CB, Bergman H, Khokhar MA, Park B, Marshall M. Intensive case management for severe mental illness. Cochrane Database Syst Rev. 2017;1:CD007906. 32. Burti L, Benson PR. Psychiatric reform in Italy: developments since 1978. Int J Law Psychiatry. 1996;19(3–4):373–390. References doi:10.1016/ S0160-2527(96)80008-2 43. Swidler RN, Seastrum T, Shelton W. Difficult hospital inpatient discharge decisions: ethical, legal and clinical practice issues. Am J Bioeth. 2007;7(3):23–28. doi:10.1080/15265160601171739 33. de Girolamo G, Polidori G, Morosini P, et al. Prevalence of common mental disorders in Italy: results from the European Study of the Epidemiology of Mental Disorders (ESEMeD). Soc Psychiatry Psychiatr Epidemiol. 2006;41(11):853–861. doi:10.1007/s00127-006- 0097-4 Dovepress guidelines, expert opinion and commentary, case reports and extended reports. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. 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РОЗРОБКА ІНФОРМАЦІЙНОЇ ТЕХНОЛОГІЇ ОПЕРАТИВНОЇ ТА КОНФІДЕНЦІЙНОЇ ДОСТАВКИ ВІДЕОІНФОРМАЦІЙНОГО РЕСУРСУ В СИСТЕМІ КРИТИЧНОЇ ІНФРАСТРУКТУРИ
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Таким чином, метою дослідження є підвищення оперативності передачі захищених відеоданих, що є актуальною науково-прикладною задачею. Таким чином, метою дослідження є підвищення оперативності передачі захищених відеоданих, що є актуальною науково-прикладною задачею. ИНФОРМАЦИОННЫЕ ТЕХНОЛОГИИ В свою чергу, завданням дослідження є розробка інформаційної технології оперативної та конфіде- нційної доставки відеоінформаційного ресурсу в системі критичної інфраструктури. ІНФРАСТРУКТУРИ БАРАННІК В.В., ГАВРИЛОВ Д.С., СОРОКУН А.Д. 2. Основна частина дослідження Розробляється інформаційна технологія оперативної та конфіденційної доставки відеоінформаційного ресурсу в системі критичної інфраструктури шляхом захисту блоків, що містять контурну інформацію. Виявляється контурна інформація за допомогою аналізу блоку роз- робленою метрикою. Показується можливість класифі- кації блоків за контурною насиченістю з метою пода- льшої обробки. Аналіз інформаційної технології JPEG показав, що даний алгоритм дозволяє зменшити об’єм вхідних даних за рахунок того, що усувається психовізуа- льна, спектральна та структурна надлишковість. Першою усувається психовізуальна надлишковість за рахунок психофізіологічної особливості зору людини, який реагує на зміни яскравості більше, ніж на зміни кольору. Маємо на увазі етап перет- ворення з колірного простору RGB в колірний простір YCrCb [9]. Слід зауважити, що найбільш важливою є компонента яскравості Y, аналіз якої вказав на можливість виявлення контурної інфор- мації. Ключові слова: інформаційна технологія, оператив- ність, конфіденційність, метрика, контурна інформація. УДК 621.397 РОЗРОБКА ІНФОРМАЦІЙНОЇ ТЕХНОЛОГІЇ ОПЕРАТИВНОЇ ТА КОНФІДЕНЦІЙНОЇ ДОСТАВКИ ВІДЕОІНФОРМАЦІЙНОГО РЕСУРСУ В СИСТЕМІ КРИТИЧНОЇ ІНФРАСТРУКТУРИ БАРАННІК В.В., ГАВРИЛОВ Д.С., СОРОКУН А.Д. Дослідження пропонується проводити на основі алгоритму обробки даних JPEG у зв’язку з широ- кою популярністю та високою ефективністю даної інформаційної технології. 1. Вступ З суцільною інформатизацією та впровадженням новітніх технологій в життя людини по всьому сві- ту, особливо в розвинутих країнах, які додержу- ються концепції Інтернету Речей, гостро постало питання контролю як внутрішніх процесів, так і зовнішніх проявів стану справ різного масштабу. Дослідження в області психофізіології за критері- єм рівня сприйняття інформації залежно від мето- ду доведення (візуально, за допомогою звуків (слова) чи кінестетично) показало, що візуальне доведення інформації про зовнішній прояв справ є найбільш ефективним. Розроблено метрику виявлення контурної інфор- мації: ; b log K 8 1 i i 2       = ∏ = ,)і( А min )і( А max bi − = де K – метрика, що визначає наявність контурної інформації в блоці; )і( А max – максимальний еле- мент і-го рядка блоку А; )і( А min – мінімальний елемент і-го рядка блоку А; ib – різниця максима- льного та мінімального елемента і-го рядка, при цьому: ∑ = = 8 1 i ib В . де K – метрика, що визначає наявність контурної інформації в блоці; )і( А max – максимальний еле- мент і-го рядка блоку А; )і( А min – мінімальний елемент і-го рядка блоку А; ib – різниця максима- льного та мінімального елемента і-го рядка, при цьому: ∑ = = 8 1 i ib В . При цьому на рівень сприйняття інформації, пред- ставленої у вигляді фото – та/чи відеоматеріалів, впливає рівень яскравості, контрасту та роздільна здатність. Варто відзначити, що відеоінформаціний ресурс збирається, обробляється та передається як на рів- ні людина-людина, так і на рівні держава-держава, цивілізація-цивілізація. Подібні відомості, як пра- вило, містять конфіденційну інформацію, тож пот- ребують криптографічного захисту. Найбільш по- мітним на зображенні є об’єкт, що виділяється (контрастує) на фоні, тобто елемент зображення, який має чіткий контур. 1. Вступ Приклад роботи даної метрики: 136 136 136 136 237 237 237 0 136 136 136 136 136 237 0 237 136 136 136 136 136 0 237 237 136 136 136 0 0 136 136 237 237 136 0 136 136 136 136 136 237 0 136 136 136 136 136 136 0 237 237 136 136 136 136 136 237 237 237 237 136 136 136 136 = А 136 136 136 136 237 237 237 0 136 136 136 136 136 237 0 237 136 136 136 136 136 0 237 237 136 136 136 0 0 136 136 237 237 136 0 136 136 136 136 136 237 0 136 136 136 136 136 136 0 237 237 136 136 136 136 136 237 237 237 237 136 136 136 136 = А 136 136 136 136 237 237 237 0 136 136 136 136 136 237 0 237 136 136 136 136 136 0 237 237 136 136 136 0 0 136 136 237 237 136 0 136 136 136 136 136 237 0 136 136 136 136 136 136 0 237 237 136 136 136 136 136 237 237 237 237 136 136 136 136 = А Отже, для забезпечення необхідного рівня захисту необхідною і достатньою умовою є криптографіч- ний захист блоків, що містять контурну інформа- цію. При цьому критичним є час на обробку та пе- редачу даних. РИ, 2018, №2 51 237 0 237 0 237 0 237 0 237 0 237 0 237 0 237 0 237 237 237 237 237 237 237 237 ; 9.9537 b 8 1 i i = ∏ = 63.1099. К = – блок з поступовим переходом кольору; Таблиця 2 )і( А max )і( А min ∏ = 8 1 i ib К 219 219 219 219 214 207 193 163 126 129 126 120 112 102 93 86 6.35512 52.496 59 120 161 194 222 248 255 255 0 1 0 0 0 0 34 52 5.4165 58.910 – блок з контурною інформацією. 1. Вступ Таблиця 3 = )і( А min = )і( А max = B – блок з поступовим переходом кольору; Таблиця 2 )і( А max )і( А min ∏ = 8 1 i ib К 219 219 219 219 214 207 193 163 126 129 126 120 112 102 93 86 6.35512 52.496 59 120 161 194 222 248 255 255 0 1 0 0 0 0 34 52 5.4165 58.910 – блок з контурною інформацією. – блок з поступовим переходом кольору; – блок з контурною інформацією. – блок з контурною інформацією. Таблиця 3 )і( А max )і( А min ∏ = 8 1 i ib К 249 237 239 231 236 222 224 241 29 23 24 25 21 19 0 0 4.91294 62.0913 255 255 255 255 249 252 255 255 14 0 77 0 1 66 36 3 7.10111 62.6228 Приклад вихідного та захищеного зображення В ході дослідження якості розробленої інформа- ційної технології встановлено можливість визна- чення класу блоку 8х8 (табл. 1-3). Виявлена особ- ливість дає змогу проводити уточнюючий та/чи додатковий аналіз фото - та/чи відеоматеріалу на наступних етапах обробки на основі JPEG- платформи. Виділено такі класи блоків: – блок без контурної інформації; Таблиця 1 )і( А max )і( А min ∏ = 8 1 i ib К 34 34 34 34 34 34 34 34 34 34 34 34 34 34 34 34 0 0 255 255 255 255 255 255 255 255 255 255 255 255 255 255 255 255 0 0 Таблиця 1 )і( А max )і( А min ∏ = 8 1 i ib К 34 34 34 34 34 34 34 34 34 34 34 34 34 34 34 34 0 0 255 255 255 255 255 255 255 255 255 255 255 255 255 255 255 255 0 0 РИ, 2018, №2 РИ, 2018, №2 РИ, 2018, №2 52 Рис. 1. Вихідне зображення Наукова новизна. Виявлення контурної інформа- ції відбувається за допомогою аналізу блоку роз- робленою метрикою. Виявлена можливість класи- фікації блоків за контурною насиченістю з метою подальшої обробки. Вирішена актуальна науково-прикладна задача підвищення оперативності передачі захищених відеоданих. Литература: 1. Баранник В.В. Метод повышения ин- формационной безопасности в системах видеомонито- ринга кризисных ситуаций / В.В. Баранник, Ю.Н. Рябуха, О.С. // Монография. Черкассы, 2015. 143 с. 2. Баранник В.В. Модель загроз безпеки відеоінфор- маційного ресурсу систем відеоконференцзв'язку / А.В. Власов, В.В. Бараннік, Р.В.Тарнополов // Наукоємні технології. 2014. № 1 (21). С. 55 – 60. 1. Вступ 3. Баранник В.В. Обоснование значимых угроз безопасности видеоин- формационного ресурса систем видеоконференцсвязи профильных систем управления / В.В. Баранник, А.В. Власов, С.А. Сидченко, А.Э. Бекиров // Информацион- но-управляющие системы на ЖД транспорте. 2014. №3. С. 24 – 31. 4. Баранник В.В. Селективний метод шифру- вання відеопотоку в телекомунікаційних системах на основі приховування базового I-кадру / В.В. Баранник, Д.І. Комолов, Ю.М. Рябуха // Наукоємні технології. № 2. 2015. С. 14 – 23. 5. Barannik V.V. The model of avalanche-relating effect in the process of images reconstruction in the combined cryptosemantic systems on the polyadic presentation / V.V. Barannik V.V. Larin, S.A. Sidchenko // Наукоємні технології. 2010. № 1(5). С. 68 – 70. 6. Гаврилов Д.С. Метод захисту низькочастотних складових в алгоритмі кодування JPEG./ Ларин В.В., Комолов Д.С., Ялівець К.В., Гаврилов Д.С. // Системи обробки інформації. 2015. № 9 (134). С. 121 – 123. 7. Гаврилов Д.С. Метод забезпечення безпеки відеоінфор- маційного ресурсу на основі багаторівневої селективної обробки в телекомунікаційних системах./ О.Г. Оксіюк, Д.С. Гаврилов, П.М. Гуржій, Б.О. Демідов// Наука і тех- ніка Повітряних Сил Збройних Сил України. 2017 № 1 С. 46 - 48. 8. Gavrulov D. The analysis of template method of video processing./ Larin V., Krasnikov P., Gavrulov D. // Proceedings of 2015 1st International Conference on Advanced Information and Communication Technologies- 2015 (AICT'2015), Lviv, Ukraine, October 29 – November 1, 2015. P. 87 – 89. 9. Ватолин Д. Методы сжатия дан- ных. Устройство архиваторов, сжатие изображений и видео / Д. Ватолин, А. Рятушняк, М. Смирнов, В. Юкин // Учебно-справочное издание. М.: ДИАЛОГ – МИФИ. 2003. 384с. 10. Гонсалес Р., Цифровая обработка изо- бражений / Гонсалес Р., Вудс Р. // М.: Техносфера, 2005. С.812-850 11. Яне Б. Цифровая обработка изображений. М.: Техносфера. 2007. С.331-356. 12. Сойфера В.А. Ме- тоды компьютерной обработки изображений. – М.: Фи- зматлит, – 2003. – С.192-203. Transliterated bibliography: 1. Barannik V.V. Metod povyshenija informacionnoj bezopasnosti v sistemah videomonitoringa krizisnyh situacij Рис. 1. Вихідне зображення Рис. 2. Зображення, захищене розробленою інформа- ційною технологією Рис. 2. Зображення, захищене розробленою інформа- ій і Рис. 2. Зображення, захищене розробленою інформа- ційною технологією Аналіз рис. 2 вказав на можливість розробленої інформаційної технології виконувати поставлене завдання по оперативній, захищеній передачі да- них з необхідною якістю. 3. Висновки Розроблено інформаційну технологію оперативної та конфіденційної доставки відеоінформаційного ресурсу в системі критичної інфраструктури шля- хом захисту блоків, що містять контурну інформа- цію. Transliterated bibliography: 1. Barannik V.V. Metod povyshenija informacionnoj bezopasnosti v sistemah videomonitoringa krizisnyh situacij 1. Barannik V.V. Metod povyshenija informacionnoj bezopasnosti v sistemah videomonitoringa krizisnyh situacij РИ, 2018, №2 53 9. Vatolin D. Metody szhatija dannyh. Ustrojstvo ar- hivatorov, szhatie izobrazhenij i video. / D. Vatolin, A. Rjatushnjak, M. Smirnov, V. Jukin // Uchebno-spravochnoe izdanie. M.: DIALOG – MIFI, 2003. 384s. 10. Gonsales R., Cifrovaja obrabotka izobrazhenij. /. Gonsales R., Vuds R. // M.: Tehnosfera, 2005. S.812-850 / V.V. Barannik, Ju.N. Rjabuha, O.S. // Monografija. Cherkassy, 2015. 143 s. / V.V. Barannik, Ju.N. Rjabuha, O.S. // Monografija. Cherkassy, 2015. 143 s. 9. Vatolin D. Metody szhatija dannyh. Ustrojstvo ar- hivatorov, szhatie izobrazhenij i video. / D. Vatolin, A. Rjatushnjak, M. Smirnov, V. Jukin // Uchebno-spravochnoe izdanie. M.: DIALOG – MIFI, 2003. 384s. 2. Barannyk V.V. Model' zagroz bezpeky videoinformacijnogo resursu system videokonferenczv'- jazku. / A.V. Vlasov, V.V. Barannik, R.V.Tarnopolov // Naukojemni tehnologii'. 2014. - № 1 (21). S. 55 – 60. 10. Gonsales R., Cifrovaja obrabotka izobrazhenij. /. Gonsales R., Vuds R. // M.: Tehnosfera, 2005. S.812-850 3. Barannik V.V. Obosnovanie znachimyh ugroz bezopas- nosti videoinformacionnogo resursa sistem videoko- nferencsvjazi profil'nyh sistem upravlenija / V.V. Barannik, 11. Jane B. Cifrovaja obrabotka izobrazhenij. – M.: Tehnosfera, 2007. S.331-356. 3. Barannik V.V. Obosnovanie znachimyh ugroz bezopas- nosti videoinformacionnogo resursa sistem videoko- nferencsvjazi profil'nyh sistem upravlenija / V.V. Barannik, A.V. Vlasov, S.A. Sidchenko, A.Je. Bekirov // Informacionno-upravljajushhie sistemy na ZhD transporte. 2014. №3. S. 24 – 31. 12. Sojfera V.A. Metody komp'juternoj obrabotki izobrazhenij. M.: Fizmatlit, 2003. S.192-203. Надійшла до редколегії 28.05.2018 A.V. Vlasov, S.A. Sidchenko, A.Je. Bekirov // Informacionno-upravljajushhie sistemy na ZhD transporte. 2014. №3. S. 24 – 31. Рецензент: д-р техн. наук, проф. Безрук В.М. Бараннік Володимир Вікторович, д-р техн. наук, професор, начальник кафедри бойового застосування та експлуатації АСУ Харківського національного універ- ситету Повітряних Сил ім. І. Кожедуба, e-mail: vvbar.off@gmail.com, orcid.org/0000-0002-2848-4524. Адреса: Україна, 61023, Харків, ул. Сумська, 77/79. 4. Barannyk V.V. Selektyvnyj metod shyfruvannja vydeopotiku v telekomunykacijnyh systemah na osnovi pryhovuvannja bazovogo I-kadru / V.V. Barannyk, D.I. Komolov, Ju.M. Rjabuha // Naukojemni tehnologii'. № 2. 2015. S. 14 - 23. 5. Barannik V.V. The model of avalanche-relating effect in the process of images reconstruction in the combined cryptosemantic systems on the polyadic presentation / V.V. Barannik V.V. Larin, S.A. 3. Висновки Sidchenko // Наукоємні техно- логії. 2010. № 1(5). С. 68 – 70. Гаврилов Дмитро Сергійович, аспірант ХНУРЕ. Нау- кові інтереси: системи, інформаційні технології, коду- вання, криптографічний захист. Адреса: Україна, 49032, Дніпро, вул. Аеродром, 10, тел. 8-066-2290463. 6. Gavrylov D.S. Metod zahystu nyz'kochastotnyh skladovyh v algorytmi koduvannja JPEG./ Laryn V.V., Komo-lov D.S., Jalivec' K.V., Gavrylov D.S. // Systemy obrobky informacii'. 2015. № 9 (134). S. 121 – 123. Сорокун Антон Дмитрович, аспірант Національного авіаційного університету. Наукові інтереси: обробка інформації. Адреса: Україна, 03058, Київ, пр. Космо- навта Комарова 1, e-mail: anton.sorokun@gmail.com Barannik Volodymyr Viktorovich, Dr. Tech. Sciences, professor, head of the military application and operation department, Kharkiv National Air University of the Air Force named after I. Kozheduba, e-mail: vvbar.off@gmail.com, orcid.org/0000-0002-2848-4524. Address: Ukraine, 61023, Kharkiv, Sumska Str., 77/79. 7. Gavrylov D.S. Metod zabezpechennja bezpeky videoinformacijnogo resursu na osnovi bagatorivnevoi' selektyvnoi' obrobky v telekomunikacijnyh systemah./ O.G. Oksijuk, D.S. Gavrylov, P.M. Gurzhij, B.O. Demidov // Nauka i tehnika Povitrjanyh Syl Zbrojnyh Syl Ukrai'ny. № 1. 2017. S. 46 - 48. Havrylov Dmytro Serhiiovych, postgraduate of Kharkov National University of Radio Electronics. Scientific inter- ests: systems, information technologies, coding, crypto- graphic protection. Address: Ukraine, 49032, Dnepr, st. Aerodrom, 10, tel. 8066-2290463. E-mail: havrylov_d@ukr.net. 8. Gavrulov D. The analysis of template method of video processing / Larin V., Krasnikov P., Gavrulov D. // Proceedings of 2015 1st International Conference on Advanced Information and Communication Technologies- 2015 (AICT'2015), Lviv, Ukraine, October 29 – November 1, 2015. P. 87 – 89. Anton D. Sorokun, PhD student of the National Aviation University. Scientific interests: information processing. Ad- dress: Ukraine, 03058, Kiev, Cosmonaut Komarov Ave. 1, e-mail: anton.sorokun@gmail.com 54 РИ, 2018, №2 РИ, 2018, №2 РИ, 2018, №2 54
https://openalex.org/W2052259167
https://europepmc.org/articles/pmc4227693?pdf=render
English
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Serum Uric Acid and Nigral Iron Deposition in Parkinson’s Disease: A Pilot Study
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Introduction Uric acid (UA), the end product of purine metabolism, is a natural antioxidant that may reduce oxidative stress [1]. In particular, higher concentrations of UA may protect against the development or progression of neurodegenerative diseases [1]. Indeed, recent studies have indicated that higher serum uric acid level is associated with lower incidence and better prognosis of Parkinson’s disease (PD) [2,3,4]. Furthermore, UA is reduced in the substantia nigra (SN) of PD patients [5]. In experimental models of PD, the administration of UA was found to suppress oxidative stress and prevent against nigral cell death [6,7]. The aim of this study was to determine whether serum UA relates to brain iron content in patients with PD. We assessed iron levels in the various brain regions by calculating phase shift values from susceptibility weighted imaging, which is proven method to measure brain iron concentration [12]. Correlative analysis between between serum UA and brain iron levels may provide further understanding of the role of these two factors in the pathogenesis of PD. Excessive iron accumulation in the brain is a major contributor of oxidative stress by means of Fenton reaction which produces toxic hydroxyl radicals [8]. Increased iron levels in the SN had been reported consistently by postmortem and in vivo studies in PD [8,9]. Iron-induced oxidative stress has been implicated in the degeneration of dopaminergic neurons [8,9]. Also, toxic iron can promote a-synuclein misfolding and aggregation contributing to the pathogenesis of PD [8,9]. Therefore, antioxidants with iron- chelating ability could be a viable neuroprotective approach for treatment of PD [10]. Abstract The funder had no role in study design, d decision to publish, or preparation of the manuscript. supported by Pusan National University Yangsan Hospital (2014). The funder had no role in study design, data collection and analysis eparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: jhlee.neuro@pusan.ac.kr * Email: jhlee.neuro@pusan.ac.kr UA has been shown to have iron chelating property by forming stable complexes with Fe3+, and diminishing the oxidizing potential of Fe3+ [11]. Consequently, manipulation of UA concentrations could be an effective disease-modifying therapy in PD. At present, however, it is unknown whether UA and brain iron deposition are related in PD patients. Tae-Hyoung Kim, Jae-Hyeok Lee* Pusan National University Yangsan Hospital, Research Institute for Convergence of Biomedical Science and Technology, Yangsan, Kore epartment of Neurology, Pusan National University Yangsan Hospital, Research Institute for Convergence of Biomedical Science and Abstract Background: Uric acid (UA) is an endogenous antioxidant which is known to reduce oxidative stress and also chelate iron ion. Recent studies have provided evidence that UA may play a neuroprotective role in Parkinson’s disease (PD). However, it is unknown whether UA relates to nigral iron deposition, which is a characteristic pathophysiological alteration in PD. The aim of this study was to determine the potential relationship of these two markers in patients with PD. Methods: A total of 30 patients of PD and 25 age- and gender- matched healthy controls underwent 3-Tesla MRI and laboratory tests including serum UA levels. We assessed iron levels by measuring phase shift values using susceptibility- weighted image. Mean phase shift values of the substantia nigra (SN), red nucleus, head of the caudate nucleus, globus pallidus, putamen, thalamus, and frontal white matter were calculated and correlated with serum UA levels. Results: Serum UA levels were significantly decreased in the PD patients than in the controls. Phase shift values in bilateral SN were significantly increased in the PD patients than in the controls. There was no significant correlation between serum UA levels and nigral phase shift values. Conclusions: As previous studies, low serum UA level and increased nigral iron content in the PD was reconfirmed in this study. However, we failed to find the relationship between these two markers. Our data suggest that serum UA may not be important determinant of nigral iron deposition in PD. Citation: Kim T-H, Lee J-H (2014) Serum Uric Acid and Nigral Iron Deposition in Parkinson’s Disease: A Pilot Study. PLoS ONE 9(11): e112512. doi:10.1371/journal. pone.0112512 Editor: Jonghan Kim, Northeastern University, United States of America Editor: Jonghan Kim, Northeastern University, United States of America Editor: Jonghan Kim, Northeastern University, United States of America Received June 25, 2014; Accepted October 6, 2014; Published November 11, 2014 Received June 25, 2014; Accepted October 6, 2014; Published November 11, 2014 Copyright:  2014 Kim, Lee. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: 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: This study was supported by Pusan National University Yangsan Hospital (2014). 2. MRI acquisition and analysis All patients and controls participating study underwent MRI using a 3-Tesla system (Siemens, Verio) equipped with a 12- channel head coil. The SWI data were acquired using flow- compensated 3D GRE sequences with an integrated Parallel Acquisition Techniques (iPAT) factor of 2. SWI images were taken parallel to the anterior–posterior commissural line with the following parameters: TR = 28 ms, TE = 20 ms, flip angle = 15u, matrix size = 3206260, field of view = 2206178 mm2, slice thickness = 2 mm. Both magnitude and phase images were acquired, but only phase data were used for analysis. 3. Statistical analysis The demographic data between PD patients and control subjects were compared using Mann-Whitney’s U test or Fisher’s exact test. Analysis of covariance was used to compare phase shift values and serum UA levels between two groups with correction for age and gender. Correlation analysis using Pearson’s correla- tion coefficient was performed to investigate the relationship between phase shift values, serum UA levels, and clinical data. Statistical significance was determined as p,0.05. All statistical analyses were performed using SPSS (Chicago, Illinois) version 18.0. 1. Subjects Severity of disease and motor symptoms were assessed by the Hoehn and Yahr (H & Y) stage and the motor section of the Unified Parkinson’s Disease Rating Scale (UPDRS III) during the practically defined ‘OFF’ state. Serum UA levels were measured by an enzymatic colorimetric test from venous blood samples. The study was carried out in accordance with the Declaration of Helsinki and the protocol for this retrospective study was approved by Institutional Review Board, Pusan National University Yangsan Hospital with waiver of consent. 1. Subjects A total 30 patients with PD and 25 age- and gender-matched healthy controls were included in this retrospective study. Data were collected from electronic medical records. All patients were diagnosed according to the UK Brain Bank criteria. Exclusion PLOS ONE | www.plosone.org November 2014 | Volume 9 | Issue 11 | e112512 1 Serum Uric Acid and Nigral Iron Table 1. Clinical characteristics and MRI data. Parkinson’s disease Controls P value N (males/females) 30 (19/11) 25 (12/13) 0.286a Age (years) 57.666.8 56.266.5 0.289b Disease duration (months) 19.8612.7 UPDRS-III motor score 24.568.4 Hoehn-Yahr stage (I/II) 9/21 BMI (kg/m2) 24.063.0 24.862.6 0.237b Serum creatinine (mg/dl) 0.8860.19 0.8760.19 0.872b Serum uric acid (mg/dl) 4.761.4 5.761.5 0.001c Phase value (radian) Frontal white matter 20.00260.002 20.00260.002 0.657c Caudate nucleus 0.03360.013 0.03160.013 0.430c Globus pallidus 0.03660.014 0.03560.015 0.725c Putamen 0.01860.012 0.01460.008 0.260c Thalamus 0.00160.004 0.00060.004 0.267c Substantia nigra 0.05060.023 0.03560.020 0.001c Red nucleus 0.06160.023 0.05260.026 0.226c ap-value is calculated from Fisher’s exact test. bp-value is calculated from Mann Whitney’s U-test. cp-value is calculated from ANCOVA with adjustment for age and gender. doi:10.1371/journal.pone.0112512.t001 calculated by averaging four measurements. Phase shift value in this study is for the left hand system [12], which is positively correlated with iron levels. ROIs drawing and calculation of phase shift values were performed using SPIN software (www. mrinnovations.com, Magnetic Resonance Innovations, Inc., De- troit, MI, USA). To verify reproducibility of the findings, phase shift values were re-measured by the same rater on the same images of 10 patients and 10 control subjects. Intraclass correlation coefficients (ICC) showed highly consistent results between two measurements (Table S1). criteria for both PD patients and healthy controls were as follows: vegetarians, taking thiazide diuretics, suffering from renal disease, gout, acute medical illness, cancer, other neurological disorders, and subjects with microvascular lesions found on the brain MRI. Severity of disease and motor symptoms were assessed by the Hoehn and Yahr (H & Y) stage and the motor section of the Unified Parkinson’s Disease Rating Scale (UPDRS III) during the practically defined ‘OFF’ state. Serum UA levels were measured by an enzymatic colorimetric test from venous blood samples. The study was carried out in accordance with the Declaration of Helsinki and the protocol for this retrospective study was approved by Institutional Review Board, Pusan National University Yangsan Hospital with waiver of consent. Results Clinical data and laboratory findings of PD patients and control subjects are summarized in Table 1. There were no statistically significant differences in age, gender, BMI and serum creatinine (Cr) levels between PD and controls. The serum UA levels were significantly lower in the PD group than in the control group (4.761.4 mg/dl vs. 5.761.5 mg/dl, p = 0.001) after adjusting for age and gender. This difference was also statistically significant when comparing each gender separately (male group, 5.361.4 mg/dl vs. 6.761.3 mg/dl, p = 0.018; female group, 3.861.0 mg/dl vs. 4.861.2 mg/dl, p = 0.011). In patients with Regions of interest (ROIs) were placed on the filtered phase images and manually drawn according to the anatomical structures, bilateral substiantia nigra, red nucleus, globus pallidus, putamen, head of caudate nucleus, thalamus, and frontal white matter (Figure S1). Polygon ROIs adjusted to the shape and size of structure were used, but the outermost pixels were avoided to minimize partial volume effect. For frontal white matter, circular ROIs were drawn. Data on each structure were obtained from two consecutive slices, except for SN, where the most well recognized slice was used. The final shift value for each structure was PLOS ONE | www.plosone.org November 2014 | Volume 9 | Issue 11 | e112512 2 Serum Uric Acid and Nigral Iron Figure 1. The correlation between the phase shift values in seven brain regions of healthy controls and postmortem iron concentrations, as reported by Hallgren and Sourander. CA = head of caudate nucleus; FWM = frontal white matter; GP = globus pallidus; PU = putamen; RN = red nucleus; SN = substantia nigra; TH = thalamus. doi:10.1371/journal.pone.0112512.g001 Figure 1. The correlation between the phase shift values in seven brain regions of healthy controls and postmortem iron concentrations, as reported by Hallgren and Sourander. CA = head of caudate nucleus; FWM = frontal white matter; GP = globus pallidus; PU = putamen; RN = red nucleus; SN = substantia nigra; TH = thalamus. doi:10.1371/journal.pone.0112512.g001 Figure 1. The correlation between the phase shift values in seven brain regions of healthy controls and postmortem iron concentrations, as reported by Hallgren and Sourander. CA = head of caudate nucleus; FWM = frontal white matter; GP = globus pallidus; PU = putamen; RN = red nucleus; SN = substantia nigra; TH = thalamus. doi:10.1371/journal.pone.0112512.g001 Figure 2. Serum Uric Acid and Nigral Iron Due to its potent antioxidant properties, UA may have a neuroprotective effect by scavenging free radicals [1]. In addition, UA has metal chelating properties [1,11]. The low UA concen- tration probably weakens the iron-chelating capacity and may expose the dopaminergic neurons to iron toxicity [6,8]. In other words, UA may protect nigral cells through an UA-iron link. Based on these observations, we proposed UA as a possible biomarker of the iron dysregulation in PD. In the present study, however, we found no significant correlation between serum UA levels and phase shift values to quantify brain iron content. Our data suggest that serum UA may not be important determinant of nigral iron deposition in PD. Further studies with larger subjects are needed to elucidate the causal role of these two factors in the pathogenesis of PD. PD, there was no significant association between serum UA and clinical parameters including UPDRS III score and disease duration. Spearman’s correlation analysis was applied to compare the phase shift values in the healthy controls and postmortem brain iron concentrations, as previously assessed by biochemical methods [13]. There was significant correlation between phase values and previously published iron concentrations (r = 0.901, p = 0.006), supporting the use of phase values as a viable method for estimating brain iron content (Figure 1). Phase shift values of bilateral SN were significantly increased in the PD patients than in the controls (0.05060.023 vs. 0.03560.020, p = 0.001) after adjusting for age and gender. In the other brain structures, there was no significant difference of phase shift value between PD patients and control subjects. In the PD patient group, phase shift values in all regions of interest had no significant correlation with clinical parameters including UPDRS III score and disease duration. There are several limitations in our study. First, sample size was relatively small to evaluate correlation between variables. Second, some of our results are different from several previous studies. Previous studies showed that iron content of SN in the PD patients was positively correlated with clinical disease severity such as UPDRS III motor score or disease duration [14,15]. However, we did not found such correlation even when contralateral SN was analyzed. Third, cerebrospinal fluid (CSF) UA levels may better reflect the microenvironment of nigral neurons, although serum and CSF UA levels were highly correlated in current study [21,22]. Supporting Information Figure S1 Illustration of phase images and the ROIs of substantia nigra, red nucleus, caudate nucleus, puta- men, globus pallidus, thalamus, and frontal white matter. (TIF) So far, numerous studies had investigated possible relationship between brain iron deposition and serum biomarkers affecting iron homeostasis [15,18]. Ceruloplasmin, a copper containing protein with ferroxidase activity, plays a significant role in iron homeo- stasis [19]. In a previous study, Jin L. et al. demonstrated that PD patients with reduced serum ceruloplasmin levels exhibited elevated nigral iron deposition as compared to those with normal serum ceruloplasmin levels [15]. Recent studies also have suggested higher serum cholesterol may be related to a lower occurrence and slower progression of PD [20]. One study found that higher serum cholesterol levels were associated with lower iron content in SN estimated from mean R2* values [18]. Table S1 Intraclass correlation coefficients in patients with Parkinson’s disease and controls. (XLSX) Discussion In this study, serum UA levels were significantly lower in the PD patients than control group. Also, phase shift values of SN which reflect iron content of brain tissue were significantly increased in patients with PD. These results are consistent with previous studies [3,14,15], suggesting both UA and iron may play an important role in the pathogenesis of PD. However, we failed to find the relationship between these two markers. There is an abundance of clinical evidence that suggests that low UA levels are associated with the development and progression of a variety of neurological diseases, such as multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease [1]. Also, associations between serum UA and brain MRI measures have been reported. In patients with multiple sclerosis, lower serum uric acid levels correlate with increased activity of disease and blood- brain barrier disruption on contrast-enhanced MRI [16,17]. These observations raised the possibility that UA may play a protective role against neuroinflammation. To our knowledge, the present study is the first attempt to assess the potential relationship of serum UA and the MRI marker for PD. In conclusion, as previous studies, low serum UA level and increased nigral iron content in the PD was reconfirmed in this study. The lack of correlation between two factors suggests that serum UA levels may not be an indirect biomarker of the nigral iron alterations in PD. Further prospective studies with larger sample sizes should be conducted to confirm such a relationship. Serum Uric Acid and Nigral Iron Moreover, serum UA levels are variable and were measured just once at the time of the study. Finally, we used manually-drawn ROIs in the selected slices, which may not reflect the actual total iron deposition in each structure. Other methodological issues such as sequence techniques, image resolution, and slice thickness may also contribute to inaccuracies in MRI-measured brain iron levels [23]. Iron measurement with phase value has some limitations. Phase values are markedly influenced by the difference in the magnetic susceptibility of the surrounding tissue besides the tissue iron content [24,25]. Phase values can be compromised by its nonlocal and orientation dependent properties [26]. For these reasons, our data are insufficient to explain the link between iron and uric acid in the brain. In patients with PD, there was no significant correlation between serum UA level and phase shift value of SN after adjustment for age, gender, UPDRS III score, and disease duration (r = 20.242, p = 0.233) (Figure 2). Furthermore, no significant correlation was found between serum UA levels and phase shift values of SN contralateral to the clinically more affected side (r = 20.272, p = 0.179). No significant correlation was found between two variables in control group after adjustment for age and gender (r = 20.159, p = 0.468). Results Scatter-plots of phase shift values of substantia nigra (A: bilateral SN, B: SN contralateral to the clinically more affected side) and serum uric acid levels in Parkinson’s disease subjects. The r and p values marked in the figure represent the results of Pearson’s partial correlation analysis adjusting for age, gender, UPDRS III score, and disease duration. doi:10.1371/journal.pone.0112512.g002 Figure 2. Scatter-plots of phase shift values of substantia nigra (A: bilateral SN, B: SN contralateral to the clinically more affected side) and serum uric acid levels in Parkinson’s disease subjects. The r and p values marked in the figure represent the results of Pearson’s partial correlation analysis adjusting for age, gender, UPDRS III score, and disease duration. doi:10.1371/journal.pone.0112512.g002 November 2014 | Volume 9 | Issue 11 | e112512 PLOS ONE | www.plosone.org 3 Serum Uric Acid and Nigral Iron Serum Uric Acid and Nigral Iron References 1. Kutzing MK, Firestein BL (2008) Altered uric acid levels and disease states. J Pharmacol Exp Ther 324: 1–7. 15. Jin L, Wang J, Zhao L, Jin H, Fei G, et al. (2011) Decreased serum ceruloplasmin levels characteristically aggravate nigral iron deposition in Parkinson’s disease. Brain 134: 50–58. p 2. Schlesinger I, Schlesinger N (2008) Uric acid in Parkinson’s disease. Mov Disord 23: 1653–1657. 16. Toncev G, Milicic B, Toncev S, Samardzic G (2002) Serum uric acid levels in multiple sclerosis patients correlate with activity of disease and blood-brain barrier dysfunction. Eur J Neurol 9: 221–226. 3. Shen L, Ji HF (2013) Low uric acid levels in patients with Parkinson’s disease: evidence from meta-analysis. BMJ Open 3: e003620. doi:10.1136/bmjopen- 2013-003620. y 17. Zoccolella S, Tortorella C, Iaffaldano P, Direnzo V, D’Onghia M, et al. (2012) Low serum urate levels are associated to female gender in multiple sclerosis patients. PLoS One 7: e40608. doi:10.1371/journal.pone.0040608. 4. Schwarzschild MA, Schwid SR, Marek K, Watts A, Lang AE, et al. (2008) Serum urate as a predictor of clinical and radiographic progression in Parkinson’s disease. Arch Neurol 65: 716–723. 18. Du G, Lewis MM, Shaffer ML, Chen H, Yang QX, et al. (2012) Serum cholesterol and nigrostriatal R2* values in Parkinson’s disease. PLoS One 7: e35397. doi:10.1371/journal.pone.0035397. 5. Church WH, Ward VL (1994) Uric acid is reduced in the substantia nigra in Parkinson’s disease: effect on dopamine oxidation. Brain Res Bull 33: 419–425. 6. Duan W, Ladenheim B, Cutler RG, Kruman II, Cadet JL, et al. (2002) Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson’s disease. J Neurochem 80: 101–110. 19. Crichton RR, Dexter DT, Ward RJ (2011) Brain iron metabolism and its perturbation in neurological diseases. J Neural Transm 118: 301–314. 20. Huang X, Auinger P, Eberly S, Oakes D, Schwarzschild M, et al. (2011) Parkinson Study Group DATATOP Investigators. Serum cholesterol and the progression of Parkinson’s disease: results from DATATOP. PLoS One 6: e22854. doi:10.1371/journal.pone.0022854. 7. Cipriani S, Desjardins CA, Burdett TC, Xu Y, Xu K, et al. (2012) Urate and its transgenic depletion modulate neuronal vulnerability in a cellular model of Parkinson’s disease. PLoS One 7: e37331. doi:10.1371/journal.pone.0037331. j p 8. Sian-Hu¨lsmann J, Mandel S, Youdim MB, Riederer P (2011) The relevance of iron in the pathogenesis of Parkinson’s disease. J Neurochem 118: 939–957. 21. Author Contributions Conceived and designed the experiments: THK JHL. Performed the experiments: THK JHL. Analyzed the data: THK JHL. Contributed reagents/materials/analysis tools: THK JHL. Contributed to the writing of the manuscript: THK JHL. PLOS ONE | www.plosone.org November 2014 | Volume 9 | Issue 11 | e112512 4 Serum Uric Acid and Nigral Iron Serum Uric Acid and Nigral Iron References Constantinescu R, Andreasson U, Holmberg B, Zetterberg H (2013) Serum and cerebrospinal fluid urate levels in synucleinopathies versus tauopathies. Acta Neurol Scand 127: e8–12. doi:10.1111/ane.12012. 9. Berg D, Hochstrasser H (2006) Iron metabolism in Parkinsonian syndromes. Mov Disord 21: 1299–1310. 22. Bowman GL, Shannon J, Frei B, Kaye JA, Quinn JF (2010) Uric acid as a CNS antioxidant. J Alzheimers Dis 19: 1331–1336. doi:10.3233/JAD-2010-1330. 10. Mounsey RB, Teismann P (2012) Chelators in the treatment of iron accumulation in Parkinson’s disease. Int J Cell Biol 2012: 983245. doi:10.1155/2012/983245. 23. Gro¨ger A, Berg D (2012) Does structural neuroimaging reveal a disturbance of iron metabolism in Parkinson’s disease? Implications from MRI and TCS studies. J Neural Transm 119: 1523–1528. 11. Davies KJ, Sevanian A, Muakkassah-Kelly SF, Hochstein P (1986) Uric acid- iron ion complexes. A new aspect of the antioxidant functions of uric acid. Biochem J 235: 747–754. 24. Pfefferbaum A, Adalsteinsson E, Rohlfing T, Sullivan EV (2009) MRI estimates of brain iron concentration in normal aging: comparison of field-dependent (FDRI) and phase (SWI) methods. Neuroimage 47: 493–500. 12. Haacke EM, Ayaz M, Khan A, Manova ES, Krishnamurthy B, et al. (2007) Establishing a baseline phase behavior in magnetic resonance imaging to determine normal vs. abnormal iron content in the brain. J Magn Reson Imaging 26: 256–264. 25. Yan SQ, Sun JZ, Yan YQ, Wang H, Lou M (2012) Evaluation of brain iron content based on magnetic resonance imaging (MRI): comparison among phase value, R2* and magnitude signal intensity. PLoS One 7: e31748. g g 13. Hallgren B, Sourander P (1958) The effect of age on the non-haemin iron in the human brain. J Neurochem 3: 41–51. 26. Li W, Wu B, Liu C (2011) Quantitative susceptibility mapping of human brain reflects spatial variation in tissue composition. Neuroimage 55: 1645–1656. 14. Zhang J, Zhang Y, Wang J, Cai P, Luo C, et al. (2010) Characterizing iron deposition in Parkinson’s disease using susceptibility-weighted imaging: an in vivo MR study. Brain Res 1330: 124–130. November 2014 | Volume 9 | Issue 11 | e112512 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 5
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Die „neue“ Borderline-Persönlichkeitsstörung: Dimensionale Klassifikation im DSM-5 und ICD-11
Psychotherapie-Forum
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übersichtsarbeit Psychotherapie Forum (2020) 24:89–99 https://doi.org/10.1007/s00729-020-00151-4 Die „neue“ Borderline-Persönlichkeitsstörung: Dimensionale Klassifikation im DSM-5 und ICD-11 Horst Mitmansgruber Online publiziert: 14. Oktober 2020 © Der/die Autor(en) 2020 Zusammenfassung Die jahrzehntelange und zunehmende Unzufriedenheit mit der bisherigen kategorialen Klassifikation der Persönlichkeitsstörungen (PS) im amerikanischen DSM-IV und in der ICD-10 der Weltgesundheitsorganisation hat u. a. mit fehlender empirischer Unterstützung vieler Kategorien, der sehr hohen Komorbidität der PS untereinander oder der großen Heterogenität von Symptomen innerhalb einer Diagnose zu tun. Sie hat in den letzten Revisionen der beiden Diagnosesysteme einen radikalen Wandel hin zu einem dimensionalen Klassifikationssystem unterstützt, das um vieles stärker in der empirischen psychologischen Forschung abgesichert ist. Im DSM-5 ist die Revolution ausgeblieben, weil das dimensionale Modell im Anhang verblieben ist, während die alte DSM-IV-Klassifikation unverändert übernommen wurde. Allerdings hat dieses „Alternative Modell der Persönlichkeitsstörungen“ (AMPD) seit seiner Publikation erhebliches Forschungsinteresse erfahren. In der ICD-11 ist eine dimensionale Einschätzung der Persönlichkeit auf den 5 Domänen „Negative Affectivity“, „Detachment“, „Dissociality“, „Disinhibition“ und „Anankastia“ als radikale Alternative zu den 10 bisherigen Kategorien bereits abgesegnet und wird ab 2022 weltweit die Diagnosestellung verändern. Während hier zunächst alle bisherigen Kategorien eliminiert wurden, erreichten kritische Stimmen am Ende, dass die Borderline-Störung als einziger Qualifier in der ICD-11 erhalten bleibt. Die beiden Systeme werden in H. Mitmansgruber () Universitätsklinik für Medizinische Psychologie, Medizinische Universität Innsbruck, Speckbacherstraße 23, 6020 Innsbruck, Österreich Arbeitsgemeinschaft für Verhaltensmodifikation – Österreich (AVM), Paris-Lodron-Straße 32, 5020 Salzburg, Österreich horst.mitmansgruber@i-med.ac.at K ihren zentralen Annahmen und im praktischen Vorgehen beleuchtet. Die Diskussionen und die bisherige empirische Befundlage zu den Dimensionen und der klinischen Nützlichkeit für Praktiker*innen werden zusammengefasst. Schlüsselwörter Borderline-Persönlichkeitsstörung · Dimensionale Klassifikation der Persönlichkeitsstörungen · ICD-11 · DSM-5 · Alternatives Modell der Persönlichkeitsstörungen (AMPD) The “new” borderline personality disorder: Dimensional classification in DSM-5 and ICD-11 Summary Decades of increasing discontent with current categorical classification systems of personality disorders (PD) in the American DSM-IV and the ICD10 of the World Health Organization are based on missing empirical support of many of the categories, high comorbidity of PDs and large heterogeneity of symptoms within a diagnosis. This discontent has paved the way for a radical change in the last revisions of these diagnostic systems to replace a categorical with a dimensional model of classification, which is strongly supported by empirical psychological research. For the DSM-5 the revolution was cancelled, as the dimensional model was placed in Section III “Emerging models” whereas the old DSM-IV categorical system was adopted without any changes. However, this “Alternative Model of Personality Disorders” (AMPD) has received much attention in research since its publication. In the ICD-11, a dimensional assessment of personality on five domains (“Negative Affectivity”, “Detachment”, “Dissociality”, “Disinhibition” and “Anankastia”) as a radical alternative to currently existing 10 categories is officially accepted and will change diagnosis of personality disorders world- Die „neue“ Borderline-Persönlichkeitsstörung: Dimensionale Klassifikation im DSM-5 und ICD-11 89 übersichtsarbeit wide from 2022. Whereas all current categories have been eliminated in the proposal, at first, critics were able to reclaim a “borderline pattern” as the one and only qualifier for a specific category in the final version of the ICD-11. Both systems are illustrated in their central assumptions and practical procedures. Discussions on dimensions, their current empirical basis and clinical utility are summarized. Keywords Borderline personality disorder · Dimensional classification of personality disorders · ICD-11 · DSM-5 · Alternative Model of Personality Disorders (AMPD) Die Borderline-Persönlichkeitsstörung im DSMIV und ICD-10: Probleme mit dem kategorialen Diagnosesystem Die kategoriale Diagnosestellung der Persönlichkeitsstörungen (PS) in den aktuell gültigen Diagnosesystemen ist uns über die vergangenen bald drei Jahrzehnte schon sehr vertraut geworden. Die aktuell noch gültige ICD-10 der Weltgesundheitsorganisation (World Health Organization 1992) und das DSM-IV bzw. DSM-51 der amerikanischen Psychiatrie (American Psychiatric Association 1994, 2013) beschreiben neun bzw. zehn spezifische PS-Diagnosen. Sie werden diagnostiziert, wenn allgemeine Kriterien einer PS (merklich abweichendes, zeitlich überdauerndes Muster von Kognitionen, Affekten, zwischenmenschlichem Verhalten oder Impulsivität, das unflexibel und tiefgreifend ist und ihren Beginn in Adoleszenz oder jungem Erwachsenenalter hat) und eine ausreichende Anzahl aus einem Satz von acht bis zehn Kriterien für eine spezifische PS erfüllt sind. Für eine Borderline-Persönlichkeitsstörung (BPS) müssen fünf von neun bzw. zehn Kriterien erfüllt sein: verzweifeltes Bemühen, tatsächliches oder vermutetes Verlassenwerden zu verhindern, emotionale Instabilität, intensive und instabile zwischenmenschliche Beziehungen mit Wechsel zwischen Idealisierung und Entwertung, Identitätsstörung, Suiziddrohungen, Selbstverletzungen, Schwierigkeiten Wut zu kontrollieren, Impulsivität in potentiell selbstschädigenden Bereichen, chronische Gefühle der Leere (Falkai und Wittchen et al. 2015; WHO 1992). Hat man mit diesen Diagnosen tatsächlich natürlich vorkommende Kategorien beschrieben und damit die Natur an ihren Gelenken getrennt („carving nature by its joints“), wenn gleichzeitig mehr als die Hälfte der Personen, die die allgemeinen Kriterien einer PS erfüllen, in keine dieser Kategorien passen (Zimmerman et al. 2005)? Wie unterschiedlich Personen mit der gleichen PSDiagnose sein können, ihre Heterogenität, wurde viel- 1 Die Kriterien der Persönlichkeitsstörungen im DSM-IV (1994) wurden im DSM-5 (2013) unverändert weitergeführt. 90 fach kritisch angemerkt (Ofrat et al. 2018; Widiger und Trull 2007). Für die BPS ergeben sich 256 verschiedene Symptomkonstellationen im DSM und bei fünf von neun Kriterien können zwei Patient*innen u. U. nur ein einziges Symptom überlappend präsentieren. Für die zwanghafte PS mit vier von acht Kriterien ist es sogar möglich, überhaupt keine Überlappung mit einer anderen Person zu haben, und dennoch die gleiche Diagnose zu bekommen (Samuel und Griffin 2015). Das ist nicht nur rechnerisch möglich, sondern zeigt sich auch in der klinischen Realität. In einer Stichprobe von 252 Patient*innen, die die Kriterien einer BPS erfüllten, waren 136 verschiedene Kombinationen einer BPS präsent (Johansen et al. 2004). Dabei ist im kategorialen System die Grenze zwischen normaler und pathologischer Persönlichkeit eine willkürliche (Ofrat et al. 2018). Im „revolutionären“ DSM-III (APA 1980), das diese Art der Diagnosestellung erstmals umsetzte, wurde bei den meisten Persönlichkeitsstörungen keinerlei Begründung für die Schwelle zwischen normal und krank angegeben (Demazeux 2015). Außerdem wurde von der letztlich nicht haltbaren Position ausgegangen, dass alle Kriterien in gleichem Maße wichtig für die Diagnosestellung seien (Balsis et al. 2011). Bei der BPS begründete man im DSM-III die fünf von damals acht Kriterien zumindest als eine versuchte Maximierung der Übereinstimmung unter Kliniker*innen. Diese Form der Entscheidungsfindung wurde humoristisch als BOGSAT-Methode bezeichnet („bunch of guys sitting around a table“; H. Pincus, zit. n. Demazeux 2015, S. 9). Damit ist die zentrale (willkürliche) Schwelle, ob jemand an einer krankheitswertigen BPS leidet, die von 4 (gesund) auf 5 (krank) erfüllte Kriterien (Samuel und Griffin 2015). Manche Autor*innen argumentieren, dass die Problematik zumindest verringert werden könnte, wenn die Anzahl der zu erfüllenden Kriterien auf mind. 7 erhöht würde (Paris 2020). Dieses strengere Vorgehen würde eine homogenere Gruppe von Personen erlauben, die eine BPS-Diagnose erhalten. Ebenfalls sehr problematisch ist das Ergebnis von Längsschnittstudien, dass diese Kriterien in recht unterschiedlichem Maß zeitlich stabil sind und damit ein postuliertes zentrales Charakteristikum einer Persönlichkeitsstörung deutlich in Frage gestellt ist. Grilo et al. (2004, 2014) haben dargestellt, dass fast 30 % einer nur über 24 Monate verfolgten Stichprobe von BPS-Patient*innen in diesem Zeitraum über 12 Monate und mehr nur 2 oder weniger der Kriterien erfüllten. Sie waren also über ein Jahr in diesen beiden Jahren sehr weit von einer BPS-Diagnose entfernt! So sind etwa Selbstverletzungen wesentlich weniger zeitlich stabil als die emotionale Instabilität. Umgekehrt würden jahrelange Selbstverletzungen, emotionale Instabilität und schwere Dissoziationen nicht für eine BPS-Diagnose ausreichen, auch wenn sie in andauernder schwerer Beeinträchtigung resultieren (Cooper und Balsis 2009; Samuel und Griffin 2015). Die „neue“ Borderline-Persönlichkeitsstörung: Dimensionale Klassifikation im DSM-5 und ICD-11 K übersichtsarbeit Tyrer et al. (2019) fassen zusammen, dass die allgemeinen Kriterien einer Persönlichkeitsstörung (als Voraussetzung für die Diagnose einer spezifischen Kategorie) schlicht ignoriert worden seien (vgl. auch First et al. 2014). Wenn Kliniker*innen Diagnosen stellten, dann reduzierte sich das verwendete Spektrum auf die drei Kategorien BPS, Antisoziale PS und kombinierte PS/Nicht Näher Bezeichnete PS. Und sie betonen, dass in Studien nur bei 8 % der psychiatrischen Patient*innen eine PS-Diagnose vergeben wurde, obwohl es sich empirisch gezeigt hatte, dass die realen Zahlen zwischen 40 % und 90 % liegen. Diese und weitere Kritikpunkte an der kategorialen Diagnostik von Persönlichkeitsstörungen haben die Aufmerksamkeit zunehmend auf dimensionale Modelle als angemessenere Alternative gerichtet (Hopwood et al. 2018; Smith et al. 2020). Hier werden Merkmale nicht dichotom als „vorhanden“ oder „nicht vorhanden“ betrachtet. Stattdessen werden kontinuierliche Dimensionen (Traits) von leicht bis extrem identifiziert, die bei Patient*innen unterschiedlich stark ausgeprägt sind (Krueger et al. 2014). Dieser Zugang entspricht einerseits den Ergebnissen der Neurobiologie, die mit dimensionalen Modellen deutlich besser vereinbar sind als mit kategorialen (Caspi et al. 2014). Er entspricht auch den dominierenden Modellen in der Normalpsychologie der Persönlichkeit. Eine umfangreiche Integration der Persönlichkeitsstörungen mit dem führenden, empirisch sehr gut abgesicherten „Big Five“-Modell der Normalpersönlichkeit (mit den Traits „Neurotizismus“, „Extraversion“, „Offenheit“, „Verträglichkeit“ und „Gewissenhaftigkeit“) wurde erstmals bereits 1994 publiziert (Costa und Widiger 1994; Widiger und Costa 2013; Miller und Widiger 2020). Dabei wurde ein Trait definiert als eine „Variable, die einer relativ stabilen Disposition für bestimmte Verhaltensmuster zugrunde liegt“ (Markon und Jonas 2015, S. 64, dt. Übers. HM). Das dimensionale Alternative Model of Personality Disorders (AMPD) im DSM-5: die Fast-Revolution In der Vorbereitung des DSM-5 arbeitete die Personality & Personality Disorder Work Group (PPDWG) unter der Leitung von Andrew Skodol klar auf die Ablöse der kategorialen Diagnostik durch ein dimensionales Modell der PS hin. Die erste konkrete Ausgestaltung (Skodol et al. 2011) sah die Streichung von fünf empirisch am schlechtesten abgesicherten Kategorien vor (paranoide, schizoide, histrionische, narzisstische und dependente PS) und beschrieb die verbleibenden PS als Prototypen auf einem Raster von Dimensionen (Krueger et al. 2014). Die Persönlichkeit von Patient*innen sollte dimensional auf 37 Facetten (z. B. emotionale Labilität, Trennungsangst, Rückzug, Feindseligkeit etc.; Skodol et al. 2011) eingeschätzt werden und diese Einschätzung sollte unabhängig von den bisherigen „Schubladen“ (antisozial, Borderline etc.) ein differen- K ziertes Profil der Person liefern, mit dem die Behandlung geplant werden kann. Die Lösung war damit ein „Hybrid-Modell“ aus Prototypen auf Persönlichkeitsdimensionen, das dimensionale und kategoriale Diagnosestellung verbinden sollte und so alle, d. h. Kritiker und Vertreter des alten Modells, zufriedenstellen und den Übergang erleichtern sollte (Krueger und Markon 2014). Das Modell löste sehr viele kritische bis negative Reaktionen aus. Sogar Widiger (2011) als langjähriger ausgewiesener Vertreter eines dimensionalen Zugangs kritisierte die PPDWG, nicht das abgesicherte Big-Five-Modell als Grundlage für das DSM-5 heranzuziehen (Huprich 2015). Folge dieser heftigen Kritik war u. a. die Wiederaufnahme bzw. Beibehaltung der narzisstischen PS (ein weiteres Beispiel der BOGSATMethode?) und neuerliche Studien, die in einem System von 5 Trait-Domänen mit insgesamt 25 Facetten mündeten. Erfassungsinstrumente für diese Facetten existieren mittlerweile sowohl als Selbstbeurteilung, Fremdbeurteilung durch Angehörige sowie als klinische Einschätzung (PID-5; Personality Inventory für DSM-5; First et al. 2018, Krueger et al. 2012; Somma et al. 2019). Die PPDWG folgte nun weitgehend dem Big Five-Modell. Nicht vertreten ist die „Offenheit“, während stattdessen eine Domäne „Psychotizismus“ eingeführt wurde, um u. a. die Symptome einer Schizotypischen Persönlichkeitsstörung abbilden zu können (die im ICD-10 als Schizotypie keine PS ist; Abb. 1). Psychotizismus wurde allerdings immer wieder als mögliche Extremvariante des Traits „Offenheit“ diskutiert (vgl. Chmielewski et al. 2014). Deutlich prominenter als im DSM-IV ist im DSM-5 die Beurteilung des Kriteriums A, die Einschätzung des Schweregrads der Beeinträchtigung der Persönlichkeit (Morey und Bender 2014). Störungen der Selbst-Funktionen (Identität und Selbststeuerung) und der zwischenmenschlichen Funktionen (Empathie und Nähe) werden differenziert und auf einem Kontinuum eingeschätzt (Tab. 1; vgl. Widiger et al. 2019 und Kommentare für eine interessante Diskussion). Erstmals wird auch die Operationalisierung der „gesunden“ Persönlichkeit inkludiert. In einer „Skala zur Erfassung des Funktionsniveaus der Persönlichkeit“ (SEFP) schätzen Kliniker*innen den Schweregrad der Beeinträchtigung von „0 = keine oder geringfügige Beeinträchtigung“ bis „4 = extreme Beeinträchtigung“ für die vier genannten Bereiche ein. Für die Diagnose einer Persönlichkeitsstörung müssen mindestens zwei von vier Bereichen als mindestens „2 = mittelgradig“ beurteilt werden. Zur Illustration ist in Tab. 1 die „mittelgradige Beeinträchtigung“ für die vier Bereiche dargestellt (Falkai et al. 2015). Ist die Diagnose einer Persönlichkeitsstörung vom Schweregrad der Beeinträchtigung gerechtfertigt, folgt in Kriterium B die inhaltliche Beschreibung der vorherrschenden Traits. Beschrieben sind die Domänen „Negative Affektivität“, „Verschlossenheit“, „Antagonismus“, „Enthemmtheit“ und „Psychotizismus“, die Die „neue“ Borderline-Persönlichkeitsstörung: Dimensionale Klassifikation im DSM-5 und ICD-11 91 übersichtsarbeit Abb. 1 Dimensionale Einschätzung einer Persönlichkeitsstörung im DSM-5 (APA 2013; Falkai und Wittchen et al. 2015) zunächst über den Schweregrad und dann über Trait-Domänen (und Facetten). Die Einschätzung des Schweregrads erfolgt in den Bereichen „Identität“, „Selbststeuerung“, „Em- pathie“ und „Nähe“ (mind. 2 Bereiche mittelgradig). Die ersten vier Trait-Domänen entsprechen dem Big Five-Modell. Die Klassifikation von 6 Prototypen (eine davon die BPS) ist hier nicht dargestellt Tab. 1 Beispiel der „mittelgradigen Beeinträchtigung“ in der Skala zur Erfassung des Funktionsniveaus der Persönlichkeit (SEFP) im AMPD des DSM-5. (Text verkürzt; Falkai et al. 2015, S. 1066) SEFP „2 = mittelgradige Beeinträchtigung“ (in zwei Bereichen notwendig für die Diagnose einer PS) Identität Übermäßige Abhängigkeit von anderen bei der Definition der eigenen Identität; beeinträchtigte Wahrnehmung von Grenzen Vulnerabler Selbstwert, Sorge um Bewertung durch andere, Wunsch nach Anerkennung, Gefühl von Unzulänglichkeit, kompensatorisch überhöhte oder verringerte Selbsteinschätzung Selbststeuerung Ziele meistens nicht selbstbestimmt, sondern Mittel, Bestätigung von anderen zu erhalten, daher ev. mangelnde Stabilität von Zielen Eigene Maßstäbe unangemessen hoch (z. B. gefallen wollen) oder niedrig (z. B. nicht mit vorherrschenden Werten übereinstimmend) Eingeschränkte Fähigkeit, eigenes Erleben zu reflektieren Empathie Übermäßig empfindsam hinsichtlich des Erlebens anderer, aber nur im Hinblick auf dessen Relevanz für die eigene Person Übermäßig selbstbezogen, deutlich eingeschränkte Fähigkeit, das Erleben anderer zu würdigen, zu verstehen, alternative Sichtweise zu berücksichtigen Ist sich der Wirkung des eigenen Verhaltens auf andere generell nicht bewusst, schätzt die Wirkung unrealistisch ein oder kümmert sich nicht darum Nähe Hat Fähigkeit und Wunsch, Beziehungen im privaten und gesellschaftlichen Umfeld aufzubauen, diese können aber größtenteils oberflächlich sein Enge Beziehungen bestehen, um Bedürfnisse der Selbstregulation und des Selbstwerts zu befriedigen, verbunden mit unrealistischer Erwartung, von anderen perfekt verstanden zu werden Tendiert dazu, Beziehungen nicht als etwas Wechselseitiges anzusehen, sondern kooperiert vorrangig aus Eigennutz nochmals in 25 Facetten differenziert werden (um Redundanzen zu vermeiden, werden in Tab. 3 nur die Domänen des für Europa wichtigeren ICD-11 genauer dargestellt, s. unten). So kann etwa Negative Affektivität in die teilweise heterogenen Facetten „Emotionale Labilität“, „Ängstlichkeit“, „Trennungsangst“, „Unterwürfigkeit“, „Feindseligkeit“, „Perseveration“, „Depressivität“, „Misstrauen“ und (Fehlen von) „Affektarmut“ differenziert werden (Falkai et al. 2015). Wie sieht in diesem System die Diagnose einer BPS aus? Das Hybrid-Modell (dimensional und kategori- 92 al) im DSM-5 wird deutlich, wenn die Kriterien für das Vorliegen einer BPS als einer von sechs möglichen Prototypen beurteilt werden sollen. Diese Form der Beurteilung ist uns wieder sehr vertraut (Tab. 2): Die BPS wird also beschrieben auf den neuen Dimensionen, die einerseits den Schweregrad der Beeinträchtigung der Persönlichkeit abbilden (Selbst und interpersonelle Beziehungen), andererseits werden jene Facetten genannt, die in den jeweiligen Trait-Domänen für die Diagnose relevant sind. Hier sind es vier von sieben Merkmalen, die erfüllt sein müssen. Die „neue“ Borderline-Persönlichkeitsstörung: Dimensionale Klassifikation im DSM-5 und ICD-11 K übersichtsarbeit Tab. 2 Hybrid-Modell der Beurteilung einer BorderlineStörung im alternativen Modell der Persönlichkeitsstörungen des DSM-5. (Text deutlich verkürzt; Falkai et al. 2015, S. 1053, 1054) A. Mittelgradige oder stärkere Beeinträchtigung im Funktionsniveau der Persönlichkeit, Schwierigkeiten in mind. 2 der folgenden Bereiche: 1. Identität: verarmtes, instabiles Selbstbild, exzessive Selbstkritik, chronische Gefühle innerer Leere, bei Belastung Dissoziation 2. Selbststeuerung: Instabilität in Zielsetzungen, Vorlieben, Wertvorstellungen und beruflichen Plänen 3. Empathie: Eingeschränkte Fähigkeit, die Gefühle und Bedürfnisse anderer Personen zu erkennen, verbunden mit zwischenmenschlicher Überempfindlichkeit 4. Nähe: Intensive, aber instabile und konfliktreiche enge zwischenmenschliche Beziehungen, die durch Misstrauen und ängstliche Beschäftigung mit Verlassenwerden gekennzeichnet sind B. Mindestens 4 der folgenden 7 Persönlichkeitsmerkmale, mindestens eines ist davon (5) Impulsivität, (6) Neigung zu riskantem Verhalten oder (7) Feindseligkeit: 1. Emotionale Labilität (Negative Affektivität) 2. Ängstlichkeit (Negative Affektivität) 3. Trennungsangst (Negative Affektivität) 4. Depressivität (Negative Affektivität) 5. Impulsivität (Enthemmtheit) 6. Neigung zu riskantem Verhalten (Enthemmtheit) 7. Feindseligkeit (Antagonismus) Sechs der bisherigen 10 Typen wurden in das Hybrid-Modell aufgenommen (antisozial, vermeidendselbstunsicher, Borderline, narzisstisch, zwanghaft, schizotyp). Sollte keiner dieser Prototypen angemessen sein, ist die Diagnose einer Persönlichkeitsstörung, Merkmalsspezifiziert (PS-MS) möglich. Sie entspricht am ehesten einem rein dimensionalen Modell: (1) allgemeine Einschätzung der Schwere der Persönlichkeitsstörung (Kriterium A; mind. zwei von vier Bereichen mind. mittelgradig) und (2) spezifische Einschätzung der fünf Traits (Kriterium B; Negativität, Verschlossenheit, Antagonismus, Enthemmtheit und Psychotizismus; Falkai et al. 2015, S. 1058). Der Schweregrad (Kriterium A) und Traits (Kriterium B) sollten damit am besten unabhängig voneinander sein, was sich empirisch wahrscheinlich nicht halten lässt (Bastiaansen et al. 2016; Berghuis et al. 2014; Zimmermann et al. 2015). Im Gegenteil bildet die Gesamtanzahl der erfüllten PS-Kriterien (A und B zusammen im Gegensatz zu A allein) am besten die gegenwärtigen und zukünftigen Belastungen in Arbeit, Freizeit und Beziehungen ab (Hopwood et al. 2011). Es schält sich also neben den spezifischen Faktoren eine Art allgemeiner Faktor heraus, auf dem alle Kriterien (A und B) laden. Interessanterweise scheinen die Kriterien der BPS auf diesem allgemeinen Faktor eher zu laden als auf spezifischen Traits (Sharp et al. 2015; Wright et al. 2016). Manche Autor*innen kritisieren daher, dass etwa die narzisstische oder die antisoziale PS sehr gut als ich-syntone Extremvarianten von Traits betrachtet werden können, was allerdings für die BPS nicht zuzutreffen scheint. Eine Reihe von BPS-Symptomen können nicht gut auf diesem System abgebildet werden. Diese Symptome werden zudem als stark ichdyston erlebt werden (z. B. chronische Suizidalität, Depersonalisation, Pseudohalluzinationen; Paris 2020). K Konkrete Anwendungsbeispiele des AMPD in Assessment und Behandlungsplanung finden sich z. B. bei Bach et al. (2015), Pincus et al. (2016) oder Waugh et al. (2017). Zum Modell entwickelte Selbstbeurteilungsinstrumente für die klinische Erfassung des Schweregrads (Levels of Personality Functioning Scale, Morey 2017; Kurzversion LPFS-BF, Bach und Hutsebaut 2018) und der Trait-Domänen (Personality Inventory für DSM-5 – PID-5 und Kurzversionen; Krueger et al. 2012; Somma et al. 2019) werden aktuell intensiv untersucht, sind aber noch nicht normiert. Diese PID-Versionen sind von der American Psychiatric Association für Forschungszwecke und klinische Evaluation kostenlos und auf Deutsch im Netz zur Verfügung gestellt. Zusammenfassende Darstellungen von Messinstrumenten zu beiden Diagnosesystemen finden sich bei Hengartner et al. (2018), Zimmermann et al. (2019) und Evans et al. (2020). Hopwood (2018) hat außerdem erstmals versucht, den Einsatz des AMPD über einzelne Fallvignetten hinaus für die Auswahl von therapeutischen Techniken zu illustrieren. Ein wesentlicher Kritikpunkt an kategorialen Modellen war ja das Fehlen von evidenzbasierten Behandlungsmaßnahmen für die meisten Persönlichkeitsstörungen mit Ausnahme der BPS (Cristea et al. 2017). Dimensionale Modelle sollten hier eine wesentlich sinnvollere (und schließlich evidenzbasierte) Basis für die Wahl von Behandlungsmethoden liefern. Hopwood (2018) hat ein noch sehr vorläufiges Modell präsentiert, das auf den ersten Blick noch ungewohnt und etwas kantig wirken mag. Aber wir werden uns vielleicht daran gewöhnen (müssen), in Zukunft Manuale mit Titeln wie „Therapie der Negativen Affektivität“, „Therapie der Verschlossenheit“ oder „Therapie der Enthemmung“ zu finden. Auch wenn es eine sehr spannende Diskussion bleibt, welche Aspekte eines (zeitlich stabilen) Traits einer Veränderung durch Therapie zugänglich sind (Ringwald et al. 2020), zeigen Reviews, dass Interventionen Wirkung auf Traits zeigen (Roberts et al. 2017), und erste Guidelines bzw. Vorschläge für den klinischen Umgang mit verschiedenen Traits wurden bereits formuliert (Bach und Presnall-Shvorin 2020). Was das Board of Trustees der American Psychiatric Association für das DSM-5 vollinhaltlich abgelehnt und in den Teil III („Emerging models“) verbannt hat, während im Teil II nach wie vor die alte kategoriale Diagnostik zu finden ist (Krueger und Markon 2014), wurde in der ICD-11 vollinhaltlich umgesetzt und ist seit Mai 2019 von der WHO abgesegnet. Wenn die ICD-11 ab dem Jahr 2022 das gültige Diagnosesystem darstellen wird, verändert sich die Diagnosestellung der Persönlichkeitsstörungen weltweit grundlegend. Damit wird die offizielle Diagnostik der PS in den USA letztlich fundamental inkonsistent mit der Diagnosestellung im Rest der Welt (Bagby und Widiger 2020). Die „neue“ Borderline-Persönlichkeitsstörung: Dimensionale Klassifikation im DSM-5 und ICD-11 93 übersichtsarbeit Paradigmenwechsel in der ICD-11: Dimensionale Klassifikation der Persönlichkeitsstörungen Die Arbeiten an der Diagnostik der Persönlichkeitsstörungen begannen im Jahr 2010 noch vor der Publikation des DSM-5 (Tyrer et al. 2019). Dabei lehnte die Arbeitsgruppe von Beginn an ein Hybrid-Modell wie im DSM-5 ab und legte stattdessen eine einzelne Dimension für den Schweregrad der PS von „normal“ bis „schwer“ fest. Die Beeinträchtigung wurde über das Maß der interpersonellen Dysfunktion, die Folgen für soziale und berufliche Rollen, kognitive und emotionale Erfahrungen und Risiko, sich selbst oder anderen zu schaden, erfasst. Hauptgrund für die herausragende Betonung einer einzelnen Dimension für den Schweregrad einer PS war die bisherige hohe Komorbidität von PS-Diagnosen, die beliebig angesetzte Schwelle zwischen „gesund“ und „krank“ und die schlechte Abdeckung der Symptome von Patient*innen in den 10 Kategorien (Ofrat et al. 2018; s. oben). Außerdem zeigte sich immer wieder, dass mit zunehmendem Schweregrad auch die Anzahl der PS-Diagnosen zunahm. Kliniker*innen wählten daher der Einfachheit halber (ev. zusätzlich) die Nicht Näher Bezeichnete PS in komplexen Fällen (Tyrer et al. 2019). Bach und First (2018) beschreiben die Anwendung des neuen Modells in der klinischen Praxis. Hier ist in einem ersten Schritt zunächst zu überprüfen, ob die allgemeinen Anforderungen an die Diagnose einer Persönlichkeitsstörung erfüllt sind. Gefordert ist eine überdauernde Störung, charakterisiert durch Probleme im Funktionsniveau von Aspekten des Selbst (z. B. Identität, Selbstwert, Akkuratheit der Selbstwahrnehmung, Selbststeuerung) und/oder interpersonelle Dysfunktion (z. B. Fähigkeit, enge und wechselseitig befriedigende Beziehungen herzustellen, die Perspektive des anderen zu verstehen und Konflikte handzuhaben). Diese Störung muss nicht mehr in der Jugend oder im jungen Erwachsenalter beginnen, sie muss nur länger als 2 Jahre andauern. Damit sind erstmals auch PS möglich, die mit dem 50. Lebensjahr ihren Anfang nehmen! Wenn vor dem 25. Lebensjahr keine Auffälligkeiten erkennbar sind, ist ein Qualifier „später Beginn“ geplant (Tyrer et al. 2015). Die Störung manifestiert sich in kognitiven Mustern, emotionalem Erleben und Ausdruck und ist über verschiedene persönliche und soziale Situationen erkennbar. Sie ist nicht entwicklungsgemäß oder durch soziale oder kulturelle Faktoren (inkl. politische Konflikte) erklärbar. Sie ist nicht Ausdruck einer Erkrankung, einer Medikamentenwirkung oder eines Entzugs und verursacht erhebliche persönliche, familiäre, soziale und berufliche Belastung. In einem zweiten zentralen Schritt wird der Schweregrad einer PS eingeschätzt. Praktiker*innen haben hier auch keine andere Wahl, zu einer Diagnose zu kommen, weil die 10 Kategorien der ICD-10 nicht länger vergeben werden können. Damit wird das Problem der Komorbidität erheblich reduziert und es findet nur mehr die Einschätzung von „mild“ (6D10.0) über „moderat“ (6D10.1) bis „schwer“ (6D10.2) statt. Zusätzlich kann auch „Schweregrad unspezifiziert“ (6D10.Z) und eine subklinische Kategorie „Schwierigkeit“ (difficulty) vergeben werden (Abb. 2). Hier gibt es also Auffälligkeiten, die ansatzweise Belastung verursachen, aber nicht so deutlich sind, dass sie die Diagnose einer Störung rechtfertigen (Tyrer et al. 2015). Das dazu entwickelte „Standardized Assess- Abb. 2 Dimensionale Einschätzung der Persönlichkeitsstörung in der ICD11 (Tyrer et al. 2019) zunächst über Schweregrad und dann die Trait-Domänen. Auf eine Beschreibung von Facetten wurde verzichtet, um das Modell möglichst einfach zu halten. Die ersten vier Domänen entsprechen dem Big FiveModell. Die Domänen „Enthemmtheit“ und „Zwangshaftigkeit“ sind möglicherweise die entgegengesetzten Pole einer einzigen bipolaren Domäne 94 Die „neue“ Borderline-Persönlichkeitsstörung: Dimensionale Klassifikation im DSM-5 und ICD-11 K übersichtsarbeit Tab. 3 Trait-Domänen der Persönlichkeitsstörungen im ICD-11 (Tyrer et al. 2015, S. 723; dt. Übers. HM) Negative Affekti- Hpts. charakterisiert durch die Tendenz, eine Bandbreite von belastenden Emotionen zu zeigen/erleben (manifest) wie Angst, Ärger, Selbstvität verachtung, Gereiztheit, Verletzlichkeit, Depression und andere negative emotionale Zustände, häufig als Reaktion auf sogar relativ kleine tatsächliche oder wahrgenommene Stressoren Dissozialität Kern dieser Trait-Domäne ist die Nicht-Beachtung von sozialen Normen und Konventionen und der Rechte und Gefühle von anderen. Traits in dieser Domäne inkludieren Gefühlskälte, Fehlen von Empathie, Feindseligkeit und Aggression, Rücksichtslosigkeit und die Unfähigkeit oder Unwilligkeit, prosoziales Verhalten aufrechtzuerhalten, oft manifestiert als ein übermäßig positives Selbstbild, Ansprüchlichkeit und einer Tendenz, andere zu manipulieren und auszunützen Verschlossenheit Kern dieser Domäne ist emotionale und zwischenmenschliche Distanz, manifestiert in deutlichem sozialem Rückzug und/oder Indifferenz anderen gegenüber, Isolation mit sehr wenigen oder keinen Bezugspersonen, inkl. Vermeidung nicht nur von intimen Beziehungen, sondern auch Freundschaften. Traits in dieser Domäne schließen Distanz oder Kälte in der Beziehung zu anderen Personen, Reserviertheit, Passivität und Fehlen von Selbstsicherheit und reduziertes Erleben und Ausdrücken von Emotionen, besonders von positiven Emotionen mit ein, in einem Ausmaß, dass die Fähigkeit, Genuss zu empfinden, verringert ist Enthemmheit Charakterisiert durch eine persistierende Tendenz, auf unmittelbare internale oder Umwelt-Stimuli impulsiv zu reagieren, ohne längerfristige Konsequenzen zu berücksichtigen. Traits in dieser Domäne schließen Verantwortungslosigkeit, Impulsivität ohne Beachtung von Risiko oder Konsequenzen, Ablenkbarkeit und Leichtsinnigkeit mit ein Zwanghaftigkeit (Anankastia) Kern dieser Trait-Domäne ist ein enger Fokus auf Kontrolle und die Regulation des eigenen und des Verhaltens Anderer, um sicherzustellen, dass Dinge nach der besonderen Vorstellung bzw. dem Ideal der Person ablaufen. Traits in dieser Domäne inkludieren Perfektionismus, Perseveration, restriktive Emotionalität und Verhalten, Starrköpfigkeit, Überlegtheit, Ordnungsliebe und Beschäftigt-sein mit dem Befolgen von Regeln und dem Nachkommen von Normen ment of Personality Disorder“ (SASPD; Olajide et al. 2018) ist ein sehr kurzes Selbstbeurteilungsinstrument mit nur 9 Items zur Erfassung des Schweregrades (vgl. auch Bach und Anderson 2020 für einen Vergleich der Instrumente für das DSM-5 und der ICD-11). In einem dritten Schritt kann der Persönlichkeitsstil einer Person beschrieben werden. Die Traits in der ICD-11 entsprechen weitgehend den DSM-5-Domänen. Ursprünglich wurden die vier Traits „Negative Affektivität“ (6D11.0), „Verschlossenheit“ (6D11.1), „Dissozialität“ (6D11.2) und „Zwanghaftigkeit (Anankastia)“ (6D11.4) vorgeschlagen. Die fünfte Dimension „Enthemmtheit“ (6D11.3) wurde erst später hinzugefügt (Tyrer et al. 2019; Tab. 3). Die Unabhängigkeit der beiden letzten Traits wird allerdings in Zweifel gezogen. Empirische Ergebnisse weisen darauf hin, dass Zwanghaftigkeit und Enthemmtheit zwei entgegengesetzte Pole der gleichen Dimension sein könnten (Bach et al. 2020; Oltmanns und Widiger 2020). Diese Beschreibung des Stils der Persönlichkeit ist für das Stellen einer Diagnose nicht notwendig (dies im Unterschied zum DSM-5; Tab. 2)! Es ist zwar wahrscheinlich, dass mit zunehmendem Schweregrad der PS auch die Anzahl der auffälligen Trait-Domänen ansteigt (Tyrer et al. 2019), die zum spezifischen Ausdruck der Dysfunktion beitragen. Aber es ist ohne weiteres möglich und zulässig, dass bei einer schweren PS nur eine oder keine Domäne markant im Vordergrund steht. Die Domänen werden als kontinuierliche Dimensionen, nicht als Kategorien verstanden. Dennoch muss für den Zweck der Kodierung die Entscheidung „vorhanden“ vs. „nicht vorhanden“ getroffen werden (Bach und First 2018). Die Autoren beschreiben in ihrer Illustration des Vorgehens fünf verschiedene Patient*innen, von denen eine dargestellte Person unter einer erheblichen Beeinträchtigung durch eine Borderline-Symptomatik leidet. Die Beeinträchtigung wurde bei ihr als „schwer“ eingeschätzt. Von den Trait-Domänen wurden (1) Negative Affektivität, (2) Dissozialität und (3) Enthemmung als „vor- K handen“ gewertet, d. h. also eine schwere Persönlichkeitsstörung geprägt durch viele negative Emotionen, wie Scham, Ärger und Misstrauen, Enthemmung als impulsive und schädliche Reaktionen auf unmittelbare Stimuli und Dissozialität als misstrauische Aggression und Tendenz andere zu manipulieren. So könnte also in Zukunft die dimensionale Beschreibung einer BPS aussehen. Während im AMPD neben den Domänen ein deutlich differenzierteres Level von 25 Facetten beschrieben wurde (Krueger und Markon 2014), hat die ICD11 Work Group bewusst auf Facetten verzichtet, um die klinische Anwendung möglichst einfach zu halten (Tyrer et al. 2011; Abb. 2). Allerdings haben Oltmanns und Widiger (2020) z. B. für die Domäne „Negative Affektivität“ klar argumentiert, dass viele der bisherigen PS-Kategorien negative Emotionen beinhalten (narzisstische, antisoziale, selbstunsichere, zwanghafte, abhängige, Borderline-PS) und eine Unterscheidung der Affekte (externalisierend: Ärger, Misstrauen vs. Internalisierend: Angst, Depression etc.; vgl. auch Smith et al. 2020) wesentlich zur klinischen Nützlichkeit des Systems beitragen würde. Daher haben sie nur 2 Jahre nach der Publikation ihres „Personality Inventory for ICD-11“ (PiCD; Oltmanns und Widiger 2018) mit dem „Five-Factor Personality Inventory for ICD11“ (FFiCD) eine Erweiterung veröffentlicht, die die Trait-Domänen auf Facetten-Niveau erfasst (Gesamt 20 Facetten, 7 für Negative Affektivität, je 3 für Dissozialität, Verschlossenheit und Zwanghaftigkeit und 4 für Enthemmung), die überwiegend auch in einer Faktorenanalyse bestätigt werden konnten (Oltmanns und Widiger 2020). Diese Erweiterung ist für eine Diagnosestellung nach ICD-11 nicht relevant, sollte sich aber als klinisch nützlich und behandlungsrelevant erweisen. Tyrer et al. (2019) betonen jedenfalls, dass auch das vorgeschlagene System ohne Facetten ausreichend vielschichtig sei, wenn bei drei Schweregraden (plus der „Schwierigkeit“ als subklinisches Phänomen) und fünf Trait-Dimensionen fast 500 unter- Die „neue“ Borderline-Persönlichkeitsstörung: Dimensionale Klassifikation im DSM-5 und ICD-11 95 übersichtsarbeit Tab. 4 Beschreibung des Borderline-Musters in der ICD11 (Tyrer et al. 2019; Bach und First 2018, dt. Übers. HM) Tiefgreifendes Muster charakterisiert von Instabilität in zwischenmenschlichen Beziehungen, Selbstbild, Affekten und Impulsivität (sichtbar durch das Vorhandensein von vielen [sic!] der folgenden Punkte): Heftige Versuche reales oder vorgestelltes Verlassenwerden zu vermeiden Muster instabiler und intensiver zwischenmenschlicher Beziehungen Identitätsstörung als deutliches und persistierendes instabiles Selbstbild Tendenz, bei starken negativen Emotionen impulsiv zu handeln, führt zu potenziell selbstschädigendem Verhalten Wiederkehrende Episoden von Selbstschädigung Emotionale Instabilität aufgrund von deutlicher Reaktivität der Stimmung Chronische Gefühle der Leere Unangemessener intensiver Ärger oder Schwierigkeiten Ärger zu kontrollieren Vorübergehende dissoziative Zustände oder psychoseähnliches Erleben in Situationen mit hoher emotionaler Anspannung Andere Manifestationen des Borderline-Musters, die nicht alle zum gegebenen Zeitpunkt präsent sein müssen: Ein Selbstbild als unzulänglich, schlecht, schuldig, abstoßend und verachtenswert Erleben des Selbst als grundlegend anders und isoliert von anderen Menschen, schmerzliches Gefühl der Entfremdung und tiefe Einsamkeit Hypersensitivität für Zurückweisung, Probleme, angemessen Vertrauen in zwischenmenschlichen Beziehungen aufzubauen bzw. aufrecht zu erhalten, häufige Fehlinterpretation von sozialen Signalen schiedliche diagnostische Ergebnisse erreicht werden können und auch die BPS im System sehr gut abgebildet werden könne. Die Akzeptanz dieses Vorgehens im ICD-11 (wie auch im AMPD) wird wesentlich davon abhängen, ob und in welcher Form in Zukunft Behandlungsentscheidungen durch die Einschätzung des Schweregrads bzw. der Domänen erleichtert werden (vgl. Bach und Presnall-Shvorin 2020). Die postulierten DSM-5 und ICD-11 Trait-Domänen scheinen jedenfalls immer wieder empirisch erhärtet werden zu können und wurden auch in verschiedenen Kulturen repliziert (Bach et al. 2017; Lotfi et al. 2018; Pires et al. 2017). Es gibt aber auch viele kritische Stimmen wie Paris (2020), der zwar grundsätzlich die Entwicklung begrüßt, jedoch Jahrzehnte von Forschung in Gefahr oder sogar verloren sieht. Dies betrifft v. a. die BPS, für die innerhalb der spezifischen PS mit Abstand die meiste Forschungsliteratur entstanden sei. Und deshalb haben gerade hier (und nur hier) bei der BPS Kritiker*innen des Modells (Herpertz et al. 2017) erreicht, dass sie als „Borderline-Muster“ (borderline pattern) im ICD-11 erhalten bleibt. Die Kritiker*innen wendeten ein, dass ein Diagnosesystem über die wissenschaftlichen Kriterien hinaus klinische Nützlichkeit haben sollte und so hilfreiche Informationen zur Wahl von Behandlungen, Prognose und Outcomes liefern sollte. Sie sollte Patient*innen helfen, informierte Zustimmung geben zu können bzw. entscheiden zu können, welche Behandlungen ihnen am ehesten hilfreich erscheinen. Es sollte Ressourcen im Gesundheitssystem sinnvoll einsetzen helfen und sollte getragen sein von robusten wissenschaftlichen Ergebnissen. Diese Aspekte sahen die Autor*innen im Vorschlag für die ICD-11 nicht wirklich erfüllt (Herpertz et al. 2017). Dabei unterstützen auch diese Kriti- 96 ker*innen grundsätzlich einen dimensionalen Ansatz, fordern aber ein Hybrid-Modell wie im DSM-5, um einen schonenderen Übergang zu gewährleisten. Tyrer et al. (2019) beschreiben die Diskussion und deren Lösung durch Aufnahme des Borderline-Qualifiers als einen politischen Kompromiss, um die Akzeptanz des neuen Systems möglichst zu stärken. Der Verlust der BPS erschiene den Kritiker*innen zu radikal, weil viele Forschungsgelder und Projekte auf dieser Basis entstanden seien und wichtige Forschung dadurch initiiert worden sei. Zwar gäbe es nur wenig Forschung in der Klassifikation der BPS, aber Forschung in ihre Behandlung sei in großem Maß entstanden und habe Optimismus aufgebaut. Im Wesentlichen ist das Borderline-Muster eine Neuformulierung und Update der Kriterien in der ICD-10 (Tab. 4). Tyrer selbst sei den Diskussionen über diese Lösung bewusst ferngeblieben, nachdem er sich mehrfach klar dagegen ausgesprochen habe. Jedoch sehe die ICD-11 Working Group die exzellente Gelegenheit, die beiden Herangehensweisen der Diagnosestellung miteinander zu vergleichen. Ihre Hoffnung ist es, dass die Möglichkeit, die BPS auf den Dimensionen gut darstellen zu können, die Bedeutung des Borderline-Musters für die Diagnose zurückgehen lässt (Tyrer et al. 2019). Der Wechsel auf ein dimensionales Modell in den Persönlichkeitsstörungen wurde wiederholt und wohl zu Recht als Paradigmenwechsel bezeichnet. Das völlige Aufgeben der sehr vertrauten Kategorien einer narzisstischen, einer ängstlich-vermeidenden oder einer dependenten PS ist ein überraschend weitreichender und gleichzeitig gut argumentierter Schritt, den man in dieser Radikalität nicht ohne weiteres für möglich gehalten hätte. Es ist ein fundamentaler Wechsel hin zu einem empirisch unterstützten diagnostischen System, der eine massive Veränderung bestehender Forschungstraditionen bedeutet, aber gleichzeitig einen generellen Übergang zu dimensionalen Modellen in der Beschreibung von Psychopathologie allgemein einläuten könnte (Kotov et al. 2017; Smith et al. 2020). Unter dem Begriff „Hierarchical Taxonomy Of Psychopathology“ (HiTOP; http:// medicine.stonybrookmedicine.edu/HiTOP) bemüht sich eine Gruppe von renommierten Forscher*innen um diesen radikalen Übergang und bereitet ein allgemeines dimensionales Klassifikationssystem für alle psychischen Symptome vor, das eine Vielzahl von empirischen Befunden zu integrieren versucht. Die Forscher*innen haben die Hoffnung, dadurch neue nosologische Forschung zu stimulieren und zu leiten (Kotov et al. 2017). Die dimensionale Diagnostik der Persönlichkeitsstörungen wird hier zur „Vorreiterin in der Post-DSM-5-Ära“ (Krueger 2013). Natürlich ist abzuwarten, wie ihre endgültige Ausgestaltung im ICD-11 aussehen wird. Aber es ist jetzt schon klar, dass die wissenschaftliche Psychologie im Wettstreit mit der klassischen psychiatrischen Nosologie markante Akzente gesetzt hat. Nun bleibt abzuwarten Die „neue“ Borderline-Persönlichkeitsstörung: Dimensionale Klassifikation im DSM-5 und ICD-11 K übersichtsarbeit und zu hoffen, dass sich dies – neben Innovationen in der Forschung – auch in einer weiteren Verbesserung der psychotherapeutischen Behandlung und Versorgung unserer (B)PS-Patient*innen niederschlagen wird. Davon wird wesentlich abhängen, wie sehr diese spannende Entwicklung an Fahrt aufnimmt. Funding Open access funding provided by University of Innsbruck and Medical University of Innsbruck. Interessenkonflikt H. Mitmansgruber gibt an, dass kein Interessenkonflikt besteht. 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SOLAR ABSORBER WITH A STRUCTURED SURFACE – A WAY TO INCREASE EFFICIENCY
Acta Polytechnica
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1. Introduction surface from the lattice of pyramid cavities. The real- ization of the proposed thin shell structure, in addition with necessary input and output threaded holes that resist the required forming pressures is certainly not a simple technological solution. Therefore, the modifi- cation of a known parallel hydroforming technology is necessary to develop, to produce solar absorbers with required structured surface (set of pyramid cavities). Moreover, theoretical calculations are also necessary to use. Solar energy is relatively low dense and volatile, there- fore, great efforts must be made to maximize the efficiency of the plant. Moreover, production and operation costs have to be taken into account. The possibilities of increasing the efficiency of the solar absorber, which converts the incident solar radiation into the thermal energy of flowing medium, have been investigated. In practice, there are a number of ap- proaches, how to optimize the thermal efficiency of flat plate solar absorbers, such as some solutions in [1–4]. One possibility is also to use a multiple reflec- tions path, thus multiple absorptions on a properly structured absorbent surface can occur. This option is outlined in [5] but has not been deeply theoretically or technologically elaborated. Therefore, it has been pro- posed to form an absorbent structure on the surface, consisting of a lattice of conical cavities, where mul- tiple reflections of the incident sunlight occur. This is the same effect as laser deep penetration welding, during which the energy of the radiation contained in the laser beam is absorbed through the multiple reflections inside the keyhole. This means that the structured surface should be formed as a lattice of con- ical cavities. Assuming the production of a structured absorbent surface by forming a steel sheet, the conical cavities are not suitable either for the production of the die blocks or for a surface area utilization because the plane cannot be completely filled with the circles. From this perspective, it is much more advantageous to form a lattice of square pyramidal cavities, whereby the entire surface of the sheet is used and the tool manufacturing is easier. For these geometrical and practical reasons, it was decided to create a structured doi:10.14311/AP.2019.59.0134 Acta Polytechnica 59(2):134–143, 2019 doi:10.14311/AP.2019.59.0134 Acta Polytechnica 59(2):134–143, 2019 © Czech Technical University in Prague, 2019 available online at http://ojs.cvut.cz/ojs/index.php/ap SOLAR ABSORBER WITH A STRUCTURED SURFACE – A WAY TO INCREASE EFFICIENCY Libor Mrňaa,b,∗, Jan Řiháčekb, Martin Šarborta, Petr Horníka Institute of Scientific Instruments of the Czech Academy of Sciences, v. v. i., Brno, Czech Republic Brno University of Technology, Faculty of Mechanical Engineering, Institute of Manufacturing Technology, B C h R bli Libor Mrňaa,b,∗, Jan Řiháčekb, Martin Šarborta, Petr Horníka a Institute of Scientific Instruments of the Czech Academy of Sciences, v. v. i., Brno, C a Institute of Scientific Instruments of the Czech Academy of Sciences, v. v. i., Brno, Czech Republic b Brno University of Technology, Faculty of Mechanical Engineering, Institute of Manufacturing Technology, Brno, Czech Republic Institute of Scientific Instruments of the Czech Academy of Sciences, v. v. i., Brno, Czech Republic b Brno University of Technology, Faculty of Mechanical Engineering, Institute of Manufacturing Technology, Brno, Czech Republic ∗corresponding author: mrna@isibrno.cz ∗corresponding author: mrna@isibrno.cz Abstract. The basic idea of a solar absorber’s thermal gain increase is the keyhole effect utilization during which the radiation is absorbed by multiple reflections on cavity walls. The lattice of pyramidal or conical cavities on the solar absorber surface can be formed to create a structured surface leading to its overall absorptivity increase and to a reduction of the surface absorptivity dependence on the solar radiation incident beam angle changes caused by the daily and annual solar cycles. This contribution concludes the results of simulations of the effect of cavity geometry, geographical position and absorber orientation on its thermal gain with respect to the technological manufacturability of cavities. Furthermore, the real construction of the absorber with a structured surface using laser welding and parallel hydroforming is briefly described. Keywords: Solar absorber, hydroforming, multiple reflections, absorption efficiency, LS-DYNA 2. Theoretical design and calculations In the theoretical verification, the key parameter will be the apex angle of the pyramid because the num- ber of reflections/absorptions and hence the thermal efficiency and energy yield of the absorber are angle- dependent. In order to study the radiation absorption process, a model in the Matlab environment was de- veloped to analyse the effect of multiple reflections within the pyramidal cavity and to help to determine the optimal apex angle [6]. The following parameters are taken into account: geographical position, slope, and orientation of the absorbent area. These parame- ters, together with the daily and annual path of the sun, define a time-varying angle of incidence of the solar radiation on the plane of the absorber. Another parameter is the mentioned pyramid apex angle, see Figure 1. The average number of reflections and the associ- ated cumulative absorbance (Figure 2, Figure 3) are calculated as a function of the apex angle. It is obvi- ous that the average number of reflections and thus 134 Solar absorber with a structured surface vol. 59 no. 2/2019 Figure 1. Input variables and a simulation procedure for cumulative absorbance and cumulative radiant exposure for a pyramidal cavity with an apex angle α. Figure 1. Input variables and a simulation procedure for cumulative absorbance and cumulative radiant exposure for a pyramidal cavity with an apex angle α. Figure 2. The number of reflections inside the pyramid depending on the pyramid’s apex angle. Figure 2. The number of reflections inside the pyramid depending on the pyramid’s apex angle. 135 L. Mrňa, J. Řiháček, M. Šarbort, P. Horník Acta Polytechnica Figure 3. Cumulative absorptance depending on the apex angle of the pyramid. Figure 3. Cumulative absorptance depending on the apex angle of the pyramid. the cumulative absorbance increases with decreasing apex angles. provides the highest efficiency in winter. When the efficiency is multiplied by the incident solar flux and we take into account the daylight time, we can also calculate the energy yield of 1 m2 of the structured absorbent surface. The result is shown in Figure 5. It is evident that the energy yield in winter is naturally lower despite the higher efficiency. However, the technological feasibility of such surface must be taken into account. Technological tests based on calculations were made concurrently to the theo- retical design. 2. Theoretical design and calculations The manufacturability of the formed surface with pyramidal cavities with an apex angle of 90° and at most 60° was demonstrated, see below. The calculations show that, for the given posi- tion and orientation of the solar absorber, the an- nual energy yield of the structured surface with the 90° pyramids is higher by 4.4% compared to the plane surface and the yield of 60° pyramids is higher by 8.4%. This is an interesting value that makes it possi- ble to increase the thermal gain or reduce the active absorbent area. Therefore, subsequent simulations were performed for the cavities only with these apex angles. The en- ergy yield also depends on the solar flux that incident on a given surface of the Earth. The model allows the total annual energy yield to be calculated after adding the solar constant (calculated for the standard atmosphere and given coordinates on the Earth’s sur- face) [7]. The results are shown in Figure 4, where the thermal efficiency is the ratio of the incident and the obtained energy (through absorbance) for each day of the year. The absorption surface is situated to the south and it is inclined by 45° towards the horizontal plane. This is a common orientation of solar collectors in most of Europe. Despite the theoretical model, it is not possible to produce pyramid cavities with a perfectly sharp apex in a real production. Some flattening will always occur. Therefore, the effect of this flattening on the absorption characteristics of the cavity has to be eval- uated. The flattening parameter b, shown in Figure 1, is taken as a fraction of the pyramid base. The results are shown in Figure 6 and Figure 7. Figure 4 shows that the efficiency fluctuates for each day of the year. This can be explained by the different sun path during the year. However, it is interesting that the efficiency curves for structured surfaces with different apex angles also differ in their course - they have different maxima. This distinction can be explained by the different character of multiple reflections in cavities with various apex angles. The positive is also the finding that the structured sur- face with an apex angle of 60° and in this orientation 4. Numerical simulation The theoretical simulation of the pillow hydroforming process uses the finite element method. For this pur- pose, LS-DYNA software was used with a support of ANSYS Workbench software, more precisely, ANSYS LS-Dyna Export module and LS-PrePost software. The material model is given by an anisotropic plastic material model using the above mentioned material data. The geometric model was created in Autodesk Inventor Professional 2016 and it was imported into the computing software using *.iges format. Then, all boundary conditions, which can be seen in Figure 12, were defined. As can be seen from the figure, a quarter model of geometry was used to simplify the simulation, which was discterized by using shell elements [10]. From the production technology point of view, a cooperation between laser welding technology and forming technology, using the pillow hydroforming technology, is expected. The principle of this tech- nology consists in forming a circumferentially welded pair of plates with a liquid medium [9]. Two different thicknesses of welded sheets are used, namely thick- ness of 1 mm for non-formed (supporting) sheet and thickness of 0.8 mm for formed sheet. In this way, the sheets are bulged into the desired shape, which is determined by an upper and a lower tool. Therefore, the manufacturing process of the solar absorber with the structured surface consists of several basic steps, which are shown in Figure 8. The pillow hydroforming tool itself uses the cassette system. Its schematic design is shown in Figure 9. As can be seen from the figures, the tool has an exchange- able matrix, which allows the production of a desired structured surface on an area of 150 × 918 mm. The pressure of the forming medium is controlled by a hydraulic pump that is connected to the input flange through a quick screw connector. The venting flange prevents an accumulation of air bubbles inside the formed part and a subsequent damage of the formed structure, especially at the beginning of the hydro- forming process. The numerical simulation, done with the LS-DYNA software, gives, most often, optimal values of the forming die radius for each edge of the pyramidal structural surface R = 2 mm and a liquid pressure of 65 MPa, for which the stamped part is without any defects. In this case, the stamping depth of structured surface elements is 4 mm (Figure 13) with a maximum material thinning of 29.2%, i.e. 4. Numerical simulation a minimum thickness of 0.557 mm, see Figure 14. 3. Manufacturing technology In the next step, the practical application of the above mentioned theory, i.e. the production of a structured surface that is the body of the solar collector absorber, was solved. Firstly, realistic production options have to be considered. The limitation of current produc- tion technologies does not allow the production of any apex angle of the structured surface. Therefore, a 136 Solar absorber with a structured surface vol. 59 no. 2/2019 Figure 4. Cumulative efficiency of solar absorber surface for three pyramidal apexes during the year. Figure 4. Cumulative efficiency of solar absorber surface for three pyramidal apexes during the year. Figure 5. Total annual energy of 1 m2 of the solar collector’s surface for three pyramidal apexes during the year. Figure 5. Total annual energy of 1 m2 of the solar collector’s surface for three pyramidal apexes during the year. 137 L. Mrňa, J. Řiháček, M. Šarbort, P. Horník Acta Polytechnica Figure 6. The influence of the alignment of the bottom of the pyramid at the annual cumulative efficiency. Figure 6. The influence of the alignment of the bottom of the pyramid at the annual cumulative efficiency. Figure 7. The influence of the different alignments of the bottom of the pyramid at cumulative radiant exposure. Figure 7. The influence of the different alignments of the bottom of the pyramid at cumulative radiant exposure. 138 Solar absorber with a structured surface vol. 59 no. 2/2019 Figure 8. The manufacturing process of the solar absorber with the structured surface. Figure 8. The manufacturing process of the solar absorber with the structured surface. i.e. 0°, 45° and 90° to the rolling direction. The result- ing dependencies are shown in the graphs in Figure 10 and Figure 11. compromise has to be sought between the smallest apex angle, which allows the greatest possible increase in the efficiency of the collector, and its manufactura- bility. Experimental studies [8] have shown that the most advantageous is the production of the structured surface with the apex angle of 60°. 5. Practical realization Hardening curve of X5CrNi18-10 Steel. formed, which can be mutually arbitrarily intercon- nected, thereby, the total heat exchange surface of the solar absorber can also be freely modified. 6. Results and discussion The theoretical analysis, which was conducted at the beginning of the article, shows the suitability of using the pyramidal structure body of the solar absorber to increase its thermal efficiency. The application of the above mentioned shape is useful namely for the maximum structurability of the absorber surface. From the point of view of the production, the par- allel hydroforming technology seems to be the most effective. 5. Practical realization The austenitic chromium-nickel stainless steel X5CrNi18-10 was chosen as the absorber material for its good weldability, formability and relatively good thermal conductivity. The basic properties of the chosen steel are shown in Table 1. Based on the proposed design, the hydroforming die was also produced, which is shown in Figure 15. The workload during the forming operation would cause problems with the tool deformation during the form- ing operation. Therefore, the hydroforming device is supported by using a hydraulic press CBJ 500-6, which prevents the die swelling, see Figure 16. For the purposes of the theoretical calculations, ma- terial analyses were carried out, which were focused on the determination of the hardening curve and the normal anisotropy coefficients for classical directions, With using the mentioned tool, segments of solar absorber with the structured surface (Figure 17) are 139 L. Mrňa, J. Řiháček, M. Šarbort, P. Horník Acta Polytechnica Figure 9. Schematic design of the hydroforming device [4]. Tensile modulus E [MPa] 1.99 · 105 Yield strength Rp0.2 [MPa] 291 Ultimate strength Rm [MPa] 700 Ductility A5 [%] 50 Density at 20 ◦C ρ [kg·m−3] 7.9 · 103 Specific heat capacity cp [J·kg−1·K−1] 500 Thermal conductivity at 20 ◦C λt [W·m−1·K−1] 14.7 Table 1. Main properties of X5CrNi18-10 steel. Table 1. Main properties of X5CrNi18-10 steel. Figure 10. Hardening curve of X5CrNi18-10 Steel. compared to a flat solar absorber. In the manufactur- ing simulation by the hydroforming technology using FEM in ANSYS LS-DYNA software, optimal parame- ters of the hydroforming die geometry (namely radii of pyramid edges R = 2 mm) and the forming pressure of 65 MPa were found, for which a defect-free production of the desired parts is secured. According to the initial simulations, it was, there- fore, possible to propose the final design of the solar absorber with the structured surface of 1000×166 mm (1 105 pyramid cavities) and realize its production. In this case, the development and production of the hydroforming equipment was realized. This concept was also experimentally tested for the desired part. The achieved result corresponds to the theoretical simulations of the forming process, both in terms of the distribution of the sheet thinning and in terms of non-infringement during forming in the region of pyramid elements. Although, currently, this concept is not much discussed or solved in other literatures in terms of the above mentioned analysis, it represents a great potential. Figure 10. 7. Conclusion The paper describes the possibility to increase the thermal efficiency of a thermal solar absorber using a structured surface. There are multiple reflections inside the pyramidal cavities, thus multiple absorp- tions of the solar radiation. Due to the complicated and time-varying nature of the absorption, a simula- tion model has been developed to study the impact of the design changes to the thermal efficiency. These solar absorbers were then manufactured using a par- allel hydroforming technology with the support of The theoretical simulation of the cumulative ab- sorbance and energy yield of the absorber, which was realized in the Matlab environment, showed that the increase in efficiency of 8.4% for the pyramidal structured surface with a 60° angle could be expected 140 Solar absorber with a structured surface vol. 59 no. 2/2019 Figure 11. Dependence of normal anisotropy coefficients on the direction of sampling. Figure 11. Dependence of normal anisotropy coefficients on the direction of sampling. Figure 12. The geometric model of numerical simulation. Figure 13. Stamping depth prediction by using numerical simulation. 141 Figure 12. The geometric model of numerical simulation. Figure 12. The geometric model of numerical simulation. Figure 13. Stamping depth prediction by using numerical simulation. Figure 13. Stamping depth prediction by using numerical simulation. 141 L. Mrňa, J. Řiháček, M. Šarbort, P. Horník Acta Polytechnica L. Mrňa, J. Řiháček, M. Šarbort, P. Horník Acta Polytechnica Figure 14. Thickness prediction by using numerical simulation. Figure 15. Upper and lower hydroforming tool. a numerical simulation of the hydroforming process by using the FEM analysis. Based on the practical experience, the influence of the flattening of the pyra- mid apex to the resulting efficiency and energy gain Figure 16. The hydroforming process in practice. termined for a specific type of the absorbent surface. It is important to note that the realization of such a thin shell, including input and output holes that resist the necessary forming pressures at 50 MPa, is certainly not a simple technological solution. This article (after necessary calculations) proved that it is possible Therefore the modification of the parallel Figure 14. Thickness prediction by using numerical simulation. Figure 14. Thickness prediction by using numerical simulation. Figure 15. Upper and lower hydroforming tool. Figure 16. The hydroforming process in practice. Figure 16. The hydroforming process in practice. Figure 15. Upper and lower hydroforming tool. termined for a specific type of the absorbent surface. Figure 17. Formed solar absorber with the structured surface. Figure 17. Formed solar absorber with the structured surface. Acknowledgements [5] J. A. Duffie, W. A. Beckman. Solar Engineering of Thermal Processes. John Wiley, 2013. The contribution was supported by TA CR project: “Devel- opment of new types of solar absorbers” no. TA04020456 and with the support of the European Commission and the Ministry of Education Youth and Sports of the Czech Republic (project no. CZ.1.05/2.1.00/01.0017) and NPU LO1212. [6] A. A. Abood. A comprehensive solar angles simulation and calculation using Matlab. International Journal of Energy & Environment 6(4):367–376, 2015. [7] F. Kasten, A. Young. Revised optical air mass tables and approximation formula. Applied Optics 28:4735–8, 1989. doi:10.1364/AO.28.004735. 7. Conclusion It is important to note that the realization of such a thin shell, including input and output holes that resist the necessary forming pressures at 50 MPa, is certainly not a simple technological solution. This article (after necessary calculations) proved that it is possible. Therefore, the modification of the parallel hydroforming technology has been developed, which is able to produce solar absorbers with the structured surface. A fully functional sample was created, which validated the calculations and the whole technology. a numerical simulation of the hydroforming process by using the FEM analysis. Based on the practical experience, the influence of the flattening of the pyra- mid apex to the resulting efficiency and energy gain was additionally simulated. It was found out that flattening less than 1/3 of the pyramid base size is negligible. The surface absorbency dependence on the angle of incidence was not included in the model. This dependence would have to be experimentally de- 142 Solar absorber with a structured surface vol. 59 no. 2/2019 Figure 17. Formed solar absorber with the structured surface. References [8] L. Mrna, J. Rihacek. Forming a structured surface of a new type of solar absorber with hydroforming. Advanced Materials Research 1127:49–54, 2015. doi:10.4028/www.scientific.net/AMR.1127.49. [1] A. C. Mintsa Do Ango, M. Medale, C. Abid. Optimization of the design of a polymer flat plate solar collector. Solar Energy 87(1):64–75, 2013. doi:10.1016/j.solener.2012.10.006. doi:10.4028/www.scientific.net/AMR.1127.49. [9] M. Koc. Hydroforming for Advanced Manufacturing. Woodhead Publishing, Cambridge, 2008. [2] S. Saha, D. K Mahanta. Thermodynamic optimization of solar flat-plate collector. Renewable Energy 23(2):181– 193, 2001. doi:10.1016/S0960-1481(00)00171-3. [10] H. H. Lee. Finite Element Simulations with ANSYS Workbench 14: Theory, Applications, Case Studies. SDC Publishing, 2012. [3] B. Kundu. Performance analysis and optimization of absorber plates of different geometry for a flat-plate solar collector: A comparative study. Applied Thermal Engineering 22(9):999–1012, 2002. doi:10.1016/S1359-4311(01)00127-2. [3] B. Kundu. Performance analysis and optimization of absorber plates of different geometry for a flat-plate solar collector: A comparative study. Applied Thermal Engineering 22(9):999–1012, 2002. doi:10.1016/S1359-4311(01)00127-2. [4] L. Mrna, J. Rihacek, K. Podany, E. Peterkova. Thermal solar collector construction with pillow absorber and its variants (Konstrukce termického solárního kolektoru s poduškovým absorbérem a jeho variant). Alternative Sources of Energy (Alternativní zdroje energie) pp. 101–107, 2016. (In Czech), doi:10.1016/S1359-4311(01)00127-2. 143
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Clinical response of broilers placed on varying levels of aqueous Cassia alata leaf extract
Journal of medicinal plant research
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Short Communication Clinical response of broilers placed on varying levels of aqueous Cassia alata leaf extract Amao E. A.*, Adeoti T. M, Ayandele B and Jimoh A. R. The Polytechnic Ibadan, Saki Campus, Oyo State, Nigeria. Received 27 March, 2013; Accepted 13 March, 2014 This study examined the clinical response of 120 Anak broiler birds to inclusion of Cassia alata aqueous extract at different levels in their water (0, 5, 10 and 15 ml/L). The birds were allotted to four treatments of 10 birds each with three replicates. They were fed ad libitum and data were collected on haematological and serum parameters (packed cell volume, haemoglobin concentration, red blood cell count, white blood cell count, total protein, urea concentration). There were significant differences (p<0.05) in all the haematological parameters across the treatments. Haematological results indicated that the health status of the birds were normal. Serum biochemistry result shows significant variation (p<0.05) across the treatments. Total protein values across the treatments shows inefficient nutrient utilization. Urea values were significantly higher (p<0.05) in birds placed on C. alata extract, this indicates renal damage in the birds. Serum electrolytes shows significant differences (p<0.05) across the treatments with potassium ion showing significantly higher values above the physiological range indicating kidney failure. Key words: Cassia alata, broiler, aqueous extract, kidney. *Corresponding author. E-mail: ayodele_amao@yahoo.com. Tel: +2348059529136. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Vol. 8(12), pp. 520-522, 25 March, 2014 DOI: 10.5897/JMPR2013.4464 ISSN 1996-0875 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR Journal of Medicinal Plant Research Vol. 8(12), pp. 520-522, 25 March, 2014 DOI: 10.5897/JMPR2013.4464 ISSN 1996-0875 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR Journal of Medicinal Plant Research Journal of Medicinal Plant Research INTRODUCTION Traditional medicine using plant extracts continues to provide health coverage for over 80% of the world’s population, especially in the developing world. Medi- cinal plants are known to owe their curative potentials to certain biological active substances, which exist in parts of the plants (Ekpo and Etim, 2009). The chemicals which are referred to as active principles or phyto- chemical substances (UNESCO, 1998). However, the more current and most effective antibiotics are very expensive and out of reach of many Africans, majority of whom reside in the rural areas. These antibiotics are also associated with some serious side effects. Emergence of resistance build up by pathogens over continuous usage of conventional antibiotics is a global concern. A medicinal plant, such as Cassia alata L. is readily available. It is used in traditional medicine mainly in the tropical areas of the world, such as Malaysia, Brazil, and Indonesia. The leaves of C. alata are used as an effective treatment against ringworm and also against other skin diseases such as eczema and chronic skin impurities. C. alata leaves contain emodin, kaempferol, aloe-emodin, chrysophanol and isochrysophanol, rhein, ellagitannin, *Corresponding author. E-mail: ayodele_amao@yahoo.com. Tel: +2348059529136. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 521 Amao et al. Table 1. Haematological parameters of broilers placed on C. alata leaf extract. Table 1. Haematological parameters of broilers placed on C. alata leaf extract. Treatment parameter 0 ml/L 5 ml/L 10 ml/L 15 ml/L SEM Red blood cell (×106/mm3) 2.43a 2.41ab 2.34ab 2.28b 0.05 White blood cell (×103/mm3) 29.65b 28.55a 28.60b 28.90b 0.81 Haemoglobin (g/dl) 11.40b 12.30a 11.40b 10.45c 0.08 Packed cell volume (%) 27.50a 27.00b 26.50ab 25.50b 0.55 SEM=Standard error of the mean. Within a row, values with different superscripts differs significantly (p<0.05). to stand for 45 min at room temperature and centrifuged at 2,000 revolutions per minute (r.p.m) for 10 min to separate the cells from the serum. Urea was determined by urease method and creatinine by Folin-Wu filtrate method (Toro and Ackermann, 1975). Total serum protein was determined using Biuret method (Reinhold, 1953), while albumin was determined using Bromocresol green method (Peters et al., 1982). Alanine transaminase (ALT) was determined using spectrophotometric method as described by Rej and Hoder (1983). phenolic acid and cassiaxanthone, amongst other substances. One of the important flavonoids of C. Extract preparation Fresh and matured C. alata leaves were collected from the school premises. 500 g of the leaves were rinsed in distilled water and was blended with a blending machine. 1000 ml of distilled water was added to the leaf paste for 6 h and the extract was obtained using a cheese cloth. The extract was kept in a bottle with lid inside a refrigerator till the period of usage. MATERIALS AND METHODS Data collected on blood and serum parameters were analysed for variance using the statistical package of SAS (SAS, 1999). Experimental birds and management One hundred and twenty (120), one day old Anak broilers fed commercial starter and finisher diet were used for the study which lasted for eight weeks. Birds were individually weighed and randomly allotted to four treatments of three replicates (10 birds per replicate). Control birds were placed on a conventional antibiotic (Neoceryl), while others were placed on 5, 10 and 15 ml/L C. alata leaf extract in water. Feed and clean water were given ad-libitum throughout the period of the study. INTRODUCTION alata leaves is astragalin (AST) (Lee et al., 2011). AST has lately aroused increased pharmaceutical inte- rest because of its potential as anti-inflammatory agent, in addition to having antimicrobial activity. This study aimed at investigating the effect of administering C. alata aqueous extract on the clinical status of broiler birds. RESULTS AND DISCUSSION Table 1 shows the values for haematological parameters of broilers placed on different levels of C. alata extract, significant differences (p< 0.05) exists among the treatment. Values for RBC range between 2.28 and 2.43; the values reduce with increase in inclusion volume of C. alata although the values fall, within the normal range of 1.58 to 3.82 (Mitruka and Rawnsely, 1977). The values for RBC indicated that the birds are not suffering from anaemic condition. The WBC was not affected by dietary treatment thereby indicating that no pathological effect was induced by the C. alata extract; hence, the health status of the birds was okay. The result of haematological indices from this research work implies that the test extract were able to combat pathogenic infection in the animal system. This agrees with the work of Lee et al. (2011) which revealed that one of the main pharmacological activities of C. alata has antimicrobial. PCV reduces with increase in the inclusion level of C. alata extract, but all the values fall, within the normal range of 24.9 to 40.7 (Mitruka and Rawnsely, 1977). Heamoglobin ranges between 10.45 and 12.30 g/dl which fall between the normal ranges of 7.40 to 12.2 g/dl indicating efficient oxygen transportation. Table 2 shows significant different (p<0.05) in all the serum parameters except in creatinine. Value for total protein was highest in birds given convectional antibiotics (3.40 g/dl). Total REFERENCES Adedeji AO (1992). Rapid Interpretation of Routine Clinical Laboratory Test, 1 st Ed., Pub. S. Asekome and Co. Samane, Zaria, Nigeria. pp. 10-16. Ekpo MA, Etim PC (2009). Antimicrobial activity of ethanolic and aqueous extracts of Sida acuta on microorganisms from skin infections. J. Med. Plants Res. 3(9):621-624. ( ) Ewuola EO, Egbunike GN (2008). Haematological and Serum biochemical response of growing rabbits bucks fed different levels of dietary fumosin B. Afr. J. Biotechnol. 7(23):4304-4309. y Lee HB, Kim EK, Park SJ, Bang SG, Kim TG, Chung DW (2011). Isolation and anti-inflammatory effect of astragalin synthesized by enzymatic hydrolysis of tea seed extract. J. Sci. Food Agric. 91:2315- 2321. Mitruka BM, Rawnsely HM (1977). Clinical biochemical and haematological reference values in normal experimental animals. Masson Publishing, USA, Inc. pp. 102-119. g ( j ) High blood levels of potassium are generally due to kidney failure or endocrine disease rather than from excessive dietary intake. The values for birds placed on C. alata were slightly above the physiological range (4.60 to 6.50 mmol/L), indicating improper functioning of the kidney. The sodium ion mean values were within the normal range (148 to 163 mmol/L). g pp Peters T, Biamonte GT, Doumas BT (1982). Protein (total protein) in serum, urine and cerebrospinal fluid; albumin in serum. Sel. Methods Clin. Chem. 9:1-7. Reinhold JG (1953). Standard methods of clinical chemistry. M. Reiner. Ed. Volume 188. Academic Press New York. Rej R, Hoder M (1983). Aspartate transaminase. Methods of enzymatic analysis. 3 rd Ed. Bergmeyer HU, Bergmeyer J, Grass M Eds. Weinheim: Verlag Chem. 3:416-433. g SAS Institute Inc (1999). SAS/STAT user’s guide. Version 8 for windows. SAS Institute Inc Cary. NC. USA. https://v8doc.sas.com/sashtml/common/images/copyrite.htm Data collection Data were collected on blood parameters (packed cell volume, haemoglobin concentration, red blood cell count, white blood cell count, serum total protein, urea concentration, creatinine concen- tration, glucose concentration, alanine transaminase, potassium ion and sodium ion concentration) blood samples for haematology were collected into sample tubes containing ethylene diamine tetra-acetic acid (EDTA) as anticoagulant, while serological samples were collected in anti-coagulant free tubes. Packed cell volume (PCV) was determined by microhaematocrit method, haemoglobin (Hb) concentration was measured spectrophotometrically using SP6-500 UV Spectrometer. The red blood cell (RBC) and white blood cell (WBC) counts were estimated using haemocytometer (Ewuola and Egbunike, 2008). Serum was obtained after the blood was allowed J. Med. Plants Res. 522 Table 2. Serum biochemistry parameters of broilers placed on C. alata leaf extract. Treatment parameter 0 ml/L 5 ml/L 10 ml/L 15 ml/L SEM Total protein (g/dl) 3.40a 1.95bc 2.91ab 1.32c 0.43 Urea (mmol/L) 15.69b 20.95ab 21.95a 18.45ab 1.79 ALT 19.50b 19.00b 19.00b 23.00a 0.18 Creatinine 2.74 2.84 2.86 2.74 0.04 Glucose 145.00ab 137.00bc 150.50a 131.00c 3.94 Potassium (mmol/L) 5.34b 6.49ab 7.51a 6.90ab 0.51 Sodium (mmol/L) 142.50a 123.50b 124.00b 138.50a 1.39 SEM=Standard error of the mean. Within a row, values with different superscripts differs significantly (p< 0.05). Table 2. Serum biochemistry parameters of broilers placed on C. alata leaf extract. Table 2. Serum biochemistry parameters of broilers placed on C. alata leaf extract. Treatment parameter 0 ml/L 5 ml/L 10 ml/L 15 ml/L SEM Total protein (g/dl) 3.40a 1.95bc 2.91ab 1.32c 0.43 Urea (mmol/L) 15.69b 20.95ab 21.95a 18.45ab 1.79 ALT 19.50b 19.00b 19.00b 23.00a 0.18 Creatinine 2.74 2.84 2.86 2.74 0.04 Glucose 145.00ab 137.00bc 150.50a 131.00c 3.94 Potassium (mmol/L) 5.34b 6.49ab 7.51a 6.90ab 0.51 Sodium (mmol/L) 142.50a 123.50b 124.00b 138.50a 1.39 SEM=Standard error of the mean. Within a row, values with different superscripts differs significantly (p< 0.05). SEM=Standard error of the mean. Within a row, values with different superscripts differs significantly (p< 0.05). Conflict of Interests protein value was lower in birds placed on cassia extract, compared to the physiological range (5.20 to 6.90) reported by Mitruka and Rawnsely (1977). The author(s) have not declared any conflict of interests This result reveals that nutrient utilization was inadequate in birds placed on the plant extract, this can be as a result of the presence of antinutritional factors in the extract. There was significant difference (p<0.05) in the values for urea across the treatment with birds on 0 ml/L C. alata having the least value of 15.69 (mmol/L). High urea content in the birds may be due to kidney damage by the increase in the level of C. alata extracts due to the presence of antinutritional factors. Inclusion of C. alata extract up to 15 ml/L in water does not pose any injury on the liver of the birds. The mean values of the electrolytes showed significant differences (p<0.05). Potassium ion concentration is a major cation of intracellular fluid and function as sodium does by influencing acid-base balance (Adedeji, 1992). Conclusion p g py Toro G, Ackermann PG (1975). Practical Clinical Chemistry. Little, Brown and Company. Boston. This study has demonstrated that aqueous C. alata extract has no deleterious effect on the clinical status of broiler birds as it elicit no adverse effect on the haematological characteristics and serum biochemistry of the birds. Optimum level of its usage should be studied as serum biochemistry shows its potential of kidney damage. Histopathological effect is recommended to elucidate this fact. United Nations Educational Scientific and Cultural Organization (1998). Terminal Report. Promotion of Ethnobotany and Sustainable use of plant Resources in Mrica. Paris. p. 129.
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Mapping Modeled Exposure of Wildland Fire Smoke for Human Health Studies in California
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Michigan Technological University Michigan Technological University Digital Commons @ Michigan Tech Digital Commons @ Michigan Tech Michigan Tech Publications 6-4-2019 Mapping modeled exposure of wildland fire smoke for human Mapping modeled exposure of wildland fire smoke for human health studies in California health studies in California Patricia Koman University of Michigan-Ann Arbor Michael Billmire Michigan Technological University, mgbillmi@mtu.edu Kirk Baker US Environmental Protection Agency Ricardo de Majo University of Michigan School of Public Health Frank Anderson University of Michigan School of Medicine See next page for additional authors Follow this and additional works at: https://digitalcommons.mtu.edu/michigantech-p Part of the Mechanical Engineering Commons Recommended Citation Recommended Citation Koman, P., Billmire, M., Baker, K., de Majo, R., Anderson, F., Hoshiko, S., Thelen, B., & French, N. H. (2019). Mapping modeled exposure of wildland fire smoke for human health studies in California. Atmosphere, 10(6), 308. http://dx.doi.org/10.3390/atmos10060308 Retrieved from: https://digitalcommons.mtu.edu/michigantech-p/339 Follow this and additional works at: https://digitalcommons.mtu.edu/michigantech-p Michigan Technological University Michigan Technological University Digital Commons @ Michigan Tech Digital Commons @ Michigan Tech See next page for additional authors Part of the Mechanical Engineering Commons Authors Authors Patricia Koman, Michael Billmire, Kirk Baker, Ricardo de Majo, Frank Anderson, Sumi Hoshiko, Brian Thelen, and Nancy H. F. French Recommended Citation Recommended Citation Recommended Citation Recommended Citation Koman, P., Billmire, M., Baker, K., de Majo, R., Anderson, F., Hoshiko, S., Thelen, B., & French, N. H. (2019). Mapping modeled exposure of wildland fire smoke for human health studies in California. Atmosphere, 10(6), 308. http://dx.doi.org/10.3390/atmos10060308 Retrieved from: https://digitalcommons.mtu.edu/michigantech-p/339 Follow this and additional works at: https://digitalcommons.mtu.edu/michigantech-p Part of the Mechanical Engineering Commons Part of the Mechanical Engineering Commons oman, Michael Billmire, Kirk Baker, Ricardo de Majo, Frank Anderson, Sumi Hoshiko, Brian nd Nancy H. F. French This article is available at Digital Commons @ Michigan Tech: https://digitalcommons.mtu.edu/mic This article is available at Digital Commons @ Michigan Tech: https://digitalcommons.mtu.edu/michigantech-p/339 Received: 15 April 2019; Accepted: 1 June 2019; Published: 4 June 2019 Abstract: Wildland fire smoke exposure affects a broad proportion of the U.S. population and is increasing due to climate change, settlement patterns and fire seclusion. Significant public health questions surrounding its effects remain, including the impact on cardiovascular disease and maternal health. Using atmospheric chemical transport modeling, we examined general air quality with and without wildland fire smoke PM2.5. The 24-h average concentration of PM2.5 from all sources in 12-km gridded output from all sources in California (2007–2013) was 4.91 µg/m3. The average concentration of fire-PM2.5 in California by year was 1.22 µg/m3 (~25% of total PM2.5). The fire-PM2.5 daily mean was estimated at 4.40 µg/m3 in a high fire year (2008). Based on the model-derived fire-PM2.5 data, 97.4% of California’s population lived in a county that experienced at least one episode of high smoke exposure (“smokewave”) from 2007–2013. Photochemical model predictions of wildfire impacts on daily average PM2.5 carbon (organic and elemental) compared to rural monitors in California compared well for most years but tended to over-estimate wildfire impacts for 2008 (2.0 µg/m3 bias) and 2013 (1.6 µg/m3 bias) while underestimating for 2009 (−2.1 µg/m3 bias). The modeling system isolated wildfire and PM2.5 from other sources at monitored and unmonitored locations, which is important for understanding population exposure in health studies. Further work is needed to refine model predictions of wildland fire impacts on air quality in order to increase confidence in the model for future assessments. Atmospheric modeling can be a useful tool to assess broad geographic scale exposure for epidemiologic studies and to examine scenario-based health impacts. Keywords: wildland fire; air quality; exposure; particulate matter; geospatial analysis; public health; chemical transport model; atmospheric modeling; epidemiology Mapping Modeled Exposure of Wildland Fire Smoke for Human Health Studies in California Patricia D. Koman 1,* , Michael Billmire 2 , Kirk R. Baker 3, Ricardo de Majo 4, Frank J. Anderson 5, Sumi Hoshiko 6 , Brian J. Thelen 2 and Nancy H.F. French 2 1 Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI 48109, U g 3 Office of Air Quality Planning & Standards, Office of Air and Radiation, U.S. Environmental Protection Agency, Research Triangle Park, NC, 27709 USA; Baker.Kirk@epa.gov g y g p g 4 Health Behavior Health Education, University of Michigan School of Public Health, Ann Arbor, MI 48109, USA; rdemajo@umich.edu g y g p g 4 Health Behavior Health Education, University of Michigan School of Public Health, Ann Arbor, MI 48109, USA; rdemajo@umich.edu 5 Obstetrics and Gynecology, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA; fwja@umich.edu j Environmental Health Investigations Branch, California Department of Public Health, Richmond, CA 94804 g p USA; Sumi.Hoshiko@cdph.ca.gov USA; Sumi.Hoshiko@cdph.ca.gov USA; Sumi.Hoshiko@cdph.ca.gov * Correspondence: tkoman@umich.edu; Tel.: +01-734-764-0552   Received: 15 April 2019; Accepted: 1 June 2019; Published: 4 June 2019 www.mdpi.com/journal/atmosphere atmosphere atmosphere atmosphere 1. Introduction An understudied and growing source of air pollution is smoke from wildland fires. Wildland fires—including unplanned wildfires, prescribed fires, and agricultural burning—have increased rapidly in the U.S. over the past three decades, accounting for 40% of PM emissions inventories in the U.S. [1,2]. Despite the growing recognition of the impact of wildfire on health and its association with Atmosphere 2019, 10, 308; doi:10.3390/atmos10060308 2 of 20 Atmosphere 2019, 10, 308 general ambient particulate matter (PM) and respiratory and cardiovascular disease, scientific evidence is lacking for wildfire smoke associations with outcomes other than asthma and chronic obstructive pulmonary disease [3–5]. More specifically, studies of single fires in California have reported limited associations between wildfire PM exposure and cardiovascular hospitalizations [6,7] and reductions in birthweight [8]. Wildland fire smoke exposure affects a broad proportion of the U.S. population and the number of those affected is increasing [2,9]. The growing wildland-urban interface includes nearly 39% of all housing units, with an estimated 46 million persons in the Western U.S. alone, including an estimated half a million pregnant women, exposed to wildfire smoke [10–12]. Ecologists recognize fire’s permanence in biologic cycles and the likely increases of its occurrence with climate change, population settlement patterns, and fire seclusion. Accordingly, novel studies examining multi-fire periods with additional wildfire exposure metrics are needed to characterize associations in vulnerable groups and to establish a scientific basis for action to minimize smoke exposure. One reason for the limited number of epidemiologic studies of wildland fire air emissions is related to the need for better exposure assessment techniques, including modeling, stationary monitoring, remote sensing, or low cost sensors [3,13,14]. The purposes of this study are to estimate county level exposures to wildland fire-PM for California and to examine the strengths and limitations of using chemical transport models for health studies. Although air quality affected by wildland fire smoke could be classified as an “exceptional event” and excluded from calculating exceedances of national ambient air quality standards, it is important to study the effects of human exposure to smoke. The link between wildland fire smoke and adverse health effects is supported by a larger body of general ambient air pollution studies [15,16]. PM and ozone are associated with a suite of cardiopulmonary health outcomes, with PM in particular associated with cardiovascular [17] and maternal/birth endpoints [18–22]. 1. Introduction Air pollution is hypothesized to induce systemic oxidative stress and inflammation, which are pathways in the pathogenesis of respiratory and cardiovascular disease [17,23]. Because fine particulate air pollution (PM2.5) is a multi-dimensional pollutant originating from a variety of sources, including wildland fire, there are gaps in our understanding of how exposure to wildland fire smoke impacts health. While wildfires produce high levels of air pollutants, the chemical signature differs from other source of ambient PM and shows differential toxicity for some endpoints [24–27]. Exposure scenarios differ as well, with substantially sharper peaks during wildfires [7]. PM2.5 concentrations during several California wildfires has been estimated between 3 to 31 times the daily PM2.5 standard concentration established by the U.S. Environmental Protection Agency (EPA) [28]. While fire-specific PM exposure is associated with pulmonary outcomes, the evidence for cardiovascular disease is mixed, and there is evidence with limitations regarding maternal and birth outcomes [3,4,8,29]. Increases in exposure to air pollutants during pregnancy have been positively associated with adverse birth outcomes and an increased risk of pregnancy-induced hypertensive disorders [22,30]. Pregnancy-induced hypertensive disorders can lead to maternal and perinatal morbidity and mortality, but the causes are not well understood [31,32] and the contribution of wildland fire smoke has not been widely studied. Associations between general ambient PM2.5 and hypertensive disorders of pregnancy (HDP) are more consistent with upper percentile concentrations and pollutants whose distributions contain geo-temporal spikes, much like wildland fire distributions, rather than the more chronic low-level exposures typifying ambient air pollution [33,34]. Some populations are disproportionately affected by wildfire smoke based on their susceptibility, geographic location, or smoke exposure characteristics. A national study showed via principal component analyses that several vulnerability factors similar to those for general air pollution were relevant for fire-PM2.5; specifically, factors included age (e.g., those >65 years); adults with respiratory disease (e.g., COPD and asthma); adults with hypertension, obesity and diabetes; children with asthma; and economic deprivation [35]. Age may also be a factor contributing to a population’s susceptibility [36–40]. 3 of 20 Atmosphere 2019, 10, 308 In addition to underlying health status of a population, health impacts of wildfire smoke may be related to characteristics of the smoke exposure. 2. Materials and Methods We used the community multiscale air quality (CMAQ; https://www.epa.gov/cmaq), photochemical transport model in order to conduct multiple annual air quality simulations [42,43]. CMAQ is a three-dimensional grid-based model that simulates chemical and physical processes in each grid cell and uses Eulerian diffusion and transport processes to move chemical species to other grid cells [44]. CMAQ combines emissions from both natural (e.g., wildfire smoke) and anthropogenic (e.g., point-source industrial, automotive) sources with weather-based atmospheric transport, dispersion, chemical transformation, and deposition using time and space variant meteorology [45]. By conducting CMAQ model runs with and without wildfire smoke emissions sources, we can characterize the relative impact of wildfire smoke on ambient air quality. For this study, all non-fire-source emissions (based on National Emission Inventories) and fire event information were modeled with the CMAQ model v5.0.1/5.0.2 [46]. Wildfire emission sources for input to CMAQ were modeled using the BlueSky (v3.5.1) framework [47] (Figure 1). The approach requires quantification of four parameters: area burned (a.k.a., fire activity), fuel loading (biomass per unit area), the fraction of biomass fuel consumed by fire, referred to also as fuel consumption or combustion completeness, and emission factors [47]. For fire date, size, type, and location data, BlueSky uses the SmartFire2 fire information system, which aggregates and reconciles a comprehensive set of disparate wildfire information sources. SmartFire2 sources include satellite detections, daily situation reports (ICS-209 reports produced by incident managers), and GEOMAC perimeters for U.S. wildland and prescribed burns as well as burns (>100 acres) [48]. Fuel Characteristic Classification System (FCCS) provides spatially-defined fuel type and loading data [49]. Fuel consumption and resulting emissions are calculated using Consume v4.1 [50] with fuel type, loading and fuel moisture (via weather information management system—WIMS; https://famit.nwcg.gov/applications/WIMS) as inputs. We did not adjust CMAQ modeling outputs using air pollution monitoring data, satellite data or other techniques; instead we compared the modeling output to speciated monitored data [51]. or other techniques; instead we compared the modeling output to speciated monitored data [51]. This study analyzed CMAQ-modeled daily PM2.5 concentration estimates for the years 2007–2013 for the state of California at 12-km spatial resolution from a national run (24-h period is from midnight to midnight, adjusted for time zone). California experienced particularly active fire seasons (>750,000 acres burned) in 2007, 2008, and 2012. 1. Introduction These characteristics include the chemical composition, pollutant concentrations, the intensity of the fire, timing, and duration of smoke exposure; land features that contribute to smoke dispersal or contact with populations; extent to which populations can protect themselves from the exposure; and the number of individuals who are exposed. Furthermore, vulnerability factors may influence exposure; these include place-based characteristics (e.g., proximity to the wildland-urban interface, housing density and ventilation, low SES [35]), and fire-based characteristics (e.g., combustion characteristics, vegetation type, prescribed vs. wildfire [41]). However, there remain serious knowledge gaps about the full effects of these vulnerability factors and wildfire smoke exposures. 2. Materials and Methods The fire-related emissions components (SmartFire2 and BlueSky v 3.5.1, orange and blue boxes) are combined with meteorology (yellow box), emissions from other sources (SMOKE) and their chemical composition (SPECIATE) (green boxes) as inputs to the Community Multiscale Air Quality (CMAQ) chemical transport models (red box). Key modeling elements include the following: SmartFire2; FCCS: Fuel Characteristic Classification System [49]; WIMS: Weather Information Management System [52]; Consume [53]; FEPS: Fire Emission Production Simulator [54]; WRF: Weather Research and Forecasting model [55]; SMOKE: Sparse Matrix Operator Kernel Emissions modeling system [56]; SPECIATE [57]; CMAQ: Community Multiscale Air Quality [42 43] Figure 1. The fire-related emissions components (SmartFire2 and BlueSky v 3.5.1, orange and blue boxes) are combined with meteorology (yellow box), emissions from other sources (SMOKE) and their chemical composition (SPECIATE) (green boxes) as inputs to the Community Multiscale Air Quality (CMAQ) chemical transport models (red box). Key modeling elements include the following: SmartFire2; FCCS: Fuel Characteristic Classification System [49]; WIMS: Weather Information Management System [52]; Consume [53]; FEPS: Fire Emission Production Simulator [54]; WRF: Weather Research and Forecasting model [55]; SMOKE: Sparse Matrix Operator Kernel Emissions modeling system [56]; SPECIATE [57]; CMAQ: Community Multiscale Air Quality [42,43]. This study analyzed CMAQ-modeled daily PM2.5 concentration estimates for the years 2007– 2013 for the state of California at 12-km spatial resolution from a national run (24-hour period is from midnight to midnight, adjusted for time zone). California experienced particularly active fire seasons (>750,000 acres burned) in 2007, 2008, and 2012. To obtain fire-specific estimates, separate CMAQ model runs were performed with and without wildfire emissions sources, and fire-PM2.5 was calculated by subtracting the former from the latter. We examined CMAQ output at the more finely resolved grid cell level and aggregated to the county level To assess model performance, daily average PM2.5 measurements of elemental and organic carbon from the interagency monitoring of protected visual environments (IMPROVE) monitor network were used to evaluate modeling system tendencies toward over- or underprediction of wildfire impacts in the different years simulated. IMPROVE monitors were chosen because this network provides speciation and is largely rural, and thus would be less impacted by biases related to the characterization of urban areas. Previous studies of CMAQ model performance have assessed PM2.5 prediction in populated urban area [43,58,59]. resolved grid-cell level and aggregated to the county level. 2. Materials and Methods To obtain fire-specific estimates, separate CMAQ model runs were performed with and without wildfire emissions sources, and fire-PM2.5 was calculated by subtracting the former from the latter. We examined CMAQ output at the more finely resolved grid-cell level and aggregated to the county level. Daily county-level mean fire-PM2.5 concentrations were calculated by averaging values of all CMAQ grid cells whose centroid fell within the county boundary (Figure 2) for that day. From these daily county averages, we calculated county-level mean annual fire-PM2.5 concentration for each year (2007–2013). From these annual county averages, we calculated quartile breaks that were used to bin county populations into fire-PM2.5 exposure classes, similar to a national study by Rappold and colleagues [35]. Because there is no conclusive evidence of a threshold for response to PM2.5 and little evidence of a demarcation of healthy v. “unhealthy” fire-related PM2.5, we used quartiles of annual 4 of 20 Atmosphere 2019, 10, 308 air pollution to compare population exposure at different levels. We compared statewide to national analyses [35]. Atmosphere 2019, 10, x FOR PEER REVIEW 4 of 20 Figure 1. The fire-related emissions components (SmartFire2 and BlueSky v 3.5.1, orange and blue boxes) are combined with meteorology (yellow box), emissions from other sources (SMOKE) and their chemical composition (SPECIATE) (green boxes) as inputs to the Community Multiscale Air Quality (CMAQ) chemical transport models (red box). Key modeling elements include the following: SmartFire2; FCCS: Fuel Characteristic Classification System [49]; WIMS: Weather Information Management System [52]; Consume [53]; FEPS: Fire Emission Production Simulator [54]; WRF: Weather Research and Forecasting model [55]; SMOKE: Sparse Matrix Operator Kernel Emissions modeling system [56]; SPECIATE [57]; CMAQ: Community Multiscale Air Quality [42,43]. Figure 1. The fire-related emissions components (SmartFire2 and BlueSky v 3.5.1, orange and blue boxes) are combined with meteorology (yellow box), emissions from other sources (SMOKE) and their chemical composition (SPECIATE) (green boxes) as inputs to the Community Multiscale Air Quality (CMAQ) chemical transport models (red box). Key modeling elements include the following: SmartFire2; FCCS: Fuel Characteristic Classification System [49]; WIMS: Weather Information Management System [52]; Consume [53]; FEPS: Fire Emission Production Simulator [54]; WRF: Weather Research and Forecasting model [55]; SMOKE: Sparse Matrix Operator Kernel Emissions modeling system [56]; SPECIATE [57]; CMAQ: Community Multiscale Air Quality [42,43]. Figure 1. 2. Materials and Methods Daily county-level mean fire-PM2.5 concentrations were calculated by averaging values of all CMAQ grid cells whose centroid fell within the county boundary (Figure 2) for that day. From these daily county averages, we calculated county-level mean annual fire-PM2.5 concentration for each year (2007–2013) From these annual county averages we calculated quartile breaks that were used to bin We matched ambient monitored measurements with the model grid cell where the monitor was located. Daily average comparisons were aggregated for each model simulation year. We calculated these comparisons for the top three quartiles (fire-impacted areas) and for the lowest quartile of fire-PM2.5 concentration (little or no fire areas) using the quartile breaks as described above. (2007–2013). From these annual county averages, we calculated quartile breaks that were used to bin county populations into fire-PM2.5 exposure classes, similar to a national study by Rappold and colleagues [35]. Because there is no conclusive evidence of a threshold for response to PM2.5 and little evidence of a demarcation of healthy v. “unhealthy” fire-related PM2.5, we used quartiles of annual air pollution to compare population exposure at different levels. We compared statewide to national analyses [35]. To assess model performance, daily average PM2.5 measurements of elemental and organic carbon from the interagency monitoring of protected visual environments (IMPROVE) monitor network were used to evaluate modeling system tendencies toward over- or underprediction of Because wildfires are typically short-lived events (e.g., several days) that often elevate PM2.5 to several orders of magnitude above ambient levels, we performed two types of analyses to the modeled air pollution. First, we plotted the mean fire-PM2.5 concentration by day for the largest 10 counties by geographic area. Second, we developed an exposure metric to reflect peak exposure patterns based on the concept of a “smokewave”, analogous to a heatwave [60]. This metric is defined here as a period when daily fire-PM2.5 concentration exceeds the NAAQS 24-h PM2.5 level of 35 µg/m3 for more than two consecutive days. The number of smokewave periods was calculated for each CMAQ grid cell and county so that the total population exposed to smokewaves could be estimated. 5 of 20 of fire- Atmosphere 2019, 10, 308 these comparisons fo M2.5 concentration (little or no fire areas) using the quartile breaks as described above. Figure 2. CMAQ 12 km grid (light green lines) overlaid onto California county boundaries (gray lines). 2. Materials and Methods County-level statistics were derived by summarizing CMAQ-derived values for each grid cell whose centroid fell within the county boundary. Figure 2. CMAQ 12 km grid (light green lines) overlaid onto California county boundaries (gray lines). County-level statistics were derived by summarizing CMAQ-derived values for each grid cell whose centroid fell within the county boundary. Figure 2. CMAQ 12 km grid (light green lines) overlaid onto California county boundaries (gray lines). County-level statistics were derived by summarizing CMAQ-derived values for each grid cell whose centroid fell within the county boundary. Figure 2. CMAQ 12 km grid (light green lines) overlaid onto California county boundaries (gray lines). County-level statistics were derived by summarizing CMAQ-derived values for each grid cell whose centroid fell within the county boundary. Because wildfires are typically short-lived events (e.g., several days) that often elevate PM2.5 to several orders of magnitude above ambient levels, we performed two types of analyses to the modeled air pollution. First, we plotted the mean fire-PM2.5 concentration by day for the largest 10 counties by geographic area. Second, we developed an exposure metric to reflect peak exposure patterns based on the concept of a “smokewave”, analogous to a heatwave [60]. This metric is defined here as a period when daily fire-PM2.5 concentration exceeds the NAAQS 24-hour PM2.5 level of 35 μg/m3 for more than two consecutive days. The number of smokewave periods was calculated for We obtained demographic and health variables from the U.S. Census for these factors at the census tract level for California for the period 2007–2013. We obtained asthma emergency department visits and hospitalizations for heart attack from the Centers for Disease Control (CDC) Environmental Public Health Tracking Network (https://ephtracking.cdc.gov/DataExplorer/#/ accessed March 3, 2018) and live births from CDC Wonder database (Source: https://wonder.cdc.gov/natality.html, accessed March 3, 2018). We compared the geospatial location of populations with these factors by quartiles of fire-PM2.5 for 2007–2013. We obtained demographic and health census tract level for California for the perio 3.1. Mean Annual Fire-PM2.5 Concentrations census tract level for California for the period 2007 2013. We obtained asthma emergency department visits and hospitalizations for heart attack from the Centers for Disease Control (CDC) Environmental Public Health Tracking Network (https://ephtracking.cdc.gov/DataExplorer/#/ accessed March 3, 2018) and live births from CDC Wonder database (Source: https://wonder.cdc.gov/natality.html, accessed March 3, 2018). We compared the geospatial location of populations with these factors by quartiles of fire-PM2.5 for 2007-2013. We summarized the CMAQ modeling output county level fire-PM2.5 by year (Table 1). Generally, PM2.5 emissions are declining during this period due to Clean Air Act regulations of stationary and mobile sources, but California has non-attainment areas that do not meet current health-based ambient air quality standards. The maps (Figure 3a–d) show geographical extent of fire-PM2.5 annual mean concentrations for selected years illustrating a high fire year (2008) and a lower fire year and low all-source PM2.5 (2013). 6 of 20 Atmosphere 2019, 10, 308 o p e e 0 9, , O EE E IE Figure 3. CMAQ-modeled mean annual PM2.5 for California by 12-km grid: (a) Year 2008, fire-only emissions sources; (b) Year 2008, all sources; (c) Year 2013, fire-only sources; and (d) Year 2013, all sources. 12 μg/m3 represents the National Ambient Air Quality Standards (NAAQS) level for mean annual all source PM2.5. Populations At Risk by Annual Mean Fire PM Concentration Quartiles Figure 3. CMAQ-modeled mean annual PM2.5 for California by 12-km grid: (a) Year 2008, fire-only emissions sources; (b) Year 2008, all sources; (c) Year 2013, fire-only sources; and (d) Year 2013, all sources. 12 µg/m3 represents the National Ambient Air Quality Standards (NAAQS) level for mean annual all source PM2.5. The 24 h average concentration of ambient modeled PM from all sources in California (2007 Figure 3. CMAQ-modeled mean annual PM2.5 for California by 12-km grid: (a) Year 2008, fire-only emissions sources; (b) Year 2008, all sources; (c) Year 2013, fire-only sources; and (d) Year 2013, all sources. 12 μg/m3 represents the National Ambient Air Quality Standards (NAAQS) level for mean annual all source PM2.5. Figure 3. CMAQ-modeled mean annual PM2.5 for California by 12-km grid: (a) Year 2008, fire-only emissions sources; (b) Year 2008, all sources; (c) Year 2013, fire-only sources; and (d) Year 2013, all sources. 12 µg/m3 represents the National Ambient Air Quality Standards (NAAQS) level for mean annual all source PM2.5. Figure 3. We obtained demographic and health census tract level for California for the perio 3.1. Mean Annual Fire-PM2.5 Concentrations CMAQ-modeled mean annual PM2.5 for California by 12-km grid: (a) Year 2008, fire-only emissions sources; (b) Year 2008, all sources; (c) Year 2013, fire-only sources; and (d) Year 2013, all sources. 12 μg/m3 represents the National Ambient Air Quality Standards (NAAQS) level for mean annual all source PM2.5. Figure 3. CMAQ-modeled mean annual PM2.5 for California by 12-km grid: (a) Year 2008, fire-only emissions sources; (b) Year 2008, all sources; (c) Year 2013, fire-only sources; and (d) Year 2013, all sources. 12 µg/m3 represents the National Ambient Air Quality Standards (NAAQS) level for mean annual all source PM2.5. 3.2. Populations At Risk by Annual Mean Fire-PM2.5 Concentration Quartiles We estimated populations living in counties with each annual fire-PM2.5 exposure quartile (2007– 2013) (Table 3). Based on the modeling, 23.5 million (63.4%) California residents lived in counties with >0.34 μg/m3 fire-PM2.5. Approximately 7.71 million residents (56.4%) were experiencing poverty (under twice the poverty level). Just over six million living in the top three quartiles of fire-PM2.5 were <18 years old (57.2%), and 2.6 million were aged 65 and older (56.5%). The 24-h average concentration of ambient modeled PM2.5 from all sources in California (2007–2013) was 4.91 µg/m3 (standard deviation 4.04 µg/m3). The yearly all-source-PM2.5 daily mean ranged from 3.74 µg/m3 (2013) to 8.90 µg/m3 (2008). The 24-h average concentration of fire-PM2.5 in California by year was 1.22 µg/m3 (standard deviation 3.78 µg/m3) accounting for about a quarter of all-source-PM2.5 concentrations. The annual average of the fire-PM2.5 daily mean ranged from 0.31 µg/m3 (2010) to 4.40 µg/m3 (2008). 7 of 20 Atmosphere 2019, 10, 308 Table 1. Mean daily PM2.5 by year for CMAQ 12-km grid cells within California (2007 – 2013). Table 1. Mean daily PM2.5 by year for CMAQ 12-km grid cells within California (2007 – 2013). Table 1. Mean daily PM2.5 by year for CMAQ 12-km grid cells within California (2007 – 2013). We obtained demographic and health census tract level for California for the perio 3.1. Mean Annual Fire-PM2.5 Concentrations Year PM2.5 Mean Daily Concentration (Standard Deviation) (µg/m3) Percent Attributable to Fire All Sources Fire Only 2007 4.62 (2.27) 0.87 (1.55) 18.9% 2008 8.90 (8.76) 4.40 (8.89) 49.4% 2009 4.77 (1.50) 0.61 (0.91) 12.7% 2010 4.60 (1.51) 0.31 (0.47) 6.8% 2011 3.90 (1.43) 0.50 (0.70) 12.8% 2012 3.84 (1.51) 0.71 (1.16) 18.4% 2013 3.74 (1.94) 1.16 (1.89) 30.9% Average 4.91 (4.04) 1.22 (3.78) 24.9% r PM2.5 Mean Daily Concentration (Standard Deviation) (µg/m3) Percent Attributable to Fire The contribution of fire-PM2.5 to ambient PM2.5 in county-level averages (2007–2013) range from 4% fire-PM2.5 (e.g., Orange county) to 70% (e.g., Trinity County) (Table 2). Table 2. Mean daily CMAQ-derived PM2.5 by county in California (2007–2013). Table 2. Mean daily CMAQ-derived PM2.5 by county in California (2007–2013). County PM2.5 Mean (std) (µg/m3) Percent Attributable to Fire All Sources Fire Only Alameda 8.50 (5.90) 0.84 (3.76) 9.9% Alpine 3.13 (7.63) 1.57 (7.55) 50.1% Amador 6.21 (7.15) 1.87 (6.78) 30.1% Butte 6.61 (13.34) 2.63 (13.14) 39.8% Calaveras 5.46 (7.06) 1.88 (6.79) 34.4% Colusa 5.17 (8.99) 1.97 (8.72) 38.1% Contra Costa 11.05 (8.29) 0.98 (4.13) 8.9% Del Norte 4.42 (12.02) 2.74 (11.88) 62.0% El Dorado 5.37 (7.90) 2.23 (7.70) 41.5% Fresno 5.23 (4.42) 1.10 (3.73) 21.1% Glenn 5.25 (9.46) 2.04 (9.21) 38.7% Humboldt 4.63 (11.22) 2.61 (11.09) 56.4% Imperial 3.46 (1.61) 0.26 (0.64) 7.6% Inyo 2.28 (2.19) 0.49 (1.43) 21.6% Kern 4.81 (3.29) 0.76 (2.41) 15.7% Kings 7.52 (6.63) 0.93 (3.48) 12.3% Lake 4.37 (10.94) 2.12 (10.83) 48.4% Lassen 3.30 (6.88) 1.61 (6.78) 48.8% Los Angeles 8.41 (4.09) 0.57 (1.68) 6.8% Madera 5.45 (4.84) 1.31 (4.39) 24.0% Marin 4.97 (5.55) 0.84 (3.80) 16.9% Mariposa 4.47 (8.33) 2.08 (8.24) 46.7% Mendocino 4.31 (11.45) 2.26 (11.35) 52.5% Merced 7.40 (6.21) 1.10 (4.36) 14.8% Modoc 2.74 (4.36) 1.25 (4.20) 45.7% Mono 2.32 (3.52) 0.82 (3.32) 35.5% Monterey 3.99 (4.06) 0.81 (3.49) 20.3% Napa 5.39 (7.85) 1.53 (7.43) 28.4% Nevada 5.64 (10.48) 2.25 (10.30) 39.9% Orange 12.09 (6.10) 0.51 (1.57) 4.2% Placer 6.99 (10.87) 2.41 (10.67) 34.5% Plumas 4.50 (11.04) 2.43 (10.93) 54.1% Riverside 4.28 (2.18) 0.34 (0.95) 8.0% Sacramento 11.83 (9.68) 1.53 (6.50) 13.0% San Benito 4.12 (3.92) 0.74 (3.12) 17.9% San Bernardino 3.42 (2.12) 0.36 (0.96) 10.7% 8 of 20 Atmosphere 2019, 10, 308 Table 2. Cont. We obtained demographic and health census tract level for California for the perio 3.1. Mean Annual Fire-PM2.5 Concentrations County PM2.5 Mean (std) (µg/m3) Percent Attributable to Fire All Sources Fire Only San Diego 5.80 (2.84) 0.40 (1.22) 7.0% San Francisco No data No data - San Joaquin 9.72 (7.78) 1.16 (5.07) 12.0% San Luis Obispo 4.42 (3.47) 0.63 (2.18) 14.4% San Mateo 6.23 (6.22) 0.70 (3.17) 11.3% Santa Barbara 3.83 (2.87) 0.64 (2.25) 16.8% Santa Clara 7.28 (5.37) 0.86 (3.84) 11.8% Santa Cruz 6.43 (5.38) 0.86 (3.62) 13.3% Shasta 4.52 (9.12) 2.24 (9.00) 49.4% Sierra 3.84 (8.48) 1.83 (8.36) 47.6% Siskiyou 4.24 (9.93) 2.63 (9.87) 62.1% Solano 8.26 (7.41) 1.25 (5.62) 15.1% Sonoma 5.37 (8.68) 1.53 (8.32) 28.5% Stanislaus 7.52 (6.57) 1.17 (5.11) 15.6% Sutter 9.03 (9.51) 1.79 (7.68) 19.9% Tehama 5.17 (12.97) 2.68 (12.88) 51.8% Trinity 5.10 (19.25) 3.57 (19.20) 70.1% Tulare 5.11 (3.63) 1.07 (3.06) 21.0% Tuolumne 4.46 (9.68) 2.26 (9.65) 50.7% Ventura 4.74 (2.95) 0.56 (1.77) 11.8% Yolo 7.30 (8.68) 1.69 (7.76) 23.1% Yuba 7.77 (9.38) 2.03 (8.70) 26.2% 3.2. Populations At Risk by Annual Mean Fire-PM2.5 Concentration Quartiles PM2.5 Mean (std) (µg/m3) 3.2. Populations At Risk by Annual Mean Fire-PM2.5 Concentration Quartiles We estimated populations living in counties with each annual fire-PM2.5 exposure quartile (2007–2013) (Table 3). Based on the modeling, 23.5 million (63.4%) California residents lived in counties with >0.34 µg/m3 fire-PM2.5. Approximately 7.71 million residents (56.4%) were experiencing poverty (under twice the poverty level). Just over six million living in the top three quartiles of fire-PM2.5 were <18 years old (57.2%), and 2.6 million were aged 65 and older (56.5%). Table 3. Population size at risk summarized by county and annual average fire-PM2.5 in California, 2007–2013. County Annual Mean Fire-PM2.5 (µg/m3) Asthma Emergency Department Visits a Births b Hospitalizations for Heart Attack a Poverty: Under Twice Poverty Line (Poor or Struggling) c Population Under 18 c Population 65 and Over c Total Population c Total 2908 591,359 1574 13.67 10.60 4.67 36.78 (0.00, 0.34] 677 241,761 350 5.73 4.44 1.92 17.45 (0.34, 0.56] 489 84,170 235 1.85 1.52 0.65 5.87 (0.56, 0.86] 626 141,995 336 3.37 2.52 1.08 9.77 (0.86, 20.3] 1079 114,496 634 2.49 2.02 0.91 7.87 Missing 38 8936 20 0.22 0.11 0.11 0.80 a Asthma emergency department visits and hospitalizations for heart attack is from CDC Environmental Public Health Tracking Network (https://ephtracking.cdc.gov/DataExplorer/#/). b Births: County-level data is recorded only for counties with populations of 100,000 persons or more. We obtained demographic and health census tract level for California for the perio 3.1. Mean Annual Fire-PM2.5 Concentrations Counties with fewer than 100,000 persons are combined together under the label “Unidentified Counties.” In order to allocate births to those counties, the total number of births of those “Unidentified Counties” were proportionally allocated according to the total population of each county. (Source: https://wonder.cdc.gov/natality.html). Counties with number of births allocated: Alpine County, Yuba County, Amador County, Calaveras County, Colusa County, Del Norte County, Glenn County, Inyo County, Lake County, Lassen County, Mariposa County, Mendocino County, Modoc County, Mono County, Nevada County, Plumas County, San Benito County, Sierra County, Siskiyou County, Sutter County, Tehama County, Trinity County, and Tuolumne County. c Population size is given in millions, averaged (2007–2013); source of population data is U.S. Census. The poverty level varies by year, size of persons in a family household, and other factors. For example, for 2019, for a family of four in California, the poverty level is $25,750 (2019 dollars), so twice the poverty level is $51,500 per year. Table 3. Population size at risk summarized by county and annual average fire-PM2.5 in California, 2007–2013. a Asthma emergency department visits and hospitalizations for heart attack is from CDC Environmental Public Health Tracking Network (https://ephtracking.cdc.gov/DataExplorer/#/). b Births: County-level data is recorded only for counties with populations of 100,000 persons or more. Counties with fewer than 100,000 persons are combined together under the label “Unidentified Counties.” In order to allocate births to those counties, the total number of births of those “Unidentified Counties” were proportionally allocated according to the total population of each county. (Source: https://wonder.cdc.gov/natality.html). Counties with number of births allocated: Alpine County, Yuba County, Amador County, Calaveras County, Colusa County, Del Norte County, Glenn County, Inyo County, Lake County, Lassen County, Mariposa County, Mendocino County, Modoc County, Mono County, Nevada County, Plumas County, San Benito County, Sierra County, Siskiyou County, Sutter County, Tehama County, Trinity County, and Tuolumne County. c Population size is given in millions, averaged (2007–2013); source of population data is U.S. Census. The poverty level varies by year, size of persons in a family household, and other factors. For example, for 2019, for a family of four in California, the poverty level is $25,750 (2019 dollars), so twice the poverty level is $51,500 per year. Atmosphere 2019, 10, 308 9 of 20 More than half of the births (2007–2013) in California occurred in counties with >0.34 µg/m3 or in the top 3 quartiles fire-PM2.5. We obtained demographic and health census tract level for California for the perio 3.1. Mean Annual Fire-PM2.5 Concentrations Likewise, in this same period, 75% of the asthma emergency department visits and 76% of hospitalizations for heart attacks in California occurred in counties with >0.34 µg/m3 fire-PM2.5. 3.3. Fire-PM2.5 Smokewaves: Geospatial Extent and Populations At-Risk Figure 4 shows the mean CMAQ-derived fire-PM2.5 concentrations by date from May to November 2008 for the ten largest California counties by land area. The pattern illustrates that for many counties, there are near zero fire-PM2.5 levels for many days followed by peaks during a fire incident that an annual average might mask. Accordingly, we analyzed populations residing in counties experiencing smokewave days. Furthermore, the timing and intensity of peak fire-PM2.5 varies across the state during this period, which relates to the challenges of siting stationary monitors. Based on the CMAQ-derived fire-PM2.5 data, 97.4% of the population of California lived in a county that experienced at least one smokewave from 2007–2013 (Figure 5). A total of 9.2 million individuals (25% of the population of California) lived in a county with at least one smokewave per year on average (i.e., at least seven smokewaves from 2007–2013). Based on a county analysis, a total of 4.5 million individuals (12.2% of the population of California) lived in counties with an average of at least 2 smokewaves per year (i.e., at least 14 from 2007–2013). Although the spatial patterns of wildfires during this period are more concentrated in the northern portion of the state during this period, fires and smokewaves occur statewide in California. Atmosphere 2019, 10, x FOR PEER REVIEW 10 of 20 Figure 4. Mean daily CMAQ-derived fire-PM2.5 for the 10 largest counties (by area) in California from May to November 2008, illustrating spatial and temporal fluctuation in fire-PM2.5 concentrations. Figure 4. Mean daily CMAQ-derived fire-PM2.5 for the 10 largest counties (by area) in California from May to November 2008, illustrating spatial and temporal fluctuation in fire-PM2.5 concentrations. Figure 4. Mean daily CMAQ-derived fire-PM2.5 for the 10 largest counties (by area) in California from May to November 2008, illustrating spatial and temporal fluctuation in fire-PM2.5 concentrations. Figure 4. Mean daily CMAQ-derived fire-PM2.5 for the 10 largest counties (by area) in California from May to November 2008, illustrating spatial and temporal fluctuation in fire-PM2.5 concentrations. 10 of 20 11 of 20 Atmosphere 2019, 10, 308 At h 2019 10 FO Figure 5. Smokewave periods per year 2007–2013 in California based on CMAQ-modeled fire-PM2.5 concentrations. Smokewave periods are defined here as periods when daily fire-PM2.5 concentration exceeds the NAAQS 24-hour PM2.5 standard of 35 μg/m3 for more than 2 consecutive days. Figure 5. 3.3. Fire-PM2.5 Smokewaves: Geospatial Extent and Populations At-Risk Smokewave periods per year 2007–2013 in California based on CMAQ-modeled fire-PM2.5 concentrations. Smokewave periods are defined here as periods when daily fire-PM2.5 concentration exceeds the NAAQS 24-h PM2.5 standard of 35 µg/m3 for more than 2 consecutive days. Figure 5. Smokewave periods per year 2007–2013 in California based on CMAQ-modeled fire-PM2.5 concentrations. Smokewave periods are defined here as periods when daily fire-PM2.5 concentration exceeds the NAAQS 24-hour PM2.5 standard of 35 μg/m3 for more than 2 consecutive days. Figure 5. Smokewave periods per year 2007–2013 in California based on CMAQ-modeled fire-PM2.5 concentrations. Smokewave periods are defined here as periods when daily fire-PM2.5 concentration exceeds the NAAQS 24-h PM2.5 standard of 35 µg/m3 for more than 2 consecutive days. 4. Discussion In this study we estimated the magnitude of potential exposures of fire-PM2.5 and all-source ambient PM2.5, and the frequency of smokewave days for fire-PM2.5 during a recent period in California. We also compared populations with factors known to modify the risk of adverse PM-related health effects (e.g., age, socioeconomic status, pre-existing conditions like asthma) and estimated population size at risk with respect to the magnitude and frequency of smoke concentrations. We computed the county level fire-PM2.5 by year (2007–2013) and from that full distribution calculated quartiles to describe the potential population exposure. Compared to national county-level multi-year average concentrations (based on cut points ratioed from national standards for all-source PM2.5 concentrations), California shows a different pattern than the national profile. Upper end distributions are higher for California (20.3 µg/m3 fire-PM2.5 compared to 4.58 µg/m3 fire-PM2.5 in the national study) [35]. 3.4. Model Performance 3.4. Model Performance Annual mean observed, predicted, and difference between observed and predicted PM2.5 carbon (organic and elemental components) are shown for each year simulated. Metrics were estimated where modeled wildland fire impacts exceed 0.34 µg/m3 (wildland impacted) and otherwise (little or no wildfire). Table 4. Annual mean observed, predicted, and difference between observed and predicted PM2.5 carbon (organic and elemental components) are shown for each year simulated. Metrics were estimated where modeled wildland fire impacts exceed 0.34 µg/m3 (wildland impacted) and otherwise (little or no wildfire). Type of Wildland Fire Impact Year N (Grid Cells) Mean Observed (µg/m3) Mean Predicted (µg/m3) Difference: Predicted–Observed (µg/m3) Wildfire impacted organic and elemental carbon components of PM2.5 2007 463 4.1 4.5 0.5 2008 721 5.2 7.1 2.0 2009 422 5.6 3.5 −2.1 2010 220 3.8 3.0 −0.8 2011 428 3.5 3.2 −0.3 2012 418 3.9 3.7 −0.1 2013 589 3.7 5.3 1.6 Little or no wildfire organic and elemental carbon components of PM2.5 2007 918 1.6 1.1 −0.5 2008 599 1.5 1.5 −0.1 2009 966 1.4 1.2 −0.2 2010 1158 1.4 1.2 −0.2 2011 947 1.3 1.0 −0.4 2012 1008 1.3 1.0 −0.4 2013 776 1.2 0.7 −0.5 3.4. Model Performance 3.4. Model Performance Model skill in replicating smoke impacts was assessed by comparing daily average speciated PM2.5 measurements with model predictions when the model predicted wildfire impacts greater than 0.34 μg/m3 of PM2.5 carbon (organic and elemental) since these components dominate smoke plume composition. Performance was also assessed when the model predicted none or little impact (less than or equal to 0.34 μg/m3) from smoke to help frame underlying biases in the modeling system unrelated to wildfires. Daily average mean observed PM2.5 carbon (organic mass and elemental), model predicted PM2.5 carbon, and the difference between the daily average predictions and observations are presented by year in Table 4 where the model predicts impacts from wildfire and little or no impact from wildfire. The modeling system tends to underestimate organic carbon when little or no wildfire impact was predicted. Performance for capturing wildfire impacts varied from year to year, with some years over-estimating PM2.5 carbon (2008 and 2013 both with a bias ~2 μg/m3 and one year (2009 with a bias of −2 μg/m3) having a notable underprediction (Table 4). CMAQ has Model skill in replicating smoke impacts was assessed by comparing daily average speciated PM2.5 measurements with model predictions when the model predicted wildfire impacts greater than 0.34 µg/m3 of PM2.5 carbon (organic and elemental) since these components dominate smoke plume composition. Performance was also assessed when the model predicted none or little impact (less than or equal to 0.34 µg/m3) from smoke to help frame underlying biases in the modeling system unrelated to wildfires. Daily average mean observed PM2.5 carbon (organic mass and elemental), model predicted PM2.5 carbon, and the difference between the daily average predictions and observations are presented by year in Table 4 where the model predicts impacts from wildfire and little or no impact from wildfire. The modeling system tends to underestimate organic carbon when little or no wildfire impact was predicted. Performance for capturing wildfire impacts varied from year to year, with some years over-estimating PM2.5 carbon (2008 and 2013 both with a bias ~2 µg/m3 and one year (2009 with a bias of −2 µg/m3) having a notable underprediction (Table 4). CMAQ has been shown to compare 11 of 20 Atmosphere 2019, 10, 308 reasonably with measurements in urban and rural areas for annual simulations [43,61] and specific episodes [46,62]. Table 4. 4.1. The Magnitude of Wildfire Smoke Exposure in California Wildfires are typically short-lived events that elevate PM2.5 to several orders of magnitude over ambient levels. Because of the distribution of many days of near-zero fire-PM2.5 and much larger peak concentrations during a fire event, an annual average may mask relevant exposures and other exposure metrics may be relevant to health studies. Our long-term research question seeks to understand to what extent peak regional exposures to wildland fire smoke is associated with increased risk of health effects. Wildland fire sources can contribute to peak values; over half of the summer time ambient measures of 24-h PM2.5 from all sources measuring greater than 35 µg/m3 in the contiguous U.S. states occur when a smoke plume is present [63]. Further, in the U.S. in situ monitors may miss peak fire-PM2.5 exposures because regulatory monitoring of PM is often conducted on a 1-in-3 or 1-in-6-day schedule and the monitoring network is sparse in fire-prone areas. California began using continuous monitors in 2006 and deploys continuous monitors to any population center of concern during a wildfire incident [64]. Atmospheric transport modeling offers the advantage of full geospatial and temporal coverage as well as the ability to isolate pollution originating from fires, but it can be biased due to limitations with key inputs such as emissions and meteorology. The CMAQ model was used to estimate smokewave days and to provide a complete spatial coverage for multiple years. CMAQ, similar to other smoke models, uses locations of known fires to inform emissions inventories – in our case SmartFire [47,48]– 12 of 20 Atmosphere 2019, 10, 308 to impose the characteristics and amount of fire emissions in place and time. In the modeling, along with emissions from other sources, the atmospheric transport of fire emissions was simulated based on modeled winds and other meteorological inputs. Simulated atmospheric chemical interactions and reactions are also modeled. While complex, these models have had extensive validation for many sources [14,43,65], including some strengths and limitations with regard to wildland fire sources [14,45], as discussed below. The atmospheric modeling methods used in this study estimate that between 2007 and 2013 population exposure to smoke in the California was extensive with 56.2% of the population living in counties with the highest three quartiles of annual mean fire-PM2.5. 4.1. The Magnitude of Wildfire Smoke Exposure in California Similarly, over half of the children (aged 18 and younger) (57.2%), over half of the elderly (aged 65 and older) (56.5%), and over half of those under twice the poverty level resided in California counties with the highest three quartiles of fire-PM2.5. Over half of the births, three quarters of the emergency department visits for asthma, and three quarters of the hospital visits for heart attacks occurred in California counties with the highest three quartiles of fire-PM2.5, although this analysis does not establish that the exposures preceded these outcomes. Future comparisons of geocoded cases could be matched with modeled concentrations or smokewave metrics to explore associations. Based on our modeling, 97.4% of California residents lived in a county with at least one smokewave during the 2007–2013 study period. A quarter of the population (24.7%) lived in a county with on average at least one smokewave per year during this period. Of those 12% resided in a county with at least two smokewaves per year during this period. Although the spatial patterns of wildfires during this period are more concentrated in the northern portion of the state during this period, fires and smokewaves occur statewide in California, so with additional years of data, it is possible that many more counties will show high smokewave exposure. 4.2. Spatiotemporal Smoke Exposure Approaches Atmospheric chemical transport models have been used for decades to understand exposures to air pollutants [15], but the use of these models for all sources and at a large geographic scale for wildland and prescribed fire pollution is relatively new [7,60,67]. Nevertheless, atmospheric modeling has been used in epidemiologic studies to represent exposure [4,67–72], and research has shown atmospheric chemical transport modeling as an effective tool for exploring the impacts and ramifications of wildfire smoke on air quality [4,7,65]. Previous health studies that use atmospheric models have been conducted at coarser geographic scales (e.g., GEOS-Chem) and have considered only fire-derived pollutants rather than fire in conjunction with PM from other sources (e.g., traffic, utilities) when assessing associations with health, which may overestimate the fire-specific exposures [60,73]. Other studies have used atmospheric modeling in conjunction with adjustments from air quality monitoring data, satellite remote sensing data or additional post-processing statistical techniques [68,71,74–79]. g p p g q All source PM concentrations for broad geographical areas can be inferred from satellite remote sensing [80,81], offering advantages over modeled approaches. The NASA-CALIPSO is an advanced satellite remote sensing system that uses LiDAR sensing to retrieve aerosol optical depth (AOD), extinction profile, and aerosol type at various altitudes; however, it does not provide reliable surface level pollutant concentrations. This and other satellite sensing systems used for characterizing surface air quality are reviewed by Martin [80]. Some researchers have augmented these datasets with ground-level air quality monitoring to address these limitations [82]. Satellite remote sensing of the atmosphere can provide a unique opportunity to understand global and regional scale presence of elevated pollution events, such as wildfires but are limited with regard to local-scale applications. Furthermore, these passive remote sensors cannot directly measure surface PM and instead record AOD, which is a measure of total column aerosol loading, a metric of aerosols from the surface to the top of the atmosphere. Knowledge of atmospheric conditions and assumptions regarding atmospheric stratification conditions provide a way to retrieve the specific metrics of interest, but with uncertainty. Additionally, clouds and other weather patterns can interfere with the measurements resulting in the retrieval algorithms that rely on broad assumptions in order to relate the satellite measurement to surface-level pollution. 4.2. Spatiotemporal Smoke Exposure Approaches Wildland fire smoke can affect air quality locally and regionally, but it can be difficult to quantify for purposes of studying health impacts [66]. Three main methods have been used in health studies to characterize exposure to wildfire emissions: 1) atmospheric chemical transport modeling, 2) air quality monitoring, and 3) satellite measures of pollutant concentration or density in the atmosphere, data often combined with in-situ monitoring or other models. A combination of these approaches through data fusion, assimilation, or machine learning have also been explored [66]. While several techniques have been assessed, a consensus on best practice has not been established. Exposure estimation techniques of fire-PM presented in the literature have strengths and limitations. Federal reference or equivalent method air quality monitoring data offer advantages of high-quality data that have been widely used in epidemiologic studies. Typically, measured air pollutant concentrations are mapped from ambient air quality monitors either through spatial interpolation to a grid or simply assigning concentrations from the nearest station(s) to populations. With the exception of the IMPROVE network, most air quality monitoring stations are located in urban and other densely populated areas creating a lack of information for more remote regions where wildland fire-derived pollution can be high. For fire-specific pollution, these gaps in spatiotemporal coverage may result in air quality monitors missing peak fire smoke concentrations that we hypothesize can be an important feature of exposure. For monitored data, source attribution to fires requires extra steps. In contrast to general air pollution, the extent to which stationary monitors represent population exposures to fire-PM has additional uncertainties due to averting behaviors during a fire event, for example. The rapid deployment of air quality monitors during a fire incident introduces logistical challenges such as high cost, siting, remoteness and extent of fire locations, power source for operation, temperature conditions, and uncertainties related to fire movement. Despite these general challenges, California has been in the forefront of rapid deployment of monitors. Alternatively, low-cost and wearable monitors may provide supplemental information, but they require a large number of study participants, lack quality Atmosphere 2019, 10, 308 13 of 20 assurance, and lack of validation for health research [13]. Low-cost monitors can provide real-time supplemental field data which may be combined with other techniques. 4.2. Spatiotemporal Smoke Exposure Approaches Optical satellite imaging (e.g., the Multi-angle Imaging SpectroRadiometer–MISR; https://www-misr.jpl.nasa.gov/) has been used to complement atmospheric sensing to derive information on smoke plume structure, including assessing plume height and density and to validate plume rise and trajectory for smoke modeling. Smoke plume images provide an avenue to improve smoke models and for validating smoke dispersion information, but they do not provide surface level pollutant concentrations by themselves [82,83]. The real value of satellite sensing for air quality lies in its use for supporting atmospheric chemical transport models. The state-of-the-art now and into the future is the use of in-situ and satellite-based measurements of PM to adjust and calibrate well-vetted air quality models, such as CMAQ. 4.3. Strengths and Limitations Our study, using the U.S. EPA’s CMAQ platform, provides a comprehensive and consistent way of assessing exposure to fire-PM2.5 over broad space and time, for many years for the entire State of California. Using CMAQ allows us to include non-wildfire PM sources, provides full geographic and fine-scale spatiotemporal coverage (e.g., hourly output), includes robust atmospheric chemistry, and isolates fire-specific pollution from primary and atmospherically transformed emissions [42,43,45]. CMAQ has been compared against the routine surface PM monitoring network and field-deployed in situ monitoring data [14,43,65]. At a regional scale, CMAQ-modeled wildfire PM2.5 has been shown to compare well to both aircraft plume transects and remotely sensed aerosol optical depth [14], though in close proximity to wildland fires a study of two large wildfires showed a tendency toward overestimation of PM2.5 in comparison to a surface monitor network [45]. Model results are limited by the quality of the emission and meteorological inputs. Our model performance results indicate 14 of 20 Atmosphere 2019, 10, 308 the modeling system tends to overestimate the magnitude of wildfire impacts on PM2.5 at routine surface monitor locations. This was most evident for years with large wildfire impacts (e.g., 2007 and 2008) although the model underestimated impacts in 2009 and showed minimal for other years (2011 and 2012) which also had notable wildfire on the landscape (Table 4). Due to lack of robust techniques to quantitatively differentiate measured PM2.5 related to wildfire smoke compared from other sources at routine monitor locations, we have not fully evaluated the model for situations when smoke concentrations are present but the model predicts zero fire impact. Further advantages of our approach are that this modeling platform provides avenues to understand exposures based on the type of fire, the location of fire, and other aspects of pollution not available with air quality monitoring or satellite data alone. Using a model opens opportunities for exploring associations on the basis of place-based and population-based variables. It provides an avenue for assessing future fire scenarios by providing the data for predictive modeling, scenario-based planning, assignment of geocoded health cases to the gridded modeled air quality for epidemiologic assessment, and the vulnerability mapping of populations [67]. Our study has several limitations. A major limitation of estimating health-relevant received dose with atmospheric modeling is that the location and behavior patterns that affect exposure of the population are generally unknown. 4.3. Strengths and Limitations During a fire event, people may modify their behavior in ways that change the relationship of modeling estimates to air pollution dose in more routine situations – people may evacuate, close windows or spend less time outdoors. Regarding the counts of asthma emergency department visits, births and cardiac hospitalizations, we did not determine if the fire-specific concentrations preceded the event or if any associations existed. This analysis represents a first step in characterizing potential population exposures for further research. Another limitation is that our CMAQ modeling outputs were not corrected with in situ monitoring data or remote sensing data, which could reduce exposure misclassification [71]. However, given the sparse nature of routine ground measurements and assumptions required for remotely sensed data a simple approach for correcting or modulating the modeled fire predictions could introduce new errors both spatially and temporally. Current studies are underway to improve smoke modeling capability [84] and advance integration of data sources through machine learning methods. Wildland fire is a permanent and at times beneficial part of the landscape, and exposures are widespread in the U.S. Because of concerns about health risk from air pollution exposures, population exposure to fire-PM should be minimized as a precaution, especially for vulnerable populations. Atmospheric modeling can play a role in the further characterization of the relationships between fire-PM and health risks. 5. Conclusions Widespread and increasing population exposure to wildland fire smoke leads to an urgent need for new techniques to characterize fire-derived pollution for epidemiologic studies. Atmospheric chemical transport modeling is an approach that allows extensive exploration of exposure to fire emissions in space and time. Using CMAQ modeling with and without wildland fire emissions, we found widespread areas in California with fire-related PM2.5 concentrations and smokewave days. The 24-h average concentration of PM2.5 from all sources in 12-km gridded output from all sources in California (2007–2013) was 4.91 µg/m3 (standard deviation 4.04 µg/m3). The average concentration of fire-PM2.5 in California by year was 1.22 µg/m3 (standard deviation 3.78 µg/m3). This represents about a quarter of the total ambient PM2.5 concentrations. The fire-PM2.5 daily mean ranged from 0.31 µg/m3 (2010) to 4.40 µg/m3 in a high fire year (2008). Although this study focused on years in which the fires were largely in the northern portion of the state, fires and smokewaves occur throughout the state as a norm. Based on the model-derived fire-PM2.5 data, 97.4% of the population of California lived in a county that experienced at least one smokewave from 2007–2013, yet the impact on health of smoke is only beginning to be understood. The strengths of our modeling study include the state-of-the-art chemical transport model and full spatial and temporal coverage of fire-PM2.5 that cannot be obtained with 15 of 20 Atmosphere 2019, 10, 308 current in situ air quality monitoring, nor satellite sensing alone. Modeling can isolate and attribute wildland fire sources of PM (including secondarily formed PM) in order to aid causal inference in future health studies. Limitations include the need for a further validation of measurements, including satellite-based PM retrievals. Atmospheric modeling can provide data at a temporal and spatial scale needed to assess exposures for epidemiologic studies, which could be utilized in future work to understand more fully how multi-temporal and broad spatial-scale epidemiological impacts relate to wildland fire smoke exposure. Author Contributions: All authors contributed substantially to the manuscript. Specifically, the following roles were completed by these authors: conceptualization, P.D.K., N.H.F.F., S.H., and M.B.; methodology, P.D.K., M.B., B.J.T., S.H., and K.B.; software, K.R.B.; validation, K.R.B.; formal analysis, M.B., B.J.T., and R.d.M.; investigation, P.D.K.; resources, P.D.K., N.H.FF., K.R.B.; data curation, M.B. References 1. Westerling, A.L.; Hidalgo, H.G.; Cayan, D.R.; Swetnam, T.W. Warming and Earlier Spring Increase Western U.S. Forest Wildfire Activity. Science 2006, 313, 940–943. [CrossRef] [PubMed] 1. Westerling, A.L.; Hidalgo, H.G.; Cayan, D.R.; Swetnam, T.W. 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Comparative Analysis of the Subventricular Zone in Rat, Ferret and Macaque: Evidence for an Outer Subventricular Zone in Rodents
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Abstract nding: This work was supported by UC Davis MIND Institute; Children’s Miracle Network; UC Davis Department of Psychiatry; and undation. 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. Competing Interests: The authors have declared that no competing interests exist. * E-mail: scnoctor@ucdavis.edu (SCN); vmartinezcerdeno@ucdavis.edu (VMC) * E-mail: scnoctor@ucdavis.edu (SCN); vmartinezcerdeno@ucdavis.edu (VMC) . These authors contributed equally to this work. VZ. In this report we refer to primary precursor cells as radial glial (RG) cells and to secondary precursor cells as intermediate progenitor (IP) cells. RG cells and IP cells can be distinguished based on several characteristics including morphology and the expression of transcription factors. RG cells are bipolar cells that have a ventricular contacting process and a long thin pial process that ascends through the cortical plate to contact the pia via endfeet [2]. RG cells divide at the surface of the ventricle, retain their pial process during division [3–5], and express the transcription factor Pax6 [6,7]. In contrast IP cells are multipolar cells [3–5], which in rodent appear to retract all processes during division [4,5], largely divide away from the surface of the ventricle [5,8], and express the transcription factor Tbr2 [7]. Comparative Analysis of the Subventricular Zone in Rat, Ferret and Macaque: Evidence for an Outer Subventricular Zone in Rodents Vero´ nica Martı´nez-Cerden˜ o1,2,6*., Christopher L. Cunningham3., Jasmin Camacho2, Jared L. Antczak4, Anish N. Prakash5, Matthew E. Cziep4, Anita I. Walker4, Stephen C. Noctor3,5,6* 1 Department of Pathology and Laboratory Medicine, School of Medicine, University of California Davis, Sacramento, California, United States of America, 2 Institute for Pediatric Regenerative Medicine, Shriners Hospital for Children of Northern California, Sacramento, California, United States of America, 3 Neuroscience Graduate Program, University of California Davis, Davis, California, United States of America, 4 Department of Biology, Brigham Young University, Rexburg, Idaho, United States of America, 5 Department of Psychiatry, School of Medicine, University of California Davis, Sacramento, California, United States of America, 6 Medical Investigations of Neurodevelopmental Disorders (M.I.N.D.) Institute, School of Medicine, University of California Davis, Sacramento, California, United States of America Abstract The mammalian cerebral cortex arises from precursor cells that reside in a proliferative region surrounding the lateral ventricles of the developing brain. Recent work has shown that precursor cells in the subventricular zone (SVZ) provide a major contribution to prenatal cortical neurogenesis, and that the SVZ is significantly thicker in gyrencephalic mammals such as primates than it is in lissencephalic mammals including rodents. Identifying characteristics that are shared by or that distinguish cortical precursor cells across mammalian species will shed light on factors that regulate cortical neurogenesis and may point toward mechanisms that underlie the evolutionary expansion of the neocortex in gyrencephalic mammals. We immunostained sections of the developing cerebral cortex from lissencephalic rats, and from gyrencephalic ferrets and macaques to compare the distribution of precursor cell types in each species. We also performed time-lapse imaging of precursor cells in the developing rat neocortex. We show that the distribution of Pax6+ and Tbr2+ precursor cells is similar in lissencephalic rat and gyrencephalic ferret, and different in the gyrencephalic cortex of macaque. We show that mitotic Pax6+ translocating radial glial cells (tRG) are present in the cerebral cortex of each species during and after neurogenesis, demonstrating that the function of Pax6+ tRG cells is not restricted to neurogenesis. Furthermore, we show that Olig2 expression distinguishes two distinct subtypes of Pax6+ tRG cells. Finally we present a novel method for discriminating the inner and outer SVZ across mammalian species and show that the key cytoarchitectural features and cell types that define the outer SVZ in developing primates are present in the developing rat neocortex. Our data demonstrate that the developing rat cerebral cortex possesses an outer subventricular zone during late stages of cortical neurogenesis and that the developing rodent cortex shares important features with that of primates. Citation: Martı´nez-Cerden˜o V, Cunningham CL, Camacho J, Antczak JL, Prakash AN, et al. (2012) Comparative Analysis of the Subventricular Zone in Rat, Ferret and Macaque: Evidence for an Outer Subventricular Zone in Rodents. PLoS ONE 7(1): e30178. doi:10.1371/journal.pone.0030178 Editor: Branden Nelson, Seattle Children’s Research Institute, United States of America Received September 9, 2011; Accepted December 12, 2011; Published January 17, 2012 Copyright:  2012 Martı´nez-Cerden˜o 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: Martı´nez-Cerden˜o V, Cunningham CL, Camacho J, Antczak JL, Prakash AN, et al. (2012) Comparative Analysis of the Subventricular Zone in Rat, Ferret and Macaque: Evidence for an Outer Subventricular Zone in Rodents. PLoS ONE 7(1): e30178. doi:10.1371/journal.pone.0030178 Editor: Branden Nelson, Seattle Children’s Research Institute, United States of America Copyright:  2012 Martı´nez-Cerden˜o 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. PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 Introduction Pax6+ cells have been described as largely restricted to the VZ in rodents [7], while Pax6+ cells are located in both the VZ and the SVZ in the prenatal cerebral cortex of humans [10,11], and carnivores such as the ferret [11]. Similarly, Tbr2+ cells are described as largely restricted to the SVZ of rodents [7], but in the human neocortex Tbr2+ cells extend further from the ventricle into the oSVZ [10]. subtypes of tRG cells. We show that rats, ferrets and macaques each possess a dense inner band of Tbr2+ cells and a diffuse outer band of Tbr2+ cells. In the macaque these bands correspond precisely with the iSVZ and oSVZ. We also show that the distribution of Tbr2+ cells is more similar in the developing cortex of lissencephalic rats and gyrencephalic ferrets, than it is in the gyrencephalic cortices of macaque and ferret. The macaque exhibits a large shift in the distribution of Tbr2+ cells away from the ventricle, and this occurs much earlier during cortical neurogenesis in macaques than in rats or ferrets. Together these data suggest that the redistribution of Pax6+ and Tbr2+ precursor cells to the oSVZ that occurs in macaque may not be a prerequisite for the development of gyrencephalic cortex since it does not occur in ferret. We show that the inner and outer fiber layers, which can be used to distinguish the boundaries of the iSVZ and oSVZ in macaque visual cortex, are not apparent in somatosensory, motor or frontal cortex. We therefore present a new method for distinguishing the iSVZ and oSVZ in any cortical area that is based on unambiguous histological and immunohistochemical methods. Finally, we propose that the developing rat cerebral cortex has an oSVZ since it possesses the cell types and the cytoarchitectural elements that define the oSVZ in primates. [ ] The oSVZ in gyrencephalic mammals is not simply an expanded zone produced by increased numbers of IP cells, but is a distinct zone that includes both IP cells and RG cells that have translocated away from the surface of the ventricle. The presence of translocating RG cells in the developing cortex was initially reported over 30 years ago by Rakic [12]. The morphological transition of RG cells into translocating radial glial cells (tRG cells) has been demonstrated in macaque [12], ferret [13], mouse [14], and human [15]. Results The following developmental ages from each species were included in our study. Rats: embryonic day (E)13, E14, E17, E18, E20, E21, E22, postnatal day (P)1, P2, P3, P7 and P10. Mice: E18. Ferrets: E23, E28, E31, E34, E38, P2, P10. Macaques: E50, E65, E80, E100 and E151. These ages included the neurogenic phase beginning with the genesis of layer VI neurons through the genesis of layer II neurons, and the post-neurogenic phase of development in each species [19–21]. We analyzed coronal sections of somatosensory cortex except where noted. The number of cells counted for each analysis is included in Table format as noted. Introduction VZ. In this report we refer to primary precursor cells as radial glial (RG) cells and to secondary precursor cells as intermediate progenitor (IP) cells. RG cells and IP cells can be distinguished based on several characteristics including morphology and the expression of transcription factors. RG cells are bipolar cells that have a ventricular contacting process and a long thin pial process that ascends through the cortical plate to contact the pia via endfeet [2]. RG cells divide at the surface of the ventricle, retain their pial process during division [3–5], and express the transcription factor Pax6 [6,7]. In contrast IP cells are multipolar cells [3–5], which in rodent appear to retract all processes during division [4,5], largely divide away from the surface of the ventricle [5,8], and express the transcription factor Tbr2 [7]. Neurons of the mammalian cerebral cortex are primarily generated before birth during a period of intense precursor cell proliferation. The number of neurons in the human cortical plate increases by about 5 billion cells between the 13th and 20th weeks of gestation [1], which indicates that on average over 1000 neurons arrive in the CP every second during that seven week period of development. Further, this data suggests that roughly 500 to 1000 precursor cells divide every second to produce cortical neurons during this stage of development. Two principal classes of neural precursor cells have been identified in the developing brain. The primary class of precursor cells resides in the ventricular zone (VZ) adjacent to the lateral ventricle; the secondary class of precursor cells resides in the subventricular zone (SVZ) just superficial to the Rodent studies of cortical development have informed our understanding of mechanisms that regulate prenatal neurogenesis, PLoS ONE 1 January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org Mammalian Outer Subventricular Zone but recent work has highlighted differences in the development of the rodent and primate cerebral cortices. The SVZ in primates and other gyrencephalic mammals is subdivided into discrete cytoarchitectural regions that are called the inner SVZ (iSVZ) and outer SVZ (oSVZ), while the SVZ in rats and mice is a comparatively thinner structure [9]. Furthermore, the distribution of Pax6+ and Tbr2+ cells is reportedly different in rodents and primates. Introduction However, the functional nature of tRG cells had not been determined. Recent studies based on time-lapse imaging of fluorescently labeled cells in live slice cultures have found that tRG cells are present in rodents and play specific functional roles. Noctor and colleagues (2004, 2008), showed time-lapse movies of mitotic translocating RG cells in the embryonic rat neocortex [4,5], and performed whole-cell patch-clamp recordings of the tRG daughter cells to show that tRG cells produce daughter cells lacking neuronal properties [5]. On the other hand, Miyata and colleagues (2004), showed time-lapse movies of mitotic translocat- ing RG cells in mouse followed by immunohistochemistry and presented evidence that mitotic translocating cells produce daughter neurons based on the expression of the Hu protein [16]. More recent studies have provided evidence that tRG cells express Pax6 and produce daughter neurons in humans [10], and in mice based on expression of the neuronal marker NeuN [17], or lack of mitotic activity [18]. To further explore the characteristics and potential of RG cells, tRG cells, and IP cells in lissencephalic and gyrencephalic mammals and what role they play in the development of gyrencephaly, we compared the cytoarchitecture of the developing cortex in rats, ferrets and macaques, quantified the distribution of Pax6+ and Tbr2+ mitotic cells in each species, and performed time-lapse imaging of green fluorescent protein (GFP) - labeled precursor cells in the embryonic rat cerebral cortex. The distribution of mitotic precursor cells in the developing cortical wall is similar in rats and ferrets, but different in macaques To investigate the relative contributions of precursor cells in the VZ and SVZ to cortical neurogenesis, we first compared the distribution of mitotic cells in the developing cerebral cortex of rat, ferret and macaque. We identified all mitoses in the dorsal somatosensory cortex in Nissl stained sections from each species and each age encompassing the period of cortical neurogenesis and early stages of gliogenesis. Mitoses were assigned to the VZ, SVZ, iSVZ or oSVZ (when present), or preplate/subplate/ cortical plate/marginal zone. We included all mitoses in our analysis, but in this manuscript we focused our attention on precursor cells in the VZ and SVZ. We therefore combined into one bin all preplate, subplate, cortical plate and marginal zone mitoses, even though meaningful differences may exist in the properties of precursor cells within these distinct structures (e.g. [22]). The SVZ first appears during early stages of macaque and ferret cortical development, we label this structure the iSVZ. As development proceeds we distinguished between the iSVZ and oSVZ. We show that most dividing cells in the developing cerebral cortex express Pax6. We show that Pax6+ mitotic cells are located both at the surface of the ventricle and away from the ventricle in each species as described by others, but we note important differences between species. For example, in both lissencephalic rat and gyrencephalic ferret neocortex the majority of mitotic Pax6+ cells are located in the VZ throughout the neurogenic period. In contrast, the distribution of Pax6+ cells in macaque shifts away from the VZ to the oSVZ early in the neurogenic period. We show that Pax6+ translocating radial glial cells are present in the rat cerebral cortex, and that Pax6+ translocating cells are present in the neocortex of each species during and after the period of cortical neurogenesis, demonstrating that Pax6+ translocating cells are not restricted in function solely to neurogenesis. We further characterized Pax6+ cells by costaining for the transcription factors Sox2 and Olig2. Nearly all Pax6+ cells also express Sox2, whereas Olig2 expression distinguishes two The distribution of mitoses in the developing macaque somatosensory cortex during neurogenesis was dramatically different than that in rat or ferret. January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org The distribution of mitotic precursor cells in the developing cortical wall is similar in rats and ferrets, but different in macaques We found that the proportion of mitoses in this zone of developing rat cortex reached 24% by E20, during neurogenesis of layer two neurons. In addition, the number of divisions occurring in the subplate/cortical plate/ marginal zone began increasing during genesis of layer 2 neurons and reached 30% in rat and ferret in the post neurogenic developing cortex. In the developing ferret somatosensory cortex the majority of divisions remained in the VZ throughout most of the neurogenic period. At E23, the beginning of cortical neurogenesis [19], the percentage of cortical divisions located in the VZ was 96%. At E28 the percentage of mitoses within the VZ was 78%, and by E34, during the production of layer 4 neurons in somatosensory cortex [19], the majority of divisions were still located in the VZ (59%). By P2, when production of layer 2 neurons is nearly complete in ferret somatosensory cortex [19], the percentage of mitoses in the VZ decreased to 32%. At P10 when neurogenesis was complete in ferret somatosensory cortex [19], the percentage of VZ mitoses had decreased to 9% (Figs. 1b & e). These data demonstrate that the distribution of mitoses in the cortical wall is more similar in the lissencephalic rat and the gyrencephalic ferret, than the distribution of mitoses in the gyrencephalic cortices of ferret compared to that of the macaque. These data suggest that the large increase in the proportion of mitoses located away from the ventricle that occurs during early stages of macaque neurogenesis may not be a prerequisite for the In the developing rat somatosensory cortex the distribution of mitoses was very similar to that of ferret. At E13 in the rat 97% of divisions were in the VZ. At E17, which is the peak of neurogenesis for layer 4 neurons in rat somatosensory cortex [21], over 75% of all mitoses were still located in the VZ. At E21, Table 1. Distribution of mitotic cells during neocortical development. Macaque age VZ iSVZ oSVZ CP (incl. PP/SP/MZ) E50 95.6% (3603/3759) 4% (141/3759) 0.4% (15/3579) 0% (0/3579) E65 57% (2009/3511) 14.7% (515/3511) 28% (977/3511) 0.3% (10/3511) E80 16% (87/551) 18% (100/551) 44% (242/551) 22% (122/551) E100 4% (22/521) 6% (32/521) 42% (217/521) 48% (250/521) E151 0.2% (2/951) 1% (13/951) 41.8% (397/951) 57% (539/951) Ferret age VZ iSVZ oSVZ CP (incl. The distribution of mitotic precursor cells in the developing cortical wall is similar in rats and ferrets, but different in macaques We noted a large shift in the distribution of mitoses away from the VZ into the SVZ during early stages of cortical neurogenesis in macaque, but not in rat or PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 2 Mammalian Outer Subventricular Zone ferret (see Table 1 for number of mitoses analyzed). During early stages of cortical development, including stages before the appearance of the SVZ, over 95% of mitoses were located at the surface of the ventricle in each species. In the E50 macaque, at the start of cortical neurogenesis [20], 96% of mitoses were located in the VZ. By E65, during production of layers 5 and 6 neurons in the macaque visual cortex [20], the percentage of mitoses located in the VZ fell to 57%, and by E80, during production of layer 4 neurons [20], the number of mitoses located in the VZ decreased even further to only 16%. In contrast, the number of divisions that were located in the oSVZ of macaque reached 57% at this stage of development. At E100, when production of layer 2 neurons is nearly complete [20], only 4% of mitoses were located in the VZ (Figs. 1a & d). when the vast majority of layer 2 neurons in rat somatosensory cortex have been generated [21], the percentage of mitoses located in the VZ had decreased to 38% and the majority of divisions were located in the SVZ. By P3, when cortical neurogenesis is complete in rat somatosensory cortex [21], the percentage of VZ mitoses had dropped to 4% (Figs. 1c & f). Rat and ferret also shared a similar pattern in the number of mitoses away from the ventricle. The percentage of mitoses in the SVZ of the rat and in the iSVZ of the ferret began increasing at the onset of neurogenesis and reached a peak of ,30% during neurogenesis of layer 2 neurons. The proportion of divisions in the ferret oSVZ reached 29% by P2. During later stages of rat cortical development we noted that a substantial proportion of divisions occurred superficial to the SVZ, as defined by the Boulder Committee [23]. These divisions were located between the SVZ and the subplate. We have previously shown that some mitotic intermediate progenitor cells produce neurons in this zone [5]. The distribution of mitotic precursor cells in the developing cortical wall is similar in rats and ferrets, but different in macaques PP/SP/MZ) E23 96% (377/394) NA NA 4% (17/394) E28 78% (538/689) 20% (138/689) 2% (10/689) 0.4% (3/689) E31 70% (447/644) 24% (157/644) 5% (32/644) 1% (8/644) E34 59% (472/802) 28% (228/802) 11% (90/802) 2% (12/802) E38 37% (429/1151) 35% (401/1151) 27% (207/1151) 1% (14/1151) P2 32% (164/509) 24% (121/509) 29% (146/509) 15% (78/509) P10 19% (183/955) 12% (112/955) 40% (386/955) 29% (274/955) Rat age VZ iSVZ oSVZ CP (incl. PP/SP/MZ) E13 97% (305/315) NA NA 3% (10/315) E15 93% (441/472) 5% (23/472) NA 2% (8/472) E17 76% (694/912) 20% (185/912) 3.9% (32/912) 0.1% (1/912) E20 (4A4) 39% (145/368) 35% (127/368) 24% (88/368) 2% (8/368) P3 (4A4) 4% (23/520) 30% (155/520) 36% (186/520) 30% (156/520) Numbers represent the proportion of mitotic cells in each compartment of the developing somatosensory cortex in each species. Cells were counted in a minimum of seven coronal sections from each age in each species. Mitoses were identified by 4A4 immunoreactivity, or condensed chromatin using DAPI or Nissl staining. Numbers in parentheses indicate the total number of mitotic cells counted in each compartment for each age in each species. doi:10.1371/journal.pone.0030178.t001 Table 1. Distribution of mitotic cells during neocortical development. Numbers represent the proportion of mitotic cells in each compartment of the developing somatosensory cortex in each species. Cells were counted in a minimum of seven coronal sections from each age in each species. Mitoses were identified by 4A4 immunoreactivity, or condensed chromatin using DAPI or Nissl staining. Numbers in parentheses indicate the total number of mitotic cells counted in each compartment for each age in each species. doi:10.1371/journal.pone.0030178.t001 January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 3 Mammalian Outer Subventricular Zone rtical development is similar in ferrets and rats, but different in macaque caque, P2 ferret and E20 rat immunostained for phosphorylated vimentin (4A4, green ges are displayed at the same scale. There are a significant number of abventricular m igure 1. The distribution of mitoses during cortical development is similar in ferrets and rats, but different in macaques. (a oronal sections of somatosensory cortex from E80 macaque, P2 ferret and E20 rat immunostained for phosphorylated vimentin (4A4, green) to la ividing cells and counterstained with DAPI (blue). Images are displayed at the same scale. There are a significant number of abventricular mitoses PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e301 Figure 1. The distribution of Tbr2-expressing cells is similar in rats and ferrets, but different in macaques We noted a significant difference in the density of Tbr2+ cells in the dense inner band versus the diffuse outer band of the SVZ. At E65 Tbr2+ cells were concentrated at 25 cells per 2500 mm2 in the dense inner band, and approximately 10 cells per 2500 mm2 in the diffuse outer band of the SVZ (Fig. 3a). The density of Tbr2+ cells in the dense inner band and diffuse outer band were fairly constant throughout much of development. But by E100, at the end of cortical neurogenesis, the dense inner band was reduced in thickness and the density of Tbr2+ cells dropped in both the dense inner band and diffuse outer band (Fig. 3a, Table 3). q The transcription factors Pax6 and Tbr2 are commonly used to identify and to distinguish VZ and SVZ precursor cells [6,7]. To characterize SVZ precursor cells in lissencephalic and gyrence- phalic mammals we examined the distribution of Tbr2+ cells in each species. Previous reports have examined Tbr2 expression in lissencephalic rodents [7], and gyrencephalic mammals including ferrets [11], and humans [10]. We first qualitatively compared the expression patterns of Tbr2 in each species. The onset of Tbr2 expression occurred prior to the appearance of the SVZ in mammalian cortex. The first Tbr2 cells were dispersed through- out the VZ, but as development proceeded Tbr2+ cells were concentrated in a dense band superficial to the VZ, thus contributing to the formation of the SVZ [5]. The dense band of Tbr2 expression remained in the dorsal cortex of rat, ferret and macaque throughout most of cortical development, and soon after a diffuse outer band of Tbr2+ cells appeared superficial to the dense inner band. The dense inner band of Tbr2 expression was located within a cell dense proliferative region that surrounds the lateral ventricle during cortical development, and can be visualized in Nissl or DAPI stained tissue (Figs. 1 and 2). The diffuse outer band of Tbr2 expression was located in a cellular region characterized by lower cell density and by tangential or obliquely oriented streams of cells that give a striated or stippled appearance to this zone of the developing cortex. To understand the dynamics of SVZ precursor cell distribution during cortical development we quantified the distribution of Tbr2+ cells across neurogenic stages in the three species. The distribution of Tbr2-expressing cells is similar in rats and ferrets, but different in macaques We identified all Tbr2+ cells in 200 mm wide radial bins of somatosensory cortex that spanned from the ventricle to the pia in coronal sections from each species and quantified the proportion of immunopositive cells located in each lamina of the developing cortex. We found more similarities in the distribution of Tbr2+ cells in the cortex of the lissencephalic rat and gyrencephalic ferret, than we did in the distribution of Tbr2+ cells in the developing gyrencephalic cortices of ferret and macaque (see Table 2 for number of Tbr2+ cells analyzed). We next asked if the dense inner band and diffuse outer band of Tbr2+ cells correspond to the inner and outer SVZ. We compared the pattern of Tbr2 expression with cortical cytoarchitecture on adjacent Tbr2/DAPI and Nissl stained sections of the macaque neocortex in the occipital, parietal and frontal lobes. The inner and outer SVZ were identified following the conventions of Smart and colleagues, namely radial organization of cells in the oSVZ and more randomly organized cells in the iSVZ [9] (Fig. 4a). Side by side comparison of Nissl and Tbr2 staining in visual cortex confirmed that the dense inner band of Tbr2 expression corresponded to the iSVZ and that the diffuse outer band of Tbr2 expression corresponded to the oSVZ (Figs. 4a & b). We also noted that it was possible to distinguish the inner and outer SVZ, and the inner and outer fiber layers by examining the pattern of DAPI staining (Fig. 4c). The outer fiber layer (OFL) serves as the upper boundary of the SVZ in macaque visual cortex and can be visualized in both Nissl and DAPI stained tissue by the striking presence of radial streams of cells (Fig. 4). Tbr2 cells do not penetrate into the macaque OFL. The inner fiber layer of macaque visual cortex lies between the iSVZ and oSVZ, and can be visualized in DAPI or Nissl stained tissue by a marked decrease in cell density. In Tbr2 stained tissue the inner fiber layer can be visualized as a gap between the dense inner band and diffuse outer band of Tbr2 expression (Fig. 4b). However, in cortical areas rostral to visual cortex, the inner fiber layer was not apparent, there was no separation between the iSVZ and oSVZ, and no separation between the dense inner band and diffuse outer band of Tbr2+ cells (Fig. 5). The distribution of mitotic precursor cells in the developing cortical wall is similar in rats and ferrets, but different in macaques The stage of development is shown at the bottom of the graphs. The approximate cortical layer generated at each stage of development is indicated along the top of the macaque graph, and applies to each graph. (d) Early in macaque neurogenesis the majority of mitoses shifted from the ventricular zone (VZ) to abventricular locations. By E80, when layer 4 neurons are being generated [20], less than 20% of mitoses remained in the VZ and 57% were located in the outer subventricular zone (oSVZ). (e) The distribution of mitoses in the gyrencephalic ferret did not shift away from the VZ during early stages of neurogenesis as in macaque. At E34 during generation of layer 4 neurons [19], greater than half of all mitoses were still located in the VZ. (f) The distribution of mitoses in the lissencephalic rat cortex was similar to that in the gyrencephalic ferret cortex. At E21, which represents the genesis of layer 2 neurons in somatosensory cortex [21], 35% of mitoses were located superficial to the SVZ in a zone that we term the oSVZ. Legend indicates histological zones: VZ: blue; inner SVZ (iSVZ): red; oSVZ: green; cortical plate (CP)/preplate (PP)/subplate (SP)/marginal zone (MZ): purple. Scale bar in (a) applies to (a–c). doi:10 1371/journal pone 0030178 g001 development of gyrencephalic cortex since it does not occur in ferret. which corresponds to the end of neurogenesis for layer 2 cortical neurons [20], 91% of Tbr2+ cells were located in the diffuse outer band and only 7% of Tbr2+ cells remained in the dense inner band (Figs. 2a & d). PLoS ONE | www.plosone.org The distribution of mitotic precursor cells in the developing cortical wall is similar in rats and ferrets, but different in macaques The distribution of mitoses during cortical development is similar in ferrets and rats, but different in macaques. (a–c) Coronal sections of somatosensory cortex from E80 macaque, P2 ferret and E20 rat immunostained for phosphorylated vimentin (4A4, green) to label dividing cells and counterstained with DAPI (blue). Images are displayed at the same scale. There are a significant number of abventricular mitoses in Figure 1. The distribution of mitoses during cortical development is similar in ferrets and rats, but different in macaques. (a–c) Coronal sections of somatosensory cortex from E80 macaque, P2 ferret and E20 rat immunostained for phosphorylated vimentin (4A4, green) to label dividing cells and counterstained with DAPI (blue). Images are displayed at the same scale. There are a significant number of abventricular mitoses in January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 4 Mammalian Outer Subventricular Zone Mammalian Outer Subventricular Zone each species. (d–f) Graphs showing changes in the distribution of mitoses during development of the neocortex. The stage of development is shown at the bottom of the graphs. The approximate cortical layer generated at each stage of development is indicated along the top of the macaque graph, and applies to each graph. (d) Early in macaque neurogenesis the majority of mitoses shifted from the ventricular zone (VZ) to abventricular locations. By E80, when layer 4 neurons are being generated [20], less than 20% of mitoses remained in the VZ and 57% were located in the outer subventricular zone (oSVZ). (e) The distribution of mitoses in the gyrencephalic ferret did not shift away from the VZ during early stages of neurogenesis as in macaque. At E34 during generation of layer 4 neurons [19], greater than half of all mitoses were still located in the VZ. (f) The distribution of mitoses in the lissencephalic rat cortex was similar to that in the gyrencephalic ferret cortex. At E21, which represents the genesis of layer 2 neurons in somatosensory cortex [21], 35% of mitoses were located superficial to the SVZ in a zone that we term the oSVZ. Legend indicates histological zones: VZ: blue; inner SVZ (iSVZ): red; oSVZ: green; cortical plate (CP)/preplate (PP)/subplate (SP)/marginal zone (MZ): purple. Scale bar in (a) applies to (a–c). doi:10 1371/journal pone 0030178 g001 each species. (d–f) Graphs showing changes in the distribution of mitoses during development of the neocortex. January 2012 | Volume 7 | Issue 1 | e30178 The distribution of Tbr2-expressing cells is similar in rats and ferrets, but different in macaques In other cortical areas it was possible to distinguish the iSVZ from the oSVZ by examining the pattern of Nissl or DAPI staining. The boundary between the iSVZ and oSVZ can be discriminated as a very sharp border created by differences in cell density (Figs. 5a & b). The VZ, iSVZ and the Tbr2 dense inner band were located in the cell dense region that surrounds the lateral ventricle. The oSVZ was located in a region that has a lower cell density compared to the iSVZ, but a higher cell density than the overlying intermediate zone (Figs. 5a & c). We noted that the diffuse outer band of Tbr2 staining/oSVZ extended dorsally two to three times farther from the lateral ventricle in somatosensory cortex than it does in visual cortex (Figs. 4 and 5). The age of onset for Tbr2 expression in macaque has not yet been determined. At E50 in the macaque 96% of Tbr2+ cells were located in the dense band of Tbr2 expression. Histologically, the oSVZ was not discernible in the cortical wall at this point of macaque development. By E65, during generation of layers 5 and 6 [20], both the dense inner band and diffuse outer band of Tbr2 expression were apparent. We found that 54% of Tbr2+ cells were located in the dense inner band, and that 37% of Tbr2+ cells were located in the diffuse outer band. The proportion of Tbr2+ cells located in the diffuse outer band of macaque cortex increased as cortical development proceeded. At E80, the majority of Tbr2+ cells were located in the diffuse outer band (59%), and by E100, January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 5 Mammalian Outer Subventricular Zone n of Tbr2+ cells during cortical development is similar in ferrets and rats, but different in macaq sensory cortex from E80 macaque, P2 ferret and E20 rat immunostained for Tbr2 (red) and counterstained at the same scale. In each species a dense inner band of Tbr2+ cells was located within the cell dense zone su osone.org 6 January 2012 | Volume 7 | Issue 1 Figure 2. The distribution of Tbr2+ cells during cortical development is similar in ferrets and rats, but different in macaques. (a–c) Coronal sections of somatosensory cortex from E80 macaque, P2 ferret and E20 rat immunostained for Tbr2 (red) and counterstained with DAPI (blue). The distribution of Tbr2-expressing cells is similar in rats and ferrets, but different in macaques Images are displayed at the same scale. In each species a dense inner band of Tbr2+ cells was located within the cell dense zone surrounding January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 6 Mammalian Outer Subventricular Zone Mammalian Outer Subventricular Zone the lateral ventricle visualized with DAPI. The diffuse outer band of Tbr2 expression was located within a striated zone marked by streams of cells organized in tangential clusters. (d–f) Graphs showing changes in the distribution of Tbr2+ cells during development of the cerebral cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. (d) In macaque the distribution of Tbr2+ cells progressively shifted to the outer subventricular zone (oSVZ) during development. By E80, during genesis of layer 4 neurons [20], 59% of Tbr2+ cells were located in the oSVZ. (e) The distribution of Tbr2+ cells in the gyrencephalic ferret shifted to the oSVZ more slowly in comparison to macaque. At P2 during genesis of layer 2 neurons [19], 62% of Tbr2+ cells remained in the inner SVZ (iSVZ). (f) The distribution of Tbr2+ cells in the lissencephalic rat was similar to that of the gyrencephalic ferret. At E20 during genesis of upper layer neurons [21], the majority of Tbr2+ cells (59%) were located in the iSVZ, and 17% were located in the oSVZ. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green. Scale bar in (a) applies to (a–c). doi:10.1371/journal.pone.0030178.g002 distribution in the rat: a dense inner band of Tbr2-expressing cells during early stages of cortical development, and the addition of a diffuse outer band of Tbr2-expressing cells at later stages. As in macaque and ferret the border between the inner dense band and outer diffuse band of Tbr2 staining could be discriminated in DAPI stained tissue as a sharp border created by differences in cell density, but only after E17. At the earliest stages of rat cortical development the diffuse outer band was either not present or contained a very small proportion of the total number of Tbr2+ cells in radial bins of the dorsal cortex. The distribution of Tbr2-expressing cells is similar in rats and ferrets, but different in macaques At E17 only 6% of Tbr2+ cells were located in the diffuse outer band of the SVZ, but by E20, when the majority of layer 2 neurons have been generated [21], the percentage of Tbr2+ cells in the diffuse outer band of the SVZ rose to 17% (Figs. 2c & f). We quantified the density of Tbr2+ cells in the dense inner band and the diffuse outer band of the rat SVZ and found that it was similar to the density of Tbr2+ cells in ferret and macaque. The density of Tbr2+ cells was 25 to 35 cells per 2500 mm2 in the dense inner band of the SVZ and five to 10 cells per 2500 mm2 in the diffuse outer band of the SVZ (Fig. 3c & f). In ferret we also noted the presence of a dense inner band of Tbr2 expression during early stages of cortical development, and the addition of a diffuse outer band of Tbr2 expression at later stages. The dense inner band and diffuse outer band of Tbr2 expression in ferret also corresponded to the iSVZ and oSVZ. The boundary between the iSVZ and oSVZ in ferret could also be discriminated in DAPI staining as a sharp border created by high cell density in the iSVZ and lower cell density in the oSVZ. However, the developmental pattern of Tbr2 expression in ferret differed from that in macaque. Whereas the majority of Tbr2+ cells shifted to the oSVZ early in macaque development, in ferret the majority of Tbr2+ cells (.60%) remained in the iSVZ throughout the entire period of cortical neurogenesis, and a much smaller proportion of Tbr2+ cells were located in the oSVZ. In ferret the proportion of Tbr2+ cells located in the oSVZ was only 2% at E31, when layer 5 neurons are generated [19], 11% at E34, when layer 4 neurons are generated [19], and 20% at P2 (Figs. 2b & e), which corresponds to the end of cortical neurogenesis in ferret somatosensory cortex [19]. We quantified the density of Tbr2+ cells in the ferret iSVZ and oSVZ and found a similar trend to the density of Tbr2+ cells in the macaque: much greater density in the iSVZ compared to the oSVZ (Fig. 3b & e). The distribution of Tbr2-expressing cells is similar in rats and ferrets, but different in macaques In the rat cortex during late stages of development we observed a cluster of Tbr2+ cells in the cingulate cortex that were located within and superficial to callosal fibers (Fig. 6). These Tbr2+ cells were apparent at P3 (Fig. 6b) but no longer present at P10. We noted Tbr2+ cells located in the same position in ferret and Inner and outer subdivisions of the SVZ have not been described in rodents as they have for macaque and ferret. Nonetheless, we also noted two distinct patterns of Tbr2 Table 2. Distribution of Tbr2+ cells during neocortical development. Macaque age Total Tbr2+ cells Radial units VZ Tbr2+ dense inner band Tbr2+ diffuse outer band E50 267 3 4% (10/267) 93% (248/267) 3% (9/267) E65 720 3 9% (66/720) 50% (361/720) 41% (293/720) E80 1213 3 2% (29/1213) 36% (433/1213) 62% (751/1213) E100 520 3 2% (10/520) 8% (42/520) 90% (468/520) Ferret age Total Tbr2+ cells Radial units VZ Tbr2+ dense inner band Tbr2+ diffuse outer band E28 290 3 29% (84/290) 68% (196/290) 3% (10/290) E31 464 3 30% (140/464) 68% (317/464) 2% (7/464) E34 456 3 28% (128/456) 60% (275/456) 12% (53/456) P2 719 3 18% (129/719) 62% (444/719) 20% (146/719) P10 81 3 5% (4/81) 6% (5/81) 89% (72/81) Rat age Total Tbr2+ cells Radial units VZ Tbr2+ dense inner band Tbr2+ diffuse outer band E14 245 3 40% (97/245) 60% (148/245) NA E17 3439 16 46% (1464/3214) 48% (1545/3214) 6% (205/3214) E20 516 3 24% (123/516) 59% (304/516) 17% (89/516) P3 243 3 0.4% (1/243) 63.6% (154/243) 36% (88/243) Tbr2+ cells were counted in a minimum of three 200 mm wide radial units from each age in each species. Radial units stretched from the ventricle to the pial surface in dorsal somatosensory cortex. Numbers represent the average percentage of Tbr2+ cells in each compartment averaged across n radial units. Numbers in parentheses indicate the total number of Tbr2+ cells counted in each compartment at each age. doi:10.1371/journal.pone.0030178.t002 Table 2. Distribution of Tbr2+ cells during neocortical development. January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 7 Mammalian Outer Subventricular Zone Figure 3. Density of Tbr2+ cells in the dense inner band and diffuse outer band of macaque, ferret and rat. (a–c) Images taken f coronal sections of E65 macaque, P2 ferret and E20 rat immunostained for Tbr2 (red) and counterstained with DAPI (blue). The distribution of Tbr2-expressing cells is similar in rats and ferrets, but different in macaques This analysis showed that there was a similar number of cells in the iSVZ of each species per 200 mm radial bin (100 to 150 cells), but that macaque possessed a far greater number of Tbr2+ cells in the oSVZ than either rats or ferrets (Fig. 7). Furthermore, the thickness of the Tbr2 diffuse band/oSVZ in developing somatosensory cortex in macaque was much greater than in either rat or ferret (Fig. 8, Table 5). At E50 in the macaque 89% of Pax6+ cells were located in the VZ. However, the distribution of Pax6+ cells in macaque quickly shifted away from the ventricle during early stages of develop- ment. By E65, during production of neurons for layers 5 and 6, only 29% of Pax6+ cells remained in the VZ while approximately 67% of Pax6+ cells had shifted to the iSVZ and oSVZ, with the majority of Pax6+ cells (57%) located in the oSVZ. At E80 the percentage of Pax6+ cells in the macaque VZ decreased to 21% while the percent in the iSVZ and oSVZ had increased to 75% (Figs. 9a & d). The proportion of Tbr2+ cells located in the diffuse outer band of the SVZ, or oSVZ, at the end of cortical neurogenesis was similar in ferret (20%) and rat (17%), but dramatically higher in macaque (.60%). These results provide two notable findings. First, the shift in the distribution of Tbr2+ cells to the outer SVZ that occurred in the macaque did not occur in the gyrencephalic ferret, suggesting that this cellular behavior is not a prerequisite for the development of gyrencephalic cortex. Second, similar to gyrencephalic ferret and macaque, lissencephalic rat cortex possesses a diffuse outer band of Tbr2+ cells during late stages of cortical development. At early stages of cortical development in rats and ferrets the distribution of Pax6+ cells was similar to that in macaques with the majority of Pax6+ cells located in the VZ. However, the majority of Pax6+ cells remained in the VZ throughout neurogenesis in rat and ferret. At the beginning of neurogenesis, over 90% of all Pax6+ cells were located in the VZ in both species. At the end of cortical neurogenesis when layer 2 neurons were being produced, 64% of Pax6+ cells were still located in the VZ of the E20 rat somatosensory cortex (Figs. The distribution of Tbr2-expressing cells is similar in rats and ferrets, but different in macaques Macaque age Total Tbr2+ cells counted Density of dense inner band Density of diffuse outer band E50 286 32 NA E65 784 25 12 E80 322 21 15 E100 157 13 5 Ferret age Total Tbr2+ cells counted Density of dense inner band Density of diffuse outer band E28 280 31 NA E31 246 27 NA E34 423 36 4 E38 390 29 6 P2 535 28 6 Rat age Total Tbr2+ cells counted Density of dense inner band Density of diffuse outer band E14 208 23 NA E17 307 27 NA E18 138 36 5 E20 803 35 6 Density measurements are average number of Tbr2+ cells in the dense inner band and diffuse outer band counted in at least three 2500 mm2 regions of interest at each age in each species. Bands were identified by the pattern of Tbr2 immunoreactivity. doi:10.1371/journal.pone.0030178.t003 Density measurements are average number of Tbr2+ cells in the dense inner band and diffuse outer band counted in at least three 2500 mm2 regions of interest at each age in each species. Bands were identified by the pattern of Tbr2 immunoreactivity. doi:10.1371/journal.pone.0030178.t003 Density measurements are average number of Tbr2+ cells in the dense inner band and diffuse outer band counted in at least three 2500 mm2 regions of interest at each age in each species. Bands were identified by the pattern of Tbr2 immunoreactivity. doi:10.1371/journal.pone.0030178.t003 macaque cingulate cortex (Figs. 6c & d). The supracallosal Tbr2+ cells in ferret and macaque appeared to be oSVZ cells that were separated from the underlying precursor cells by growing callosal axons. The position of Tbr2+ cells in the rat cingulate cortex appeared remarkably similar to that in ferret and macaque. revealed a similar pattern. Pax6 staining initially appeared in a dense band in the VZ, and as development proceeded an additional diffuse band of Pax6+ cells appeared in the SVZ. As with Tbr2 expression, we found more similarities in the distribution pattern of Pax6+ cells in the developing cortex of lissencephalic rat and gyrencephalic ferret, than we did between gyrencephalic ferret and macaque cortices (see Table 6 for number of Pax6+ cells analyzed). We compared the total number of Tbr2+ cells in each lamina of the developing cortex of each species within 200 mm wide radial sections (see Table 4 for numbers). The distribution of Tbr2-expressing cells is similar in rats and ferrets, but different in macaques 9b & e) and 52% of Pax6+ cells remained in the VZ of the P2 ferret somatosensory cortex (Figs. 9c & f). In both rat and ferret the proportion of Pax6+ cells in the VZ dropped rapidly after neurogenesis was complete. The distribution of Tbr2-expressing cells is similar in rats and ferrets, but different in macaques The dense inner band diffuse outer band of Tbr2+ cells are indicated. (d–f) Histograms displaying the number of Tbr2+ cells per 2500 mm2 in the dense inner band (b and diffuse outer band (red) for each species. The diffuse outer band of Tbr2+ cells appears at an early age in macaque in comparison to ferret rat. In macaque the diffuse outer band was present at E65 during generation of deep layer neurons while the diffuse outer band first appeare later stages of development in ferret and rat. Nonetheless, the relative density of Tbr2+ cells in the dense and diffuse bands was similar in species. Scale bar in (a) applies to (a–c). doi:10.1371/journal.pone.0030178.g003 Figure 3. Density of Tbr2+ cells in the dense inner band and diffuse outer band of macaque, ferret and rat. (a–c) Images taken from coronal sections of E65 macaque, P2 ferret and E20 rat immunostained for Tbr2 (red) and counterstained with DAPI (blue). The dense inner band and diffuse outer band of Tbr2+ cells are indicated. (d–f) Histograms displaying the number of Tbr2+ cells per 2500 mm2 in the dense inner band (blue) and diffuse outer band (red) for each species. The diffuse outer band of Tbr2+ cells appears at an early age in macaque in comparison to ferret and rat. In macaque the diffuse outer band was present at E65 during generation of deep layer neurons while the diffuse outer band first appeared at later stages of development in ferret and rat. Nonetheless, the relative density of Tbr2+ cells in the dense and diffuse bands was similar in each species. Scale bar in (a) applies to (a–c). doi:10.1371/journal.pone.0030178.g003 January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 8 Mammalian Outer Subventricular Zone Table 3. Density of Tbr2+ cells per 2500 mm2 in dense inner band and diffuse outer band. The distribution of Pax6-expressing cells is similar in rats and ferrets, but different in macaques Pax6 is expressed by VZ precursor cells in the dorsal cortex, and recent work has described Pax6+ cells in the SVZ of primates and ferrets [10,11]. We quantified the distribution of Pax6+ cells in the developing cortex during neurogenesis in each species. We quantified the number of Pax6+ cells in 200 mm wide radial bins of somatosensory cortex and compared Pax6 expression between species following the method described above. Qualitative assessment of Pax6 staining in each species We compared the total number of Pax6+ cells in each lamina of the developing cortex within 200 mm wide radial bins. This analysis showed that there were similar numbers of Pax6+ cells in the VZ and in the iSVZ per 200 mm wide bin in each species. However, macaque possessed a far greater number of Pax6- expressing cells in the oSVZ (Fig. 10, Table 7). PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 9 Mammalian Outer Subventricular Zone Figure 5. Inner and outer fiber layers are not apparent in macaque cortical areas rostral to the occipital lobe. (a) Nissl stained coronal section of E80 macaque frontal lobe at the level of the genu of the corpus callosum (CC). (b–c) An adjacent section taken from the E80 macaque frontal lobe stained with DAPI (blue) and Tbr2 (red). (b) DAPI staining highlights the different cell densities within the inner subventricular zone (iSVZ) and outer SVZ (oSVZ). The cell dense region surrounding the lateral ventricle includes the very thin ventricular zone and the iSVZ. Tangential streams of cells can be visualized within the oSVZ in DAPI staining. Inner and outer fiber layers are not apparent. (c) The same section showing Tbr2 (red) and DAPI. As in visual cortex, the dense inner band of Tbr2 cells corresponds to the iSVZ and the diffuse outer band of Tbr2+ cells corresponds to the oSVZ. Scale bar in (c) applies to (b). IC, internal capsule. doi:10.1371/journal.pone.0030178.g005 Figure 4. The dense inner band of Tbr2+ cells corresponds to the inner subventricular zone (iSVZ) and the diffuse outer band of Tbr2+ cells corresponds to the outer SVZ (oSVZ) in macaque visual cortex. (a) Nissl stained E80 macaque visual cortex. (b) An adjacent section immunostained for Tbr2 (red). The dense inner band and diffuse outer band are indicated. The distribution of Pax6-expressing cells is similar in rats and ferrets, but different in macaques quantified the proportion of mitotic cells that express 4A4 during cortical neurogenesis and compared that to the proportion of mitotic cells that express phosphohistone H3 (PH3) in rat. We double immunostained sections of embryonic rat cortical tissue with 4A4 and PH3 antibodies and counterstained the tissue with DAPI to label all nuclei. We identified mitoses based on the pattern of DAPI-labeled chromatin and then determined whether they expressed 4A4 or PH3. Using this straightforward approach one can readily distinguish interphase cells with dispersed chromatin from M-phase cells in prophase, metaphase, anaphase and telophase, which have condensed chromatin. We included all cells in prophase through telophase for analysis. We analyzed over 100 mitoses in the embryonic rat at E15, E16, E17 and E18, including both surface and non-surface dividing cells. All mitoses The distribution of Pax6-expressing cells is similar in rats and ferrets, but different in macaques (c) Image of the same adjacent section showing Tbr2 (red) and DAPI stain (blue). The different compartments, such as the outer fiber layer (OFL) can be visualized with DAPI stain. VZ, ventricular zone; IFL, inner fiber layer. Scale bar in (b) applies to (a–c). doi:10.1371/journal.pone.0030178.g004 Figure 4. The dense inner band of Tbr2+ cells corresponds to the inner subventricular zone (iSVZ) and the diffuse outer band of Tbr2+ cells corresponds to the outer SVZ (oSVZ) in macaque visual cortex. (a) Nissl stained E80 macaque visual cortex. (b) An adjacent section immunostained for Tbr2 (red). The dense inner band and diffuse outer band are indicated. (c) Image of the same adjacent section showing Tbr2 (red) and DAPI stain (blue). The different compartments, such as the outer fiber layer (OFL) can be visualized with DAPI stain. VZ, ventricular zone; IFL, inner fiber layer. Scale bar in (b) applies to (a–c). doi:10.1371/journal.pone.0030178.g004 Figure 5. Inner and outer fiber layers are not apparent in macaque cortical areas rostral to the occipital lobe. (a) Nissl stained coronal section of E80 macaque frontal lobe at the level of the genu of the corpus callosum (CC). (b–c) An adjacent section taken from the E80 macaque frontal lobe stained with DAPI (blue) and Tbr2 (red). (b) DAPI staining highlights the different cell densities within the inner subventricular zone (iSVZ) and outer SVZ (oSVZ). The cell dense region surrounding the lateral ventricle includes the very thin ventricular zone and the iSVZ. Tangential streams of cells can be visualized within the oSVZ in DAPI staining. Inner and outer fiber layers are not apparent. (c) The same section showing Tbr2 (red) and DAPI. As in visual cortex, the dense inner band of Tbr2 cells corresponds to the iSVZ and the diffuse outer band of Tbr2+ cells corresponds to the oSVZ. Scale bar in (c) applies to (b). IC, internal capsule. doi:10.1371/journal.pone.0030178.g005 These data show that the distribution of Pax6+ cells in the developing cortex of two gyrencephalic mammals is dramatically different. In macaque the distribution of Pax6+ cells shifts away from the ventricle into the oSVZ at an early stage of neurogenesis, but the majority of Pax6+ cells remain in the VZ of the gyrencephalic ferret and in the lissencephalic rat throughout neurogenesis. This finding supports the idea that the shift of Pax6+ cells to the oSVZ is not a prerequisite for the development of gyrencephalic cortex. PLoS ONE | www.plosone.org The distribution of Tbr2+ mitoses shifts to the oSVZ during early stages of cortical development in macaque, but not in ferret or rat We next investigated where Pax6+ or Tbr2+ cells undergo division during cortical development. For this purpose we needed a marker to unequivocally identify all cortical mitoses. We previously used the anti-phosphorylated vimentin antibody (4A4, [24]) in rat [25], and human [26], and found that it labels all mitoses in the proliferative zones of the prenatal cerebral cortex [25]. In this study we retested whether 4A4 would reliably label all mitotic cells in the developing cerebral cortex of each species. We PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 10 Mammalian Outer Subventricular Zone Figure 6. Tbr2+ cells are located superficial to the developing white matter near the corpus callosum (CC) in the prenatal medial cortex of macaque, ferret and rat. (a–d) Coronal sections from rat, ferret, and macaque immunostained for Tbr2 (red) and counterstained with DAPI (blue). (a) Image from E20 rat showing clusters of Tbr2+ cells superficial to developing callosal fibers along the medial wall of the cortex (arrowheads). (b) Image from P3 rat showing an isolated pocket of Tbr2+ cells (arrowheads) located deep within the developing cingulate cortex (Cg Cx) superficial to the developing white matter. (c) Tbr2+ cells located superficial to the developing callosal fibers along the medial wall of the P2 ferret cortex (arrowheads). (d) Tbr2+ cells positioned above callosal fibers (arrowheads) in the cingulate cortex of E80 macaque. The supracallosal Tbr2+ cells appear to be oSVZ cells that were separated from underlying precursor cells by growing callosal axons. LV, lateral ventricle. doi:10.1371/journal.pone.0030178.g006 precursor cells actively migrate throughout the developing cortex (Fig. S1). We next examined where Tbr2+ cells undergo division in the developing cortex. Sections of neocortical tissue were stained with 4A4 and Tbr2 antibodies, and counterstained with DAPI. We analyzed the distribution of Tbr2+ mitoses within a 300 mm wide radial bin of cerebral cortex stretching from the ventricle to the pial surface at several developmental stages in each species. We performed analysis through two approaches. For approach 1 we quantified the proportion of mitotic cells that expressed Tbr2 in each layer of the developing cortex (i.e.: what percentage of mitoses in the VZ express Tbr2, see Table 9 for number of cells analyzed). The distribution of Tbr2+ mitoses shifts to the oSVZ during early stages of cortical development in macaque, but not in ferret or rat For approach 2 we quantified the percentage of Tbr2+ mitoses that were located in each zone of the developing cortex expressed as a proportion of the total number of Tbr2+ mitoses per radial bin (i.e.: of all Tbr2+ mitoses in the cortex, what proportion are located in the VZ, what proportion are located in the SVZ, etc., see Table 10 for number of cells analyzed). The first approach revealed that greater than 80% of mitotic cells in the dense inner band of the SVZ in rat and ferret expressed Tbr2, and that a lower percentage of mitotic cells in the diffuse outer band of the SVZ expressed Tbr2 during peak neurogenic stages (approx- imately 30–40%). The percentage of mitotic cells that expressed Tbr2 in the iSVZ was lower in the macaque (,50%) than in rat or ferret (.80%), but the percentage of mitoses that expressed Tbr2 in macaque oSVZ was comparable to that in the diffuse outer band of rat and ferret (Table 9). To better understand the radial distribution of Tbr2+ mitoses across development, we adopted approach 2 and focused our analysis on the distribution of Tbr2+ mitoses in each zone expressed as a proportion of the total number of Tbr2+ mitoses in a 300 mm wide radial bin of the neocortex. In macaque we found that 100% of Tbr2 mitoses were located in the iSVZ at E50. However, the distribution of Tbr2+ mitoses quickly shifted away from the ventricle and by E80 only 21% remained in the iSVZ while 76% were located in the oSVZ (Fig. 11a & d). Figure 6. Tbr2+ cells are located superficial to the developing white matter near the corpus callosum (CC) in the prenatal medial cortex of macaque, ferret and rat. (a–d) Coronal sections from rat, ferret, and macaque immunostained for Tbr2 (red) and counterstained with DAPI (blue). (a) Image from E20 rat showing clusters of Tbr2+ cells superficial to developing callosal fibers along the medial wall of the cortex (arrowheads). (b) Image from P3 rat showing an isolated pocket of Tbr2+ cells (arrowheads) located deep within the developing cingulate cortex (Cg Cx) superficial to the developing white matter. (c) Tbr2+ cells located superficial to the developing callosal fibers along the medial wall of the P2 ferret cortex (arrowheads). (d) Tbr2+ cells positioned above callosal fibers (arrowheads) in the cingulate cortex of E80 macaque. PLoS ONE | www.plosone.org The distribution of Tbr2+ mitoses shifts to the oSVZ during early stages of cortical development in macaque, but not in ferret or rat The supracallosal Tbr2+ cells appear to be oSVZ cells that were separated from underlying precursor cells by growing callosal axons. LV, lateral ventricle. doi:10.1371/journal.pone.0030178.g006 In the E31 ferret, when layer 5 neurons are generated [19], 100% of Tbr2+ mitoses were located in the inner SVZ. The proportion of Tbr2+ mitoses located in the ferret inner SVZ (iSVZ) did not decrease dramatically across development as occurred in the macaque. By P2 71% of Tbr2+ mitoses in the ferret were still located in the iSVZ. We first noted Tbr2+ mitoses in the diffuse outer band of the SVZ/oSVZ at E34, during genesis of layer 4 neurons [19], when 11% of Tbr2+ mitoses were located in the oSVZ. The proportion of Tbr2+ mitoses in the ferret outer SVZ rose to 29% by P2, when layer 2 neurons are generated [19] (Fig. 11b & e). The developmental distribution of Tbr2+ mitoses in the rat was similar to that in ferret. At E14 the SVZ had not formed as a recognizable structure, but Tbr2+ mitoses were present and 93% of the Tbr2+ mitoses were located in the upper portion of the VZ at this age. At E17 87.5% of Tbr2+ mitoses were located in the dense inner band of the SVZ and the remaining 12.5% were located in the diffuse outer band of the SVZ. At E20 the proportion of Tbr2+ mitoses located in the dense inner band was 73%, while the proportion of Tbr2+ mitoses located in the diffuse outer band of the SVZ grew to 24%. We noted that Tbr2+ mitoses were located up to 300 mm from the ventricle, which corresponds to the upper boundary of the diffuse outer band. After cortical neurogenesis at P3, the proportion of Tbr2+ mitoses located in the diffuse outer band fell to approximately 9% (Fig. 11c & f). m in rat cortex were strongly labeled with 4A4, but only ,70% were labeled with the PH3 antibody. The lower proportion of PH3+ mitoses most likely reflects the lack of PH3 labeling in telophase cells [26]. We quantified the proportion of ferret and macaque cortical mitoses that express 4A4 and found that 100% of surface and non-surface mitoses were 4A4+ in both species throughout the period of neurogenesis. January 2012 | Volume 7 | Issue 1 | e30178 The distribution of Tbr2+ mitoses shifts to the oSVZ during early stages of cortical development in macaque, but not in ferret or rat At E17 only 37% of Pax6+ mitoses were located in the dense inner and diffuse outer subdivisions of the SVZ, but near the end of neurogenesis at E20 this proportion rose to 58% (Fig. 12c & f). The distribution of Tbr2+ mitoses shifts to the oSVZ during early stages of cortical development in macaque, but not in ferret or rat Since 4A4 labeling is apparent through a broader portion of M-phase it labels a larger cell population, and since 4A4 more clearly labels the somal outline of mitotic cells we used 4A4 to label cortical precursor cells for analysis of protein and transcription factor expression by precursor cells in each species (see Table 8 for number of 4A4+ precursor cells analyzed). in rat cortex were strongly labeled with 4A4, but only ,70% were labeled with the PH3 antibody. The lower proportion of PH3+ mitoses most likely reflects the lack of PH3 labeling in telophase cells [26]. We quantified the proportion of ferret and macaque cortical mitoses that express 4A4 and found that 100% of surface and non-surface mitoses were 4A4+ in both species throughout the period of neurogenesis. Since 4A4 labeling is apparent through a broader portion of M-phase it labels a larger cell population, and since 4A4 more clearly labels the somal outline of mitotic cells we used 4A4 to label cortical precursor cells for analysis of protein and transcription factor expression by precursor cells in each species (see Table 8 for number of 4A4+ precursor cells analyzed). We observed tangential and ventricular oriented 4A4+ mitotic cells in the SVZ of each species, as we previously noted in the embryonic mouse [27]. These 4A4+ mitotic cells were Pax6+ and many had a single process up to 100 mm long that resembled the leading process of a migrating neuron, suggesting that some These data demonstrate that the lissencephalic rat and the gyrencephalic ferret share similar developmental distribution patterns of Tbr2+ mitoses, with the proportion of Tbr2+ mitoses January 2012 | Volume 7 | Issue 1 | e30178 11 Mammalian Outer Subventricular Zone Table 4. Average number of Tbr2+ cells in the VZ, iSVZ and oSVZ/200 mm radial unit during neocortical development. Table 4. Average number of Tbr2+ cells in the VZ, iSVZ and oSVZ/200 mm radial unit during neocortical development. The distribution of Pax6+ mitoses shifts to the oSVZ during early stages of cortical development in macaque, but not in ferret or rat We next examined the distribution of Pax6+ mitoses in the developing rat. As in the ferret we noted a steady increase in the proportion of Pax6+ mitoses that occurred away from the ventricle in the dense inner band and diffuse outer band of the SVZ as development proceeded. At E17 only 37% of Pax6+ mitoses were located in the dense inner and diffuse outer subdivisions of the SVZ, but near the end of neurogenesis at E20 this proportion rose to 58% (Fig. 12c & f). but not in ferret or rat We next examined where Pax6-expressing cells undergo division during cortical development. We stained cortical tissue with 4A4 and Pax6 antibodies and counterstained with DAPI (see Tables 11 and 12 for the number of Pax6+ mitotic cells analyzed). We found that Pax6 was expressed by 100% of surface dividing precursor cells and by the overwhelming majority of all mitotic cells undergoing division away from the ventricle in each species. In the macaque 100% of surface mitoses were Pax6+ at each age and a smaller proportion of mitotic cells expressed Pax6 away from the ventricle, particularly at later stages of development. We examined the distribution of Pax6+ mitoses in macaque and found that the distribution of Pax6+ mitoses shifted away from the ventricle as development proceeded. At E50 80% of Pax6+ mitoses were located in the VZ, but by E65, during production of layer 5 and 6 neurons [20], the majority (53%) were located in the iSVZ and oSVZ. The shift in the distribution of Pax6+ mitoses from the VZ to the SVZ continued through development. By E80 the proportion of Pax6+ mitoses in the SVZ was 79%, and at E100 93% of Pax6+ mitoses were located in the SVZ, with the majority of Pax6+ mitotic precursor cells (79%) located in the oSVZ (Fig. 12a & d). We next examined where Pax6-expressing cells undergo division during cortical development. We stained cortical tissue with 4A4 and Pax6 antibodies and counterstained with DAPI (see Tables 11 and 12 for the number of Pax6+ mitotic cells analyzed). We found that Pax6 was expressed by 100% of surface dividing precursor cells and by the overwhelming majority of all mitotic cells undergoing division away from the ventricle in each species. Mitotic precursor cells coexpress Tbr2 and Pax6 in the oSVZ The distribution of Tbr2+ mitoses shifts to the oSVZ during early stages of cortical development in macaque, but not in ferret or rat Macaque age Average Tbr2+ cells/200 mm bin Radial units VZ iSVZ oSVZ E50 89 3 3 83 3 E65 240 3 22 120 98 E80 404 3 10 144 250 E100 173 2 3 14 156 Ferret age Average Tbr2+ cells/200 mm bin Radial units VZ iSVZ oSVZ E28 97 3 28 66 3 E31 155 3 47 106 2 E34 152 3 43 92 17 P2 240 2 43 148 49 P10 27 3 1 2 24 Rat age Average Tbr2+ cells/200 mm bin Radial units VZ iSVZ oSVZ E14 82 3 32 50 0 E17 202 12 92 97 13 E20 172 3 41 101 30 P3 81 3 0 51 30 Numbers represent the average number of Tbr2+ cells in each compartment of developing somatosensory cortex per radial unit averaged across n radial units. Cells were counted in a minimum of two 200 mm wide radial units from each age in each species. Cells were identified through Tbr2 immunoreactivity. doi:10.1371/journal.pone.0030178.t004 Numbers represent the average number of Tbr2+ cells in each compartment of developing somatosensory cortex per radial unit averaged across n radial units. Cells were counted in a minimum of two 200 mm wide radial units from each age in each species. Cells were identified through Tbr2 immunoreactivity. doi:10.1371/journal.pone.0030178.t004 Pax6+. We noted a steady increase in the proportion of Pax6+ divisions that occurred away from the ventricle in the iSVZ and oSVZ as development proceeded. However, in comparison to macaque, the shift in the distribution of Pax6+ mitoses away from the ventricle in the ferret occurred more slowly, at a later stage of cortical development and was not as pronounced. At E34, during production of layer 4 neurons [19], the majority of Pax6+ mitoses (53%) were located in the iSVZ and oSVZ. At P2, near the end of neurogenesis in ferret somatosensory cortex [19], the proportion of Pax6+ mitoses in the iSVZ and oSVZ reached 66% (Fig. 12b & e). We next examined the distribution of Pax6+ mitoses in the developing rat. As in the ferret we noted a steady increase in the proportion of Pax6+ mitoses that occurred away from the ventricle in the dense inner band and diffuse outer band of the SVZ as development proceeded. PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 The distribution of Tbr2+ mitoses shifts to the oSVZ during early stages of cortical development in macaque, but not in ferret or rat At E17 only 37% of Pax6+ mitoses were located in the dense inner and diffuse outer subdivisions of the SVZ, but near the end of neurogenesis at E20 this proportion rose to 58% (Fig. 12c & f). Pax6+. We noted a steady increase in the proportion of Pax6+ divisions that occurred away from the ventricle in the iSVZ and oSVZ as development proceeded. However, in comparison to macaque, the shift in the distribution of Pax6+ mitoses away from the ventricle in the ferret occurred more slowly, at a later stage of cortical development and was not as pronounced. At E34, during production of layer 4 neurons [19], the majority of Pax6+ mitoses (53%) were located in the iSVZ and oSVZ. At P2, near the end of neurogenesis in ferret somatosensory cortex [19], the proportion of Pax6+ mitoses in the iSVZ and oSVZ reached 66% (Fig. 12b & e). located in the outer SVZ reaching a maximum of approximately 25% during neurogenesis of layer 2 cells. In contrast, a much larger proportion of Tbr2+ mitoses (.85%) were located in the macaque oSVZ during neurogenesis of layer 2 neurons. These data are consistent with the idea that the shift in the distribution of Tbr2+ mitoses to the oSVZ that occurs in macaque is not a prerequisite for the development of gyrencephalic cortex since it does not occur in ferret. Pax6+. We noted a steady increase in the proportion of Pax6+ divisions that occurred away from the ventricle in the iSVZ and oSVZ as development proceeded. However, in comparison to macaque, the shift in the distribution of Pax6+ mitoses away from the ventricle in the ferret occurred more slowly, at a later stage of cortical development and was not as pronounced. At E34, during production of layer 4 neurons [19], the majority of Pax6+ mitoses (53%) were located in the iSVZ and oSVZ. At P2, near the end of neurogenesis in ferret somatosensory cortex [19], the proportion of Pax6+ mitoses in the iSVZ and oSVZ reached 66% (Fig. 12b & e). We next examined the distribution of Pax6+ mitoses in the developing rat. As in the ferret we noted a steady increase in the proportion of Pax6+ mitoses that occurred away from the ventricle in the dense inner band and diffuse outer band of the SVZ as development proceeded. Mitotic precursor cells coexpress Tbr2 and Pax6 in the oSVZ In the macaque 100% of surface mitoses were Pax6+ at each age and a smaller proportion of mitotic cells expressed Pax6 away from the ventricle, particularly at later stages of development. We examined the distribution of Pax6+ mitoses in macaque and found that the distribution of Pax6+ mitoses shifted away from the ventricle as development proceeded. At E50 80% of Pax6+ mitoses were located in the VZ, but by E65, during production of layer 5 and 6 neurons [20], the majority (53%) were located in the iSVZ and oSVZ. The shift in the distribution of Pax6+ mitoses from the VZ to the SVZ continued through development. By E80 the proportion of Pax6+ mitoses in the SVZ was 79%, and at E100 93% of Pax6+ mitoses were located in the SVZ, with the majority of Pax6+ mitotic precursor cells (79%) located in the oSVZ (Fig. 12a & d). We found that in rat, ferret and macaque most Tbr2+ mitoses also expressed Pax6 at either weak or strong levels. In the macaque 90% of abventricular Tbr2 mitoses were also Pax6+; in the ferret 95% of abventricular Tbr2+ mitoses were also Pax6+; and in the rat 95% of Tbr2+ mitoses were also Pax6+ (Fig. 13, Table 13). We noted that in most cases the level of Pax6 expression was lower in Tbr2-positive mitoses than the level of Pax6 expression in Tbr2- negative mitoses. A small subset of Tbr2+ mitoses also expressed strong levels of Pax6. We also examined the proportion of Pax6+ mitoses that expressed Tbr2 and found that it was much lower. Approximately 50% of Pax6+ precursor cells expressed Tbr2 in macaque and in rat, while roughly one third of Pax6+ precursor cells expressed Tbr2 in the ferret (Table 13). These data indicate that there are at least two distinct populations of Pax6+ precursor cells in the developing brain based on Tbr2 expression. Previous In the ferret we found that 90 to 100% of all mitoses expressed Pax6 in each lamina of the developing cortex. Few 4A4+ cells were found beyond the oSVZ, but most of these mitoses were also PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 12 Mammalian Outer Subventricular Zone Figure 7. Mitotic precursor cells coexpress Tbr2 and Pax6 in the oSVZ Graphs showing the total number of Tbr2+ cells the ventricular zone (VZ), inner subventricular zone (iSVZ) an outer SVZ (oSVZ) within a 200 mm wide radial unit of macaqu ferret and rat somatosensory cortex. (a–c) There was a similar number of Tbr2+ cells in the iSVZ of each species, but macaque had a much larger number of Tbr2+ cells in the oSVZ. The stage of development is shown at the bottom of each graph, and the appro- ximate cortical layer generated during each stage of development is indicated along the top of each graph. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green. doi:10.1371/journal.pone.0030178.g007 studies that examined co-expression of the Pax6 and Tbr2 transcription factors reported that 85% of Pax6+ interphase cells in the SVZ expressed Tbr2 in the E14.5 mouse [28], or that 11% of cells in the VZ, SVZ and IZ of E14.5 mouse coexpressed Pax6 and Tbr2 [7], or that there was no expression of Pax6 by precursor cells outside of the VZ in the E16.5 mouse [6]. Differences in co- expression studies may result from different antibodies or staining protocols, from examination of different developmental stages, or because our analysis focused exclusively on mitotic cells. Of note, our data shows that Tbr2+ mitoses co-express Pax6 in the diffuse outer band of the SVZ in lissencephalic rat, and in the oSVZ of the gyrencephalic ferret and macaque. In addition, we also find Pax6+ mitoses that do not co-express Tbr2 in the diffuse outer band of the rat SVZ, and in the oSVZ of ferret and macaque. These data provide further evidence that the outer portion of the SVZ comprises similar cell types in rat, ferret and macaque. tRG cells are present in embryonic rat neocortex During embryonic cortical development in rats and mice, radial glial cells undergo divisions that produce translocating radial glial (tRG) daughter cells that inherit the pial process and translocate away from the VZ toward the cortical plate [4,16]. The tRG cells in rat share features with radial glial cells including similar electrophysiological membrane properties, as well as vimentin and nestin expression [4,5]. The tRG cells remain mitotic as they translocate toward the cortical plate [4,5,16]. The daughter cells produced by dividing tRG may either be non-neuronal, based on electrophysiological recordings [5], or in the neuronal lineage, based on expression of the Hu protein [16], or NeuN [17]. Studies have shown that Pax6+ tRG cells in the human oSVZ produce Tbr2+ intermediate progenitor cells [10]. The 4A4 antibody labels mitotic RG with intact pial fibers in the embryonic rat cerebral cortex [25]. We found that 4A4 also labels numerous cells in the outer SVZ of each species that possessed the characteristic morphology of tRG cells, including a long pial process extending toward the cortical plate. We counterstained tissue with 4A4 and Pax6 antibodies and found that all 4A4+ mitotic cells with tRG morphology expressed Pax6 (Fig. 14a). To more closely examine the morphology of apparent tRG cells, we performed retroviral injections in the E16 to E20 rat neocortex and allowed embryos to continue developing in utero several days after the retroviral injections, at which point embryos were perfused, sectioned and immunostained with Pax6 antibod- ies. We found that all retrovirally labeled GFP+ cells with tRG morphology in the rat neocortex expressed Pax6 (Fig. 14b). Similar experiments performed in developing mice produced the same result. In addition we found that the murine Pax6+ tRG cells also expressed the precursor cell marker Sox2 (Fig. 14c). To determine if cells with apparent tRG morphology in the developing rat cortex were actually translocating cells, we performed retroviral injections in E16 to E20 embryos and prepared organotypic slices 24–72 hours after injections for time- lapse imaging of GFP-labeled cells in the embryonic cortex as described previously [3]. We imaged RG cells undergoing division at the surface of the ventricle and recorded the movements of Figure 7. January 2012 | Volume 7 | Issue 1 | e30178 tRG cells are present in embryonic rat neocortex Graphs showing the total number of Tbr2+ cells in the ventricular zone (VZ), inner subventricular zone (iSVZ) and outer SVZ (oSVZ) within a 200 mm wide radial unit of macaque, PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 13 Mammalian Outer Subventricular Zone Figure 8. Histograms comparing the thickness of the subventricular zone in the developing cortex of rat, ferret, and macaque. Measurements of the thickness of the dense inner band (blue) and diffuse outer band (red) of Tbr2+ cells were made in radial bins stretching from the ventricle to the pial surface in the dorsal somatosensory cortex of rat (a), ferret (b), and macaque (c). In each species the dense inner band appeared first in development followed by the diffuse outer band. In macaque the dense inner band and diffuse outer band were both significantly thicker than in ferret or rat. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. doi:10.1371/journal.pone.0030178.g008 Mammalian Outer Subventricular Zone Figure 8. Histograms comparing the thickness of the subventricular zone in the developing cortex of rat, ferret, and macaque. Measurements of the thickness of the dense inner band (blue) and diffuse outer band (red) of Tbr2+ cells were made in radial bins stretching from the ventricle to the pial surface in the dorsal somatosensory cortex of rat (a), ferret (b), and macaque (c). In each species the dense inner band appeared first in development followed by the diffuse outer band. In macaque the dense inner band and diffuse outer band were both significantly thicker than in ferret or rat. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. doi:10 1371/journal pone 0030178 g008 Table 5. Radial thicknesses of the SVZ, the Tbr2+ dense inner band and the Tbr2+ diffuse outer band. tRG cells are present in embryonic rat neocortex Macaque age Total SVZ Dense inner band Diffuse outer band E50 440 mm 440 mm NA E65 960 mm 750 mm 210 mm E80 2590 mm 500 mm 2090 mm E100 2870 mm 440 mm 2430 mm Ferret age Total SVZ Dense inner band Diffuse outer band E28 97 mm 97 mm NA E31 105 mm 105 mm NA E34 263 mm 103 mm 160 mm E38 545 mm 220 mm 325 mm P2 563 mm 156 mm 407 mm Rat age Total SVZ Dense inner band Diffuse outer band E14 27 mm 27 mm NA E17 103 mm 103 mm NA E18 237 mm 120 mm 117 mm E20 257 mm 117 mm 140 mm Radial thickness from ferret and rat are averages obtained from a minimum of two radial units per age. Radial thicknesses from macaque are from one radial unit per age. The dense inner band and diffuse outer band were identified by the pattern of Tbr2 immunoreactivity. doi:10.1371/journal.pone.0030178.t005 Table 5. Radial thicknesses of the SVZ, the Tbr2+ dense inner band and the Tbr2+ diffuse outer band. Table 5. Radial thicknesses of the SVZ, the Tbr2+ dense inner band and the Tbr2+ diffuse outer band. Radial thickness from ferret and rat are averages obtained from a minimum of two radial units per age. Radial thicknesses from macaque are from one radial unit per age. The dense inner band and diffuse outer band were identified by the pattern of Tbr2 immunoreactivity. doi:10 1371/journal pone 0030178 t005 January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 14 Mammalian Outer Subventricular Zone Table 6. Distribution of Pax6+ cells during neocortical development. Macaque age Total Pax6+ cells Radial units VZ iSVZ oSVZ CP (incl. PP/SP/MZ) E50 1023 3 89% (910/1023) 6.7% (68/1023) 4% (42/1023) 0.3% (3/1023) E65 2098 3 33.9% (704/2098) 11% (239/2098) 55% (1154/2098) 0.1% (1/2098) E80 1915 3 20% (381/1915) 26% (493/1915) 51% (974/1915) 3% (67/1915) E100 1043 2 24% (247/1043) 6% (61/1043) 67% (701/1043) 3% (34/1043) Ferret age Total Pax6+ cells Radial units VZ iSVZ oSVZ CP (incl. tRG cells are present in embryonic rat neocortex 15a) shared some characteristics with neurons: it migrated through locomotion, exhibited retrograde migratory movements toward the ventricle, did not divide and was Pax6-negative after three days of time-lapse imaging. Nonetheless, this daughter cell did not express NeuN although 78 hours had passed after it was generated (Fig. 15c). The second daughter cell produced by the tRG cell (t = 47 h, indicated with a white arrow in Fig. 15a) was a presumed intermediate progenitor cell that subsequently divided at t = 74 h, and produced two daughter cells that expressed weak levels of Pax6. The tRG cell produced a third daughter cell at the end of the time-lapse experiment (t = 78 h, indicated by a white arrow with a red border) that expressed higher levels of Pax6 than the previous generations of daughter cells, but lower levels of Pax6 expression than its tRG mother cell (Fig. 15c). These data suggest a pattern of Pax6 expression during neurogenic divisions whereby actively dividing RG and tRG cells retain high levels of Pax6 expression while the RG daughter cells slowly lose Pax6 expression as they differentiate into neurons or intermediate progenitor cells that express Tbr2. This idea is consistent with our data showing that many Tbr2+ cells express low levels of Pax6 (Fig. 13). We have previously shown that tRG daughter cells in the embryonic cortex lack the physiological membrane properties of neurons, consistent with the idea that tRG cells in the embryonic cortex can generate glial daughter cells [4,5]. We presume that astroglial daughter cells produced during prenatal development would retain Pax6 expression. Importantly, this data confirms that tRG cells express Pax6 in the rat as they do in ferret and macaque. tRG cells are present in embryonic rat neocortex PP/SP/MZ) E28 847 3 95% (803/847) 3% (29/847) 2% (15/847) 0% (0/847) E31 1171 3 91% (1070/1171) 5% (54/1171) 3.9% (46/1171) 0.1% (1/1171) E34 1185 3 75% (894/1185) 17% (201/1185) 8% (90/1185) 0% (0/1185) E38 627 2 58.9% (369/627) 22% (141/627) 19% (116/627) 0.1% (1/627) P2 1066 3 51% (547/1066) 32% (337/1066) 16% (168/1066) 1% (14/1066) P10 3121 3 15% (476/3121) 2% (60/3121) 78% (2443/3121) 5% (142/3121) Rat age Total Pax6+ cells Radial units VZ iSVZ oSVZ CP (incl. PP/SP/MZ) E14 474 3 91% (431/474) 8.6% (41/474) 0.4% (2/431) 0% (0/474) E17 3965 12 87% (3463/3965) 12% (484/3965) 1% (18/3965) 0% (0/3965) E20 499 3 64% (318/499) 27% (134/499) 9% (45/499) 0.4% (2/499) P3 855 3 18% (155/855) 35% (300/855) 31% (266/855) 16% (134/855) Numbers represent the average percentage of Pax6+ cells in each compartment of the developing somatosensory cortex per radial unit, averaged across n radial units. Cells were identified through Pax6 immunoreactivity in a minimum of two 200 mm wide radial unit sections from each age in each species. Numbers in parentheses indicate the total number of Pax6+ cells counted in each compartment. doi:10.1371/journal.pone.0030178.t006 Numbers represent the average percentage of Pax6+ cells in each compartment of the developing somatosensory cortex per radial unit, averaged across n radial units. Cells were identified through Pax6 immunoreactivity in a minimum of two 200 mm wide radial unit sections from each age in each species. Numbers in parentheses indicate the total number of Pax6+ cells counted in each compartment. doi:10.1371/journal.pone.0030178.t006 daughter cells, paying special attention to the daughter cell that inherited the pial process. During later stages of embryonic rat cortical development, RG daughter cells that inherited the pial process either remained in the VZ as RG cells or acquired tRG cell morphology, detached from the lateral ventricle and translocated away from the VZ following the radial trajectory of the pial process. We noted that many of the tRG cells continued dividing as they translocated toward the cortical plate, as we have previously shown [4,5]. After the tRG cells had migrated away from the VZ we fixed and immunostained the cultured slices with Pax6 antibodies. We found that rat tRG cells expressed Pax6 (Fig. 15). Mitotic RG/tRG cells exhibited strong levels of Pax6 expression and we noted that the level of Pax6 expression appeared to decrease over time in the daughter cells produced by radial glia and tRG cells. tRG cells are present in embryonic rat neocortex For example, Figure 15 shows an example of an RG cell that transitioned into a tRG cell. We followed the tRG cell and three generations of daughter cells. The first daughter cell produced (indicated with a white arrowhead in Fig. 15a) shared some characteristics with neurons: it migrated through locomotion, exhibited retrograde migratory movements toward the ventricle, did not divide and was Pax6-negative after three days of time-lapse imaging. Nonetheless, this daughter cell did not express NeuN although 78 hours had passed after it was generated (Fig. 15c). The second daughter cell produced by the tRG cell (t = 47 h, indicated with a white arrow in Fig. 15a) was a presumed intermediate progenitor cell that subsequently divided at t = 74 h, and produced two daughter cells that expressed weak levels of Pax6. The tRG cell produced a third daughter cell at the end of the time-lapse experiment (t = 78 h, indicated by a white arrow with a red border) that expressed higher levels of Pax6 than the previous generations of daughter cells, but lower levels of Pax6 expression than its tRG mother cell (Fig. 15c). These data suggest daughter cells, paying special attention to the daughter cell that inherited the pial process. During later stages of embryonic rat cortical development, RG daughter cells that inherited the pial process either remained in the VZ as RG cells or acquired tRG cell morphology, detached from the lateral ventricle and translocated away from the VZ following the radial trajectory of the pial process. We noted that many of the tRG cells continued dividing as they translocated toward the cortical plate, as we have previously shown [4,5]. After the tRG cells had migrated away from the VZ we fixed and immunostained the cultured slices with Pax6 antibodies. We found that rat tRG cells expressed Pax6 (Fig. 15). Mitotic RG/tRG cells exhibited strong levels of Pax6 expression and we noted that the level of Pax6 expression appeared to decrease over time in the daughter cells produced by radial glia and tRG cells. For example, Figure 15 shows an example of an RG cell that transitioned into a tRG cell. We followed the tRG cell and three generations of daughter cells. The first daughter cell produced (indicated with a white arrowhead in Fig. January 2012 | Volume 7 | Issue 1 | e30178 Mitotically active Pax6+ tRG Cells are present during and after cortical neurogenesis g We found that Pax6-expressing mitotically active tRG cells in each species were present both during and after cortical neurogenesis (Fig. 16), which suggests that tRG cells may be both neurogenic and gliogenic. We double immunostained tissue with Pax6 and Sox2 antibodies to determine if we could distinguish two subtypes of tRG cells. However, we found that nearly all Pax6+ mitotic cells in the SVZ also expressed Sox2, both during and after neurogenesis (Fig. 17, Table 14). This may not be surprising since Sox2 is expressed by both neuronal and glial precursor cells in the developing dorsal telencephalon [29]. At both E17 and P3 over 95% of Pax6+ mitotic cells expressed Sox2. We next tested the possibility that the Pax6+ tRG cells in the postnatal rat (e.g.: P3 rat) generate the last compliment of superficial layer 2 neurons. We pulsed rats with two injections of BrdU (80 mg/kg) at P1, P3, and P7. Animals were sacrificed on P10 and neocortical tissue was co-immunostained with antibodies against NeuN and BrdU. P1 January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 15 PLoS ONE | www.plosone.org Mammalian Outer Subventricular Zone ribution of Pax6+ cells during cortical development is similar in ferrets and rats, but different in macaques. somatosensory cortex from E80 macaque, P2 ferret and E20 rat immunostained for Pax6 (red) and counterstained with splayed at the same scale. In each species a dense inner band of Pax6+ cells was colocalized with the ventricular zone (VZ), 6-expressing cells extended outward through the inner subventricular zone (iSVZ) and the outer SVZ (oSVZ). The iSVZ and ed on the pattern of DAPI staining as described above. (d–f) Graphs showing changes in the distribution of Pax6+ cells d e somatosensory cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortica tage of development is indicated along the top of each graph. (d) In macaque the distribution of Pax6+ cells rapidly shifted 90% of Pax6+ cells were located in the VZ. But by E65 during production of layer 5 neurons [20], the majority of Pax6+ cells oSVZ. (e) The distribution of Pax6+ cells in the gyrencephalic ferret shifted to the oSVZ much more slowly than in macaque. www.plosone.org 16 January 2012 | Volume 7 | Issue 1 | e Figure 9. Mitotically active Pax6+ tRG Cells are present during and after cortical neurogenesis The distribution of Pax6+ cells during cortical development is similar in ferrets and rats, but different in macaques. (a–c) Coronal sections of somatosensory cortex from E80 macaque, P2 ferret and E20 rat immunostained for Pax6 (red) and counterstained with DAPI (blue). Images are displayed at the same scale. In each species a dense inner band of Pax6+ cells was colocalized with the ventricular zone (VZ), and a diffuse band of Pax6-expressing cells extended outward through the inner subventricular zone (iSVZ) and the outer SVZ (oSVZ). The iSVZ and oSVZ were identified based on the pattern of DAPI staining as described above. (d–f) Graphs showing changes in the distribution of Pax6+ cells during development of the somatosensory cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. (d) In macaque the distribution of Pax6+ cells rapidly shifted to the oSVZ. At E50 nearly 90% of Pax6+ cells were located in the VZ. But by E65 during production of layer 5 neurons [20], the majority of Pax6+ cells (60%) were located in the oSVZ. (e) The distribution of Pax6+ cells in the gyrencephalic ferret shifted to the oSVZ much more slowly than in macaque. At P2 January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 16 Mammalian Outer Subventricular Zone during genesis of layer 2 neurons [19], 52% of Pax6+ cells remained in the VZ. (f) The distribution of Pax6+ cells in the lissencephalic rat was similar to that of the gyrencephalic ferret. At the end of neurogenesis on E20 during production of layer 2 neurons [21], the majority of Pax6+ cells (64%) were still located in the VZ. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green. Cortical plate (CP)/preplate (PP)/subplate (SP)/marginal zone (MZ): purple. Scale bar in (a) applies to (a–c). doi:10.1371/journal.pone.0030178.g009 which suggests that Olig2+ precursor cells are restricted to producing glial progeny. BrdU injections produced a very small number of BrdU+ nuclei that appeared to be weakly positive for NeuN in the medial cingulate cortex. However, no BrdU+ nuclei expressed NeuN in the dorsolateral cortex. P3 and P10 injections did not produce BrdU+/NeuN+ cells in the cerebral cortex of any animals, consistent with previous data on rat neurogenesis [21]. Identifying the oSVZ The iSVZ and oSVZ were identified and subdivided into discrete proliferative zones in the macaque visual cortex based on cytoarchitectural features, particularly the inner and outer fiber layers [9]. The inner fiber layer becomes apparent in macaque visual cortex by E72 [9]. We compared cortical cytoarchitecture in Nissl stained coronal sections at three rostro-caudal levels and at several developmental stages in each species. We examined coronal sections of tissue at the genu of the corpus callosum, at the level of the anterior commissure, which includes primary somatosensory cortex in the ferret [19] and macaque [31], and in the occipital lobe. In Nissl stained sections of macaque tissue the inner and outer fiber layers were present in the occipital lobe (Fig. 4), as previously described [9]. However, these structures did not have the same appearance or were not apparent in somatosensory cortex or frontal cortical areas, and did not present obvious cues for distinguishing the iSVZ from the oSVZ (Figs. 19 and 20). At E65 a slight decrease in cell density was observed between the iSVZ and oSVZ in somatosensory cortex, but this was not apparent at E80. We also noted that the macaque oSVZ was much thicker in somatosensory cortex compared to visual cortex. One explanation for the difference in thickness may be that the somatosensory oSVZ generates cortical cells that are destined for wide spread cortical areas, including neocortex directly superficial to the somatosensory oSVZ and neocortical areas in the lateral cerebral wall (see Fig. 20a). In contrast, in the visual cortex there is a more strict ‘‘one-to-one’ radial relationship so that the oSVZ appears to serve only the developing cortex that is directly superficial (see Fig. 20c). In the developing macaque we found that the majority of mitotic Pax6+ tRG cells (94%) were Olig2 negative at E65 and that only 2% expressed Olig2. At E80 57% of mitotic Pax6+ tRG cells were Olig2 negative and 38% expressed Olig2. By E100, representing the end of macaque cortical neurogenesis [20], the proportion of mitotic Pax6+ tRG cells that were Olig2 negative was 24% and the proportion that were Olig2+ increased to 43% (Fig. 18a & d). We further examined the possibility that tRG cell subtypes exist in the developing macaque neocortex by counter- staining tissue with 4A4 and GFAP, but qualitatively found that all 4A4+ tRG cells were GFAP+. Mitotically active Pax6+ tRG Cells are present during and after cortical neurogenesis In these experiments testing for Olig2 expression by tRG cells relied on 4A4 identification of tRG cells. To test the validity of this approach we labeled tRG cells through in utero retroviral injections and electroporations of GFP-expressing plasmids. tRG cells were identified based on morphology, i.e.: cells in the SVZ that lacked a ventricular contacting process but that possessed an intact pial contacting process. We tested whether the tRG cells expressed Olig2 through immunostaining and found similar proportions of Olig2+ and Olig2-negative cells as we found among 4A4 identified tRG cells (data not shown). These data show that tRG cells in the rodent SVZ express Pax6 as they do in ferret and macaque and that the diffuse outer band of the embryonic SVZ in rodents consists in part of Pax6+ tRG cells, as does the oSVZ of ferret and macaque (Fig. 16), and the human oSVZ [10,11]. Furthermore, these data strongly suggest that Pax6+ tRG cells that are present in the postnatal cortex are not neurogenic. These data are consistent the idea that two subtypes of tRG cells exist in the developing cortex during cortical neurogenesis, one subtype that expresses Olig2 and produces glial progeny, and a second subtype that does not express Olig2 and produces neuronal progeny. Furthermore, these data provide more evidence that the same cellular subtypes are present in the outer SVZ of lissencephalic rats that are present in the oSVZ of gyrencephalic ferrets and macaques. Olig2 expression distinguishes two distinct subtypes of tRG cells in the developing cerebral cortex Our data demonstrate that Pax6 expression alone is not enough to identify neurogenic precursor cells and that Pax6+ mitotic cells are not neurogenic in the postnatal cortex. Since we previously presented data showing that tRG cells can produce non-neuronal daughter cells [4,5], we explored the possibility that a subset of Pax6+ tRG cells in the embryonic neocortex are gliogenic. Since Olig2 directs astrocyte formation in the postnatal SVZ [30], we used 4A4 immunostaining to identify mitotic tRG cells in the outer SVZ. Presumed 4A4+ tRG cells possessed a process that was directed toward the pia. We asked whether these cells expressed Pax6 and Olig2. We noted strong Olig2 expression in a subset of Pax6+ tRG cells during late stages of embryonic rat cortical development (Fig. 18a, Table 15). PLoS ONE | www.plosone.org Identifying the oSVZ In the developing ferret we found that the majority of mitotic Pax6+ tRG cells (92%) were Olig2 negative at E34 and only 2% expressed Olig2. At P2 81% of mitotic Pax6+ tRG cells were Olig2 negative and 18% expressed Olig2. By P10, the end of ferret cortical neurogenesis [19], the proportion of mitotic Pax6+ tRG cells that were Olig2 negative was 31% and the proportion that were Olig2+ increased to 62% (Figs. 18b & e). In developing rat cortex mitotic Pax6+ tRG cells did not express Olig2 at E14. At E17 78% of mitotic Pax6+ tRG cells were Olig2 negative and 22% expressed Olig2. At P3, after cortical neurogenesis [21], the proportion of mitotic Pax6+ tRG cells that were Olig2 negative was only 3% and the proportion that were Olig2+ increased to 77% (Figs. 18c & f). We did not observe morphological differences between Pax6+ tRG cells that expressed Olig2 versus those that did not express Olig2. We noted that the inner fiber layer was not apparent at any age in somatosensory cortex in Nissl stained tissue prepared from ferret. In some areas of the ferret visual cortex, cells in the oSVZ were organized in radial palisades that resembled the organization of the macaque OFL (Fig. 21, see Fig. 4). However, this ‘OFL-like’ structure in ferret differed from the macaque OFL since it is superficial to the iSVZ, not the oSVZ, and because Tbr2+ cells penetrate through the ferret ‘OFL-like’ structure, whereas Tbr2+ We tested if the Olig2+ cells might be neurogenic by performing an Olig2/Tbr2 double immunostaining in the E21 rat but did not find any double positive cells (194 Tbr2+ cells, 432 Olig2+ cells), PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 17 January 2012 | Volume 7 | Issue 1 | e30178 Mammalian Outer Subventricular Zone Figure 10. Graphs showing the total number of Pax6+ cells in the ventricular zone (VZ), inner subventricular zone (iSVZ), outer SVZ (oSVZ), and cortical plate (CP) within a 200 mm wide radial unit of macaque, ferret and rat somatosensory cortex. (a–c) There was a similar number of Pax6+ cells in the iSVZ of each species, but macaque had a much larger number of Pax6+ cells in the oSVZ. Identifying the oSVZ The stage of development is shown at the bottom of each graph, and the approximate cortical layer generated at each stage of develop- ment is indicated along the top of each graph. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green; CP: purple. PP, preplate; SP, subplate; MZ, marginal zone. doi:10.1371/journal.pone.0030178.g010 cells are not present in the macaque OFL (Figs. 21 and Fig. 4). The radially oriented streams of cells in the OFL-like structure of ferret visual cortex likely represent cell migration along a radial trajectory. The oSVZ in many cortical areas of ferret and rat was characterized by streams of cells that appear to cross the oSVZ along oblique or tangential trajectories (Figs. 19, 20, 21, 22). The organization of these cells is likely influenced by a combination of factors including the migratory trajectory of newly generated cells as well as the ingrowth of axonal fibers. Nevertheless, the IFL and OFL were not apparent in any cortical areas of the developing rat cortex (Fig. 22). We asked if the inner and outer fiber layers could be better visualized in other cortical regions of macaque, ferret, and rat neocortex through immunohistochemistry using Tau-1 antibodies that label axonal fibers. Tau-1 immunostaining in the macaque produced dense immunoreactivity in the cortical plate and developing white matter, a striated pattern of staining in the oSVZ, and scarce labeling in the iSVZ and VZ (Fig. 23a & b). The same pattern was present in rats and ferrets (Fig. 23c & d). Tau-1 immunostaining did not provide evidence for an inner or outer fiber layer in frontal/somatosensory sections of macaque cortex, or in any regions of ferret and rat cortex. However, in sections that were co-stained with Tau-1 and Tbr2 antibodies we noticed striking similarities in the pattern of staining in rat, ferret and macaque. In each species the dense inner band of Tbr2 expression was located in the Tau-free zone. In macaque, ferret, and rat the diffuse band of Tbr2 expression was located in the Tau-striated zone. In some regions of macaque cortex the diffuse Tbr2+ cells extended beyond the horizontally oriented fibers in the Tau- striated zone. Identifying the oSVZ PP/SP/MZ) E14 159 3 144 14 1 0 E17 321 12 282 38 1 0 E20 167 3 106 45 15 1 P3 286 3 52 100 89 45 Numbers in each cell represent the average number of Pax6+ cells in each compartment of the developing somatosensory cortex per radial unit averaged across n radial units. Cells were identified through Pax6 immunoreactivity in a minimum of two 200 mm wide radial units from each age in each species. doi:10.1371/journal.pone.0030178.t007 Numbers in each cell represent the average number of Pax6+ cells in each compartment of the developing somatosensory cortex per radial unit averaged across n radial units. Cells were identified through Pax6 immunoreactivity in a minimum of two 200 mm wide radial units from each age in each species. doi:10 1371/journal pone 0030178 t007 proliferative region of the VZ/iSVZ (Fig. 24). In macaque the dense band of Tbr2+ cells nearly fills the Tau-free/cell dense proliferative region. However, in ferret and rat the inner dense band of Tbr2+ cells does not completely fill the iSVZ, and the diffuse band of Tbr2+ cells is located in the outer portion of the staining identified the cell dense proliferative zone around the ventricle that includes the VZ and the iSVZ, and a lower density proliferative zone that includes the oSVZ. Finally, Tau-1 immunostaining distinguished the border between iSVZ from the oSVZ since Tau-1+ fibers do not enter the cell dense Table 8. Proportion of mitotic cells that are 4A4+ in macaque, ferret and rat. Macaque age Percentage of total mitoses that are 4A4+ Surface mitoses Abventricular mitoses E50 100% (617/617) 100% (476/476) 100% (141/141) E65 100% (796/796) 100% (358/358) 100% (438/438) E80 100% (474/474) 100% (170/170) 100% (304/304) E100 100% (287/287) 100% (57/57) 100% (230/230) Ferret age Percentage of total mitoses that are 4A4+ Surface mitoses Abventricular mitoses E28 100% (134/134) 100% (93/93) 100% (41/41) E31 100% (243/243) 100% (173/173) 100% (70/70) E34 100% (213/213) 100% (123/123) 100% (90/90) E38 100% (172/172) 100% (68/68) 100% (104/104) P2 100% (238/238) 100% (65/65) 100% (173/173) P10 100% (147/147) 100% (27/27) 100% (120/120) Rat age Percentage of total mitoses that are 4A4+ Surface mitoses Abventricular mitoses E14 100% (163/163) 100% (127/127) 100% (36/36) E17 100% (146/146) 100% (87/87) 100% (59/59) E20 100% (169/169) 100% (67/67) 100% (102/102) P3 100% (135/135) 100% (12/12) 100% (123/123) Cells were identified by condensed chromatin using DAPI staining. Identifying the oSVZ The Tau-free zone corresponded precisely with the cell dense proliferative region that surrounds the ventricle in rats, ferrets, and macaques, and the Tau-striated zone corresponded to the more superficial zone that has a lower cell density, as seen in DAPI, Nissl or other cellular stains (Fig. 23). The changes in cell density between the iSVZ and oSVZ, the absence of Tau-1+ fibers in the iSVZ, and the presence of Tau-1+ fibers in the oSVZ of each species demonstrate that the iSVZ and oSVZ subdivisions are histologically distinct structures in macaque, ferret, and rat. Thus, Tau-1 immunostaining can be used in conjunction with DAPI, and Tbr2/Pax6 to distinguish the boundaries between the VZ, iSVZ and oSVZ proliferative zones in these three species (Figs. 23 and 24). It is important to note that although Tbr2 staining in the E17 rat shows the presence of a dense inner band and diffuse outer band, the distinction between iSVZ and oSVZ based on cell density alone is not discernible until E18, when perceptible changes in cell density between the iSVZ and oSVZ become readily apparent. In summary, Pax6 immunostaining presented as a dense band that corresponds to the VZ, and a diffuse band of labeled cells in the iSVZ and oSVZ, with a few scattered cells in the CP and MZ. Tbr2 immunostaining presented as a dense band that corresponds to the iSVZ and a diffuse band of labeled cells in the oSVZ. DAPI January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 18 Mammalian Outer Subventricular Zone Table 7. Total number of Pax6+ cells/200 mm radial unit during neocortical development. Macaque age Average Pax6+ cells/200 mm bin Radial units VZ iSVZ oSVZ CP (incl. PP/SP/MZ) E50 342 3 303 23 15 1 E65 700 3 235 80 385 0 E80 638 3 127 164 325 22 E100 523 2 124 31 351 17 Ferret age Total Pax6+ cells/200 mm bin Radial units VZ iSVZ oSVZ CP (incl. PP/SP/MZ) E28 283 3 268 10 5 0 E31 390 3 357 18 15 0 E34 395 3 298 67 30 0 E38 315 2 185 71 58 1 P2 355 3 182 112 56 5 Rat age Total Pax6+ cells/200 mm bin Radial units VZ iSVZ oSVZ CP (incl. Identifying the oSVZ Numbers in parentheses indicate the total number of mitotic cells counted at each age in each species. doi:10.1371/journal.pone.0030178.t008 Table 8. Proportion of mitotic cells that are 4A4+ in macaque, ferret and rat. January 2012 | Volume 7 | Issue 1 | e30178 19 Mammalian Outer Subventricular Zone Table 9. Proportion of mitotic cells in each zone that are Tbr2+. Macaque age Percentage of total mitoses that are Tbr2+ VZ iSVZ oSVZ CP (incl PP/SP/MZ) E50 14% (47/325) 0% (0/236) 53% (47/89) NA NA E65 24% (91/378) 0% (0/123) 56% (54/97) 23% (37/158) NA E80 35% (98/278) 6% (3/52) 44% (24/54) 43% (71/164) 0% (0/8) E100 18% (44/249) 8% (2/26) 28% (8/29) 20% (34/166) 0% (0/28) Ferret age Percentage of total mitoses that are Tbr2+ VZ iSVZ oSVZ CP (incl PP/SP/MZ) E31 19% (29/153) 0% (0/114) 94% (29/31) 0% (0/8) NA E34 24% (36/152) 2% (2/100) 91% (30/33) 21% (4/19) NA E38 36% (33/91) 0% (0/31) 80% (24/30) 30% (9/30) NA P2 26% (36/136) 0% (0/31) 44% (27/61) 21% (9/43) 0% (0/1) P10 19% (12/63) 0% (0/19) 0% (0/1) 34% (12/35) 0% (0/8) Rat age Percentage of total mitoses that are Tbr2+ VZ iSVZ oSVZ CP (incl PP/SP/MZ) E14 26% (29/110) 3% (2/81) 93% (27/29) NA NA E17 39% (24/62) 0% (0/37) 100% (21/21) 75% (3/4) NA E20 41% (38/93) 3% (1/37) 85% (28/33) 43% (9/21) 0% (0/2) P3 16% (11/70) 13% (1/8) 28% (9/32) 6% (1/17) 0% (0/13) Numbers represent the proportion of mitotic cells in each compartment of the developing somatosensory cortex that are Tbr2+. Cells were identified through Tbr2 immunoreactivity in a minimum of three 200 mm radial units from each age in each species. Mitoses were identified with 4A4 immunoreactivity or by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the number of Tbr2+ mitotic cells counted in each compartment from each age in each species. doi:10.1371/journal.pone.0030178.t009 Numbers represent the proportion of mitotic cells in each compartment of the developing somatosensory cortex that are Tbr2+. Cells were identified through Tbr2 immunoreactivity in a minimum of three 200 mm radial units from each age in each species. Mitoses were identified with 4A4 immunoreactivity or by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the number of Tbr2+ mitotic cells counted in each compartment from each age in each species. doi:10.1371/journal.pone.0030178.t009 iSVZ and in the oSVZ. Identifying the oSVZ We define the rat oSVZ as the proliferative region where Tbr2+ cells are localized superficial to the cell dense iSVZ (Fig. 24a). We propose that in later stages of rat cortical development, the SVZ is divided into an iSVZ and oSVZ, as it is in macaque and ferret. The boundary between iSVZ and oSVZ can be identified based on cell density in Nissl or DAPI Table 10. Distribution of Tbr2+ mitotic cells during neocortical development. Macaque age Total Tbr2+ mitoses VZ iSVZ oSVZ E50 19 0% (0/19) 100% (19/19) 0% (0/19) E65 52 0% (0/52) 67% (35/52) 33% (17/52) E80 72 3% (2/72) 21% (15/72) 76% (55/72) E100 21 0% (0/21) 14% (3/21) 86% (18/21) Ferret age Total Tbr2+ mitoses VZ iSVZ oSVZ E31 29 0% (0/29) 100% (29/29) 0% (0/29) E34 36 6% (2/36) 83% (30/36) 11% (4/36) E38 33 0% (0/33) 73% (24/33) 27% (9/33) P2 36 0% (0/36) 75% (27/36) 25% (9/36) P10 12 0% (0/12) 0% (0/12) 100% (12/12) Rat age Total Tbr2+ mitoses VZ iSVZ oSVZ E14 29 7% (2/29) 93% (27/29) 0% (0/29) E17 24 0% (0/24) 88% (21/24) 13% (3/24) E20 38 2% (1/38) 74% (28/38) 24% (9/38) P3 11 9% (1/11) 82% (9/11) 9% (1/11) Numbers represent the proportion of Tbr2+ mitotic cells that were located in each compartment of the developing somatosensory cortex expressed as a percentage of the total number of Tbr2+ mitoses in a 200 mm wide radial unit. Cells were identified by Tbr2 immunoreactivity in a minimum of three 200 mm radial unit sections from each age in each species. Mitoses were identified with 4A4 immunoreactivity or by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the total number of Tbr2+ mitotic cells counted in each compartment at each age. doi:10.1371/journal.pone.0030178.t010 Table 10. Distribution of Tbr2+ mitotic cells during neocortical development. Numbers represent the proportion of Tbr2+ mitotic cells that were located in each compartment of the developing somatosensory cortex expressed as a percentage of the total number of Tbr2+ mitoses in a 200 mm wide radial unit. Cells were identified by Tbr2 immunoreactivity in a minimum of three 200 mm radial unit sections from each age in each species. Mitoses were identified with 4A4 immunoreactivity or by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the total number of Tbr2+ mitotic cells counted in each compartment at each age. Numbers represent the proportion of Tbr2+ mitotic cells that were located in each compartment of the developing somatosensory cortex expressed as a percentage of the total number of Tbr2+ mitoses in a 200 mm wide radial unit. Cells were identified by Tbr2 immunoreactivity in a minimum of three 200 mm radial unit sections from each age in each species. Mitoses were identified with 4A4 immunoreactivity or by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the total number of Tbr2+ mitotic cells counted in each compartment at each age. doi:10.1371/journal.pone.0030178.t010 Identifying the oSVZ doi:10.1371/journal.pone.0030178.t010 Numbers represent the proportion of Tbr2+ mitotic cells that were located in each compartment of the developing somatosensory cortex expressed as a percentage of the total number of Tbr2+ mitoses in a 200 mm wide radial unit. Cells were identified by Tbr2 immunoreactivity in a minimum of three 200 mm radial unit sections from each age in each species. Mitoses were identified with 4A4 immunoreactivity or by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the total number of Tbr2+ mitotic cells counted in each compartment at each age. doi:10.1371/journal.pone.0030178.t010 January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 20 Mammalian Outer Subventricular Zone Figure 11. The distribution of Tbr2+ mitoses shifts to the outer subventricular zone (oSVZ) during early stages of co development in macaque, but not in ferret or rat. (a–c) Images taken from coronal sections of the E80 macaque, P2 ferret, and E20 rat Tissue was immunostained for 4A4 (green), Tbr2 (red) and counterstained with DAPI (blue). Boxes to the right highlight examples of 4A4+ m cells that either do not express Tbr2 (#1) or that do express Tbr2 (#2). (d–f) Graphs showing changes in the distribution of Tbr2+ mitoses d development of the somatosensory cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortica generated at each stage of development is indicated along the top of each graph. (d) At E50 in the macaque all Tbr2+ mitoses were located inner SVZ (iSVZ). There was a steady decrease in the proportion of Tbr2+ divisions occurring in the iSVZ. By E80 the majority of Tbr2+ mitoses Figure 11. The distribution of Tbr2+ mitoses shifts to the outer subventricular zone (oSVZ) during early stages of cortical development in macaque, but not in ferret or rat. (a–c) Images taken from coronal sections of the E80 macaque, P2 ferret, and E20 rat oSVZ. Tissue was immunostained for 4A4 (green), Tbr2 (red) and counterstained with DAPI (blue). Boxes to the right highlight examples of 4A4+ mitotic cells that either do not express Tbr2 (#1) or that do express Tbr2 (#2). (d–f) Graphs showing changes in the distribution of Tbr2+ mitoses during development of the somatosensory cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. (d) At E50 in the macaque all Tbr2+ mitoses were located in the inner SVZ (iSVZ). There was a steady decrease in the proportion of Tbr2+ divisions occurring in the iSVZ. By E80 the majority of Tbr2+ mitoses (76%) Figure 11. The distribution of Tbr2+ mitoses shifts to the outer subventricular zone (oSVZ) during early stages of cortical development in macaque, but not in ferret or rat. (a–c) Images taken from coronal sections of the E80 macaque, P2 ferret, and E20 rat oSVZ. Tissue was immunostained for 4A4 (green), Tbr2 (red) and counterstained with DAPI (blue). Boxes to the right highlight examples of 4A4+ mitotic cells that either do not express Tbr2 (#1) or that do express Tbr2 (#2). (d–f) Graphs showing changes in the distribution of Tbr2+ mitoses during development of the somatosensory cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. (d) At E50 in the macaque all Tbr2+ mitoses were located in the inner SVZ (iSVZ). There was a steady decrease in the proportion of Tbr2+ divisions occurring in the iSVZ. By E80 the majority of Tbr2+ mitoses (76%) January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 21 Mammalian Outer Subventricular Zone had shifted to the oSVZ. (e) In ferret the majority of Tbr2+ mitoses were located in the iSVZ throughout neurogenesis in the somatosensory cortex. At P2, during neurogenesis of layer 2 neurons [19], 73% of Tbr2+ mitoses were still located in the iSVZ. Discussion We provide evidence that the lissencephalic rat cortex possesses an outer SVZ. We show that the embryonic SVZ in rat, ferret and macaque shares key cellular and cytoarchitectural features. We demonstrate a simple novel approach for discriminating the boundaries of the VZ, iSVZ and oSVZ in mammals based on costaining with DAPI, Tbr2/Pax6, and Tau-1. We also describe the presence of two subtypes of tRG cells that co-exist in the perinatal neocortex based on the expression of Olig2. We propose a definition for the iSVZ and oSVZ based on the following criteria: 1) The iSVZ is located within the cell dense proliferative region that surrounds the lateral ventricle, while the oSVZ is located in the cell-diffuse zone that is superficial to the iSVZ; 2) The iSVZ appears homogeneous in terms of cell density, while the oSVZ is often marked by a striated or stippled appearance created by clusters of cells organized into oblique or tangential streams that appear to cross the oSVZ; 3) The iSVZ is localized to the dense inner band of Tbr2+ cells and the oSVZ is localized to the diffuse outer band of Tbr2+ cells; 4) The iSVZ is localized to the Tau-free zone that surrounds the ventricle, the (f) The pattern observed in rat was similar to that of the ferret. The majority of Tbr2+ mitoses were located in the iSVZ throughout the period of cortical neurogenesis. At P2, during production of layer 2 neurons [21], there was a similar number of Tbr2+ mitoses located in the rat iSVZ (74%) as was observed in the ferret. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green. doi:10 1371/journal pone 0030178 g011 had shifted to the oSVZ. (e) In ferret the majority of Tbr2+ mitoses were located in the iSVZ throughout neurogenesis in the somatosensory cortex. At P2, during neurogenesis of layer 2 neurons [19], 73% of Tbr2+ mitoses were still located in the iSVZ. (f) The pattern observed in rat was similar to that of the ferret. The majority of Tbr2+ mitoses were located in the iSVZ throughout the period of cortical neurogenesis. At P2, during production of layer 2 neurons [21], there was a similar number of Tbr2+ mitoses located in the rat iSVZ (74%) as was observed in the ferret. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green. doi:10.1371/journal.pone.0030178.g011 stained tissue, and/or Tau-1 immunostaining. These data support the hypothesis that the developing rat cerebral cortex possesses distinct iSVZ and oSVZ compartments. number of neurons in the ferret neocortex has not been determined, but based on brain weight one might predict that ferret numbers lie much closer to rat than to macaque values. The oSVZ in macaque is considerably larger than the oSVZ in ferret or rat, up to 20 times thicker than the rat oSVZ in developing somatosensory cortex. In addition, the macaque oSVZ is populated by a significantly greater number of precursor cells than the oSVZ of ferret or rat (Figs. 7 and 10). Thus, based on a number of measures the macaque oSVZ and resulting neocortex are significantly larger than in rats or ferrets. However, the determination of whether or not the oSVZ is present in a given species should be based on a defined set of characteristics rather than sheer size or number of cells. Defining the outer SVZ: does size matter? boundary between the iSVZ and oSVZ is marked by the boundary between the Tau-free and Tau-striated zones, and the oSVZ is localized in the Tau-striated zone; 5) The iSVZ and oSVZ are marked by the presence of mitotic Pax6+ tRG cells; and 6) The iSVZ and oSVZ are sites of neurogenesis. We have shown that the iSVZ and oSVZ of macaque, ferret and rat meet these cytoarchitectural criteria. proportion of mitotic cells in each compartment that express markers such as Tbr2 differs in the iSVZ and oSVZ (see Table 9), demonstrating that each zone comprises a different cohort of precursor cells. However, whether or not neural progenitor cells in the iSVZ and oSVZ are functionally different remains to be determined. Neurogenesis in the macaque and ferret oSVZ has not been directly visualized but is widely accepted based on the presence of Tbr2+ cells in the oSVZ. We show here that Tbr2+ mitoses, the majority of which are presumed to be neurogenic divisions [4,16,38], are located throughout the macaque and ferret iSVZ and oSVZ (Fig. 11). Evidence for neurogenesis in the rodent iSVZ has been conclusively demonstrated by multiple laboratories over the past decade [4,5,16,38–40]. Evidence for neurogenesis in the structure we term oSVZ in the rat has been reported. We previously showed through time-lapse imaging of in vitro slices prepared from embryonic rats that intermediate progenitor cells migrate up to 175 mm from the ventricle before undergoing divisions that produce neurons that we identified through electrophysiological recordings [5]. Miyata and colleagues have shown cell divisions occurring at similar distances from the ventricle in developing mouse cortex [16]. In the current study we show in vivo that Tbr2+ mitoses are located up to 300 mm from the ventricle. These mitotic cells are superficial to the cell dense proliferative zone that surrounds the lateral ventricle and are located within the structure we term the oSVZ (see Fig. 22). We conclude that the rodent cortex is subdivided into an iSVZ and an oSVZ during later stages of cortical neurogenesis. In rat the oSVZ can be distinguished from the iSVZ starting at E18, which is the peak of neurogenesis for layer 3 neurons in the rat somatosensory cortex [21]. An important point that must be addressed concerns the nature of the iSVZ versus the oSVZ. Are these structures functionally distinct? Histologically the iSVZ and oSVZ are distinct (see Figs. 20, 21, 22, 23, 24). Defining the outer SVZ: does size matter? The cerebral cortex of macaques is much more extensive than that of rats or ferrets. This difference is reflected in total brain weight. The adult rat brain weighs approximately 2 grams, the adult ferret brain weighs approximately 7 grams and the adult macaque brain weighs 90–100 grams [32]. Furthermore, there are approximately 100 times more neurons in the macaque neocortex than in the rat (approximately two billion neurons in macaque [33,34], versus 20–30 million neurons in rat [35–37]). The Table 11. Proportion of mitotic cells in each zone that are Pax6+. Macaque age Proportion of total mitoses that are Pax6+ VZ iSVZ oSVZ CP (incl PP/SP/MZ) E50 95% (295/309) 99% (250/251) 78% (45/58) NA NA E65 94% (238/253) 98% (58/59) 93% (79/85) 93% (101/109) NA E80 88% (235/268) 100% (40/40) 98% (40/41) 87% (154/178) 11% (1/9) E100 86% (77/90) 100% (8/8) 100% (5/5) 93% (64/69) 0% (0/8) Ferret age Proportion of total mitoses that are Pax6+ VZ iSVZ oSVZ CP (incl PP/SP/MZ) E28 96% (128/134) 99% (92/93) 87% (34/39) 100% (2/2) NA E31 99% (89/90) 100% (59/59) 96% (26/27) 100% (4/4) NA E34 96% (99/103) 100% (46/46) 98% (41/42) 80% (12/15) NA E38 95% (77/81) 100% (37/37) 86% (18/21) 95% (20/21) 100% (2/2) P2 96% (99/103) 100% (34/34) 100% (41/41) 92% (24/26) 0% (0/2) P10 86% (72/84) 100% (8/8) 100% (4/4) 89% (54/61) 55% (6/11) Rat age Proportion of total mitoses that are Pax6+ VZ iSVZ oSVZ CP (incl PP/SP/MZ) E14 100% (53/53) 100% (46/46) 100% (7/7) NA NA E17 98% (80/82) 100% (50/50) 91% (20/22) 100% (10/10) NA E20 97% (74/76) 100% (30/30) 100% (34/34) 91% (10/11) 0% (0/1) P3 94% (61/65) 100% (4/4) 97% (28/29) 91% (21/23) 89% (8/9) Numbers represent the proportion of mitotic cells in each compartment of the developing somatosensory cortex that are Pax6+. Cells were identified by Pax6 immunoreactivity in a minimum of three 200 mm radial units from each age in each species. Mitoses were identified with 4A4 immunoreactivity or by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the number of Pax6+ mitotic cells and total mitotic cells counted in each compartment from each age in each species. doi:10.1371/journal.pone.0030178.t011 Table 11. Proportion of mitotic cells in each zone that are Pax6+. Table 11. Proportion of mitotic cells in each zone that are Pax6+. January 2012 | Volume 7 | Issue 1 | e30178 22 Mammalian Outer Subventricular Zone Table 12. Defining the outer SVZ: does size matter? Distribution of Pax6+ mitotic cells during neocortical development. Macaque age Total Pax6+ mitoses VZ iSVZ oSVZ CP (incl. PP/SP/MZ) E50 99 80% (79/99) 20% (20/99) 0% (0/99) 0% (0/99) E65 123 47% (58/123) 33% (40/123) 20% (25/123) 0% (0/123) E80 132 10% (13/132) 28% (37/132) 61% (81/132) 1% (1/132) E100 39 8% (3/39) 13% (5/39) 79% (31/39) 0% (0/39) Ferret age Total Pax6+ mitoses VZ iSVZ oSVZ CP (incl. PP/SP/MZ) E28 128 72% (92/128) 27% (34/128) 1% (2/128) 0% (0/128) E31 89 66% (59/89) 29% (26/89) 5% (4/89) 0% (0/89) E34 99 47% (46/99) 41% (41/99) 12% (12/99) 0% (0/99) E38 77 48% (37/77) 23% (18/77) 26% (20/77) 3% (2/77) P2 99 34% (34/99) 42% (41/99) 24% (24/99) 0% (0/99) Rat age Total Pax6+ mitoses VZ iSVZ oSVZ CP (incl. PP/SP/MZ) E14 53 87% (46/53) 13% (7/53) 0% (0/53) 0% (0/53) E17 80 63% (50/80) 25% (20/80) 12% (10/80) 0% (0/80) E20 74 41% (30/74) 46% (34/74) 13% (10/74) 0% (0/74) P3 61 7% (4/61) 46% (28/61) 34% (21/61) 13% (8/61) Numbers represent the proportion of Pax6+ mitotic cells in each compartment of the developing somatosensory cortex in each species. Cells were identified by Pax6 immunoreactivity in a minimum of three 200 mm radial unit sections from each age in each species. Mitoses were identified by 4A4 immunoreactivity and/or condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the total number of Pax6+ mitotic cells counted in each compartment from each age. doi:10.1371/journal.pone.0030178.t012 Numbers represent the proportion of Pax6+ mitotic cells in each compartment of the developing somatosensory cortex in each species. Cells were identified by Pax6 immunoreactivity in a minimum of three 200 mm radial unit sections from each age in each species. Mitoses were identified by 4A4 immunoreactivity and/or condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the total number of Pax6+ mitotic cells counted in each compartment from each age. doi:10.1371/journal.pone.0030178.t012 boundary between the iSVZ and oSVZ is marked by the boundary between the Tau-free and Tau-striated zones, and the oSVZ is localized in the Tau-striated zone; 5) The iSVZ and oSVZ are marked by the presence of mitotic Pax6+ tRG cells; and 6) The iSVZ and oSVZ are sites of neurogenesis. We have shown that the iSVZ and oSVZ of macaque, ferret and rat meet these cytoarchitectural criteria. January 2012 | Volume 7 | Issue 1 | e30178 Defining the outer SVZ: does size matter? In addition, the Does the outer SVZ produce gyrencephalic cortex? We examined the expression characteristics and distribution of precursor cells in the developing somatosensory cortex of mammals representing 3 orders: rodents (rat), carnivores (ferret), and primates (macaque). We expected to find more commonalities in the developmental organization of the SVZ in the gyrencephalic ferret and macaque, but instead found that lissencephalic rat and gyrencephalic ferret shared more in common. Our analyses showed that the distribution of Tbr2+ cells and Pax6+ cells, and mitotic cells in general, shift away from the ventricle to the oSVZ during early stages of macaque cortical neurogenesis. In contrast, the shift of precursor cells away from the ventricle to the oSVZ occurred much later in rats and ferrets and was not as pronounced. For example, in macaque the majority of Pax6+ cells shift to the oSVZ prior to genesis of layer 5 neurons, but in rats and ferrets the majority of Pax6+ cells remain in the VZ throughout cortical neurogenesis (Fig. 9). These results suggest that the shift of precursor cells to the oSVZ that occurs in macaque may not be a prerequisite for the development of gyrencephalic cortex since it does not occur in ferrets. The size of oSVZ is unquestionably a key determinant for the large number of cortical neurons in primate neocortex. In fact, we find that the macaque oSVZ is much thicker and has considerably more Tbr2+ cells than the ferret or rat oSVZ (see Fig. 8). However, it does not necessarily follow that the oSVZ is a crucial factor in the development of gyrencephaly, especially when considered in light of the differences we found between characteristics of oSVZ in ferret and macaque. Regional January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 23 Mammalian Outer Subventricular Zone Figure 12. The distribution of Pax6+ mitoses shifts from the ventricular zone (VZ) to the subventricular zone (oSVZ) during early stages of cortical development in macaque, but not in ferret or rat. (a–c) Images taken from coronal sections of the E80 macaque, P2 ferret and E20 rat oSVZ. Tissue was immunostained for 4A4 (green), Pax6 (red) and counterstained with DAPI (blue). Boxes to the right highlight examples of 4A4+ mitotic cells that express Pax6. Figure 12. The distribution of Pax6+ mitoses shifts from the ventricular zone (VZ) to the subventricular zone (oSVZ) during early stages of cortical development in macaque, but not in ferret or rat. (a–c) Images taken from coronal sections of the E80 macaque, P2 ferret and E20 rat oSVZ. Tissue was immunostained for 4A4 (green), Pax6 (red) and counterstained with DAPI (blue). Boxes to the right highlight examples of 4A4+ mitotic cells that express Pax6. (d–f) Graphs showing changes in the distribution of Pax6+ mitoses during development of the somatosensory cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. (d) At E50 in the macaque nearly 90% of Pax6+ mitoses were located in the VZ. There was a steady shift in the distribution of mitoses to the outer SVZ (oSVZ). By E80 the majority of Pax6+ mitoses (61%) were located in the oSVZ. (e) In ferret the largest proportion of Pax6+ mitoses were located in the VZ until late stages of neurogenesis. At E38, during neurogenesis of layer 2 neurons [19], 48% of Pax6+ mitoses were still located in the VZ, 23% were located in the iSVZ and 26% in the oSVZ. (f) A similar pattern was observed in the rat. The majority of Pax6+ mitoses were located in the VZ until E20. At E20 40% of Pax6+ mitoses were located in the VZ, 45% were in the iSVZ and 13% in the oSVZ. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green; CP: purple. PP, preplate; SP, subplate; MZ, marginal zone. doi:10.1371/journal.pone.0030178.g012 associated with gyrus and sulcus formation. In support of this idea, Reillo and colleagues (2011) reported that proliferation in the oSVZ is two to four times greater in regions of the developing ferret neocortex that produce gyri versus regions underlying sulci [42]. Mechanisms that could harness and regulate the proliferative output of precursor cells in the VZ and/or SVZ on a regional basis differences in the thickness of the SVZ have been proposed as one mechanism that could play a factor in gyrencephaly [41]. This hypothesis, which remains to be thoroughly tested, posits that gyri form over regions of the SVZ that are thicker while sulci form over regions of the SVZ that are thinner. Defining the outer SVZ: does size matter? (d–f) Graphs showing changes in the distribution of Pax6+ mitoses during development of the somatosensory cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. (d) At E50 in the macaque nearly 90% of Pax6+ mitoses were located in the VZ. There was a steady shift in the distribution of mitoses to the outer SVZ (oSVZ). By E80 the majority of Pax6+ mitoses (61%) were located in the oSVZ. (e) In ferret the largest proportion of Pax6+ mitoses were located in the VZ until late stages of neurogenesis. At E38, during neurogenesis of layer 2 neurons [19], 48% of Pax6+ mitoses were still located in the VZ, 23% were located in the iSVZ and 26% in the oSVZ. (f) A similar pattern was observed in the rat. The majority of Pax6+ mitoses were located in the VZ until E20. At E20 40% of Pax6+ mitoses were located in the VZ, 45% were in the iSVZ and 13% in the oSVZ. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green; CP: purple. PP, preplate; SP, subplate; MZ, marginal zone. doi:10.1371/journal.pone.0030178.g012 January 2012 | Volume 7 | Issue 1 | e30178 Alternatively, changes in the level of cell production in the proliferative zones could be January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 24 Mammalian Outer Subventricular Zone Figure 13. A subpopulation of Pax6+ mitotic cells coexpress Tbr2 in the outer subventricular zone (oSVZ) of macaque, ferret, and rat dorsal somatosensory cortex. (a) Image from E80 macaque oSVZ immunostained for Pax6 (green) and Tbr2 (red), and counterstained with DAPI (blue). Boxes highlight examples of Pax6+ mitoses. (b) Examples of Pax6+ mitotic cells from insets in (a) demonstrating two examples of Pax6+ mitoses that coexpress Tbr2 (1 and 2) and one example that does not (3). (c) E34 ferret oSVZ. An example of a Pax6+ mitosis that coexpresses Tbr2 and a second example that does not. (d) E20 rat oSVZ. An example of a Pax6+ mitosis that coexpresses Tbr2 and a second example that does not. In all three species most Tbr2+ mitotic cells express Pax6, although in most cases the level of Pax6 expression is low in Tbr2+ cells. There are significant numbers of Pax6+ mitotic cells in the oSVZ of each species that do not express Tbr2 (see Table 13). Scale bar applies to all images. doi:10.1371/journal.pone.0030178.g013 Figure 13. A subpopulation of Pax6+ mitotic cells coexpress Tbr2 in the outer subventricular zone (oSVZ) of macaque, ferret, and rat dorsal somatosensory cortex. (a) Image from E80 macaque oSVZ immunostained for Pax6 (green) and Tbr2 (red), and counterstained with DAPI (blue). Boxes highlight examples of Pax6+ mitoses. (b) Examples of Pax6+ mitotic cells from insets in (a) demonstrating two examples of Pax6+ mitoses that coexpress Tbr2 (1 and 2) and one example that does not (3). (c) E34 ferret oSVZ. An example of a Pax6+ mitosis that coexpresses Tbr2 and a second example that does not. (d) E20 rat oSVZ. An example of a Pax6+ mitosis that coexpresses Tbr2 and a second example that does not. In all three species most Tbr2+ mitotic cells express Pax6, although in most cases the level of Pax6 expression is low in Tbr2+ cells. There are significant numbers of Pax6+ mitotic cells in the oSVZ of each species that do not express Tbr2 (see Table 13). Scale bar applies to all images. doi:10.1371/journal.pone.0030178.g013 Table 13. Coexpression of Pax6 and Tbr2 in mitotic cells. Macaque age Total Pax6+ mitoses Total Tbr2+ mitoses Total Pax6+ Tbr2+ mitoses Percentage of Pax6+ mitoses that are Tbr2+ Percentage of Tbr2+ mitoses that are Pax6+ E50 176 47 30 17% (30/176) 64% (30/47) E65 244 122 120 49% (120/244) 98% (120/122) E80 72 37 37 51% (37/72) 100% (37/37) E100 38 20 17 45% (17/38) 85% (17/20) Ferret age Total Pax6+ mitoses Total Tbr2+ mitoses Total Pax6+ Tbr2+ mitoses Percentage of Pax6+ mitoses that are Tbr2+ Percentage of Tbr2+ mitoses that are Pax6+ E28 55 22 19 35% (19/55) 86% (19/22) E31 52 8 8 15% (8/52) 100% (8/8) E34 115 36 34 30% (34/115) 94% (34/36) P2 66 17 17 26% (17/66) 100% (17/17) P10 26 6 6 23% (6/26) 100% (6/6) Rat age Total Pax6+ mitoses Total Tbr2+ mitoses Total Pax6+ Tbr2+ mitoses Percentage of Pax6+ mitoses that are Tbr2+ Percentage of Tbr2+ mitoses that are Pax6+ E20 108 58 55 51% (55/108) 95% (55/58) Numbers represent the coexpression of Pax6 and Tbr2 by mitotic cells in the developing somatosensory cortex in each species. Cells were identified by Pax6 and/or Tbr2 immunoreactivity in a minimum of three 200 mm radial unit sections from each age in each species. Mitoses were identified by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the total number of mitotic cells of each type (Pax6 or Tbr2) that coexpress both proteins. doi:10.1371/journal.pone.0030178.t013 Numbers represent the coexpression of Pax6 and Tbr2 by mitotic cells in the developing somatosensory cortex in each species. Cells were identified by Pax6 and/or Tbr2 immunoreactivity in a minimum of three 200 mm radial unit sections from each age in each species. Mitoses were identified by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the total number of mitotic cells of each type (Pax6 or Tbr2) that coexpress both proteins. doi:10.1371/journal.pone.0030178.t013 January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 25 Mammalian Outer Subventricular Zone Figure 14. Mitotic translocating radial glial (tRG) cells in rodent cortex express Pax6. (a) Example of a mitotic tRG cell labeled with 4A4 (green). The pial fiber of the dividing cell is seen coursing to the pial surface. All mitotic tRG cells coexpressed Pax6 (red). DAPI stained nuclei are shown (blue). (b) An example of a GFP+ tRG cell (green) labeled through in utero retroviral injection at E16. The tRG cell (arrowhead) expresses Pax6 (red). PLoS ONE | www.plosone.org A daughter cell can be seen migrating along the pial fiber (arrow). All tRG cells labeled with retroviral injections expressed Pax6. (c) tRG cells labeled in the E18 mouse through in utero electroporation with a GFP expressing plasmid (green) expressed both Pax6 (red) and Sox2 (blue). Left panel shows a low magnification merged image of the tRG cell. Middle images show high magnification individual images of GFP (green), Pax6 (red) and Sox2 (blue). Right image shows high magnification merged image of GFP (green), Pax6 (red) and Sox2 (blue). All GFP+ cells with tRG morphology in mouse were Pax6+ and Sox2+. doi:10 1371/journal pone 0030178 g014 Figure 14. Mitotic translocating radial glial (tRG) cells in rodent cortex express Pax6. (a) Example of a mitotic tRG cell labeled with 4A4 (green). The pial fiber of the dividing cell is seen coursing to the pial surface. All mitotic tRG cells coexpressed Pax6 (red). DAPI stained nuclei are shown (blue). (b) An example of a GFP+ tRG cell (green) labeled through in utero retroviral injection at E16. The tRG cell (arrowhead) expresses Pax6 (red). A daughter cell can be seen migrating along the pial fiber (arrow). All tRG cells labeled with retroviral injections expressed Pax6. (c) tRG cells labeled in the E18 mouse through in utero electroporation with a GFP expressing plasmid (green) expressed both Pax6 (red) and Sox2 (blue). Left panel shows a low magnification merged image of the tRG cell. Middle images show high magnification individual images of GFP (green), Pax6 (red) and Sox2 (blue). Right image shows high magnification merged image of GFP (green), Pax6 (red) and Sox2 (blue). All GFP+ cells with tRG morphology in mouse were Pax6+ and Sox2+. doi:10.1371/journal.pone.0030178.g014 doi:10.1371/journal.pone.0030178.g014 have been proposed (e.g.: [43]), but remain to be fully understood. Other mechanisms may work in parallel to shape the formation of gyri and sulci. For example, Van Essen’s tension-based hypothesis posits that axonal connections between cortical areas define the patterns of sulci and gyri [44]. We believe that cell proliferation plays an important role in the formation of gyri but cannot rule out the possibility that multiple mechanisms work simultaneously in the developing forebrain to produce the complex pattern of sulci and gyri that characterize gyrencephalic neocortex. January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 26 Mammalian Outer Subventricular Zone Figure 15. Translocating RG (tRG) cells in the embryonic rat oSVZ divide and express Pax6. (a) Time lapse recording of a GFP-labeled tRG cell in the rat somatosensory cortex after an E16 retroviral injection. At the start of the experiment the labeled cell (red arrowhead) has RG morphology including a ventricular process and a pial process. The first timepoints of this movie focused on the RG cell body in the VZ and did not capture the pial process. An RG daughter cell (white arrowhead) is in contact with the pial process. The RG cell lost contact with the ventricle at t = 17 h, acquired tRG morphology, and began translocating toward the cortical plate. The first daughter cell (white arrowhead) remained in close proximity to its parent tRG cell for the duration of the experiment and did not divide. The tRG cell divided again at approximately t = 47 h (asterisk), producing a self-renewed tRG cell (red arrowhead) and a second daughter cell (white arrow). The tRG cell continued to translocate and divided once again at approximately t = 78 h, producing a third daughter cell (white arrowhead with red border). In addition, the second daughter cell also divided at approximately t = 78 h, producing two daughter cells with similar morphology. After an additional two hours the section was fixed in 4% PFA, sectioned on a cryostat and immunostained for Pax6 and NeuN. The clonal cells are shown on the far right image at higher magnification after fixation. (b) Lineage tree depicting the progeny of the RG cell in this time-lapse recording. (c) Immunostaining shows that the tRG cell expresses Pax6. The first daughter cell did not express Pax6. The daughter cells that were produced by division of the tRG second daughter cell (white arrow) expressed weak levels of Pax6. The tRG third daughter cell (white arrowhead with red border) expressed a moderate level of Pax6 that was lower in comparison to the level of Pax6 expression by the tRG mother cell. All cells in this clone were NeuN-negative. White arrowheads indicate the locations of clonal cells in each panel. This data shows that mitotically active RG cells and tRG cells maintain high levels of Pax6 expression, and suggests that non-RG daughter cells slowly downregulate Pax6 expression as they migrate toward the cortical plate. Scale bar in the right panel of (a) applies to all images in (c). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone. doi:10.1371/journal.pone.0030178.g015 Figure 15. Translocating RG (tRG) cells in the embryonic rat oSVZ divide and express Pax6. (a) Time lapse recording of a GFP-labeled tRG cell in the rat somatosensory cortex after an E16 retroviral injection. At the start of the experiment the labeled cell (red arrowhead) has RG morphology including a ventricular process and a pial process. The first timepoints of this movie focused on the RG cell body in the VZ and did not capture the pial process. An RG daughter cell (white arrowhead) is in contact with the pial process. The RG cell lost contact with the ventricle at t = 17 h, acquired tRG morphology, and began translocating toward the cortical plate. The first daughter cell (white arrowhead) remained in close proximity to its parent tRG cell for the duration of the experiment and did not divide. The tRG cell divided again at approximately t = 47 h (asterisk), producing a self-renewed tRG cell (red arrowhead) and a second daughter cell (white arrow). The tRG cell continued to translocate and divided once again at approximately t = 78 h, producing a third daughter cell (white arrowhead with red border). In addition, the second daughter cell also divided at approximately t = 78 h, producing two daughter cells with similar morphology. After an additional two hours the section was fixed in 4% PFA, sectioned on a cryostat and immunostained for Pax6 and NeuN. The clonal cells are shown on the far right image at higher magnification after fixation. (b) Lineage tree depicting the progeny of the RG cell in this time-lapse recording. (c) Immunostaining shows that the tRG cell expresses Pax6. The first daughter cell did not express Pax6. The daughter cells that were produced by division of the tRG second daughter cell (white arrow) expressed weak levels of Pax6. The tRG third daughter cell (white arrowhead with red border) expressed a moderate level of Pax6 that was lower in comparison to the level of Pax6 expression by the tRG mother cell. All cells in this clone were NeuN-negative. White arrowheads indicate the locations of clonal cells in each panel. This data shows that mitotically active RG cells and tRG cells maintain high levels of Pax6 expression, and suggests that non-RG daughter cells slowly downregulate Pax6 expression as they migrate toward the cortical plate. Scale bar in the right panel of (a) applies to all images in (c). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone. doi:10.1371/journal.pone.0030178.g015 Gliogenic and neurogenic tRG cells are present in the developing rat neocortex postnatal SVZ [30]. We show that during neurogenesis in the macaque, ferret and rat the majority of mitotic tRG cells are Pax6+ and Olig2-negative, but that by the end of neurogenesis the majority of mitotic tRG cells are Pax6+/Olig2+ (Fig. 18). We propose that these subtypes represent neurogenic and gliogenic precursor cells, respectively. We found that Pax6-expressing tRG cells are present in the developing rat neocortex as in ferret and macaque (Figs. 14, 15, 16). We have previously shown that translocating radial glial cells are present in the embryonic rat, that individual tRG cells in the embryonic brain divide multiple times and produce daughter cells that lack neuronal membrane properties [4,5]. Other groups have also investigated mitotic tRG cells and reported that they produce neuronal progeny based on marker expression [16,17]. We show that tRG cells express Pax6 and Sox2 in the rats and mice as they do in ferret and macaque (Fig. 17), and as has been reported in the mouse [17,18]. We further these findings by showing that mitotic Pax6-expressing tRG cells are not restricted in function to neurogenesis since they are present in the neocortex after cortical neurogenesis is complete (Fig. 16). We also show that tRG cells comprise multiple subtypes based on the expression of Olig2, which has been shown to direct astrocyte formation in the PLoS ONE | www.plosone.org G cells are a common cell type in mammalian We suggest that these cells, meticulously described by different laboratories in several mammalian species, belong to a single cell class and we propose the simplified term ‘tRG’ to encompass members of this cell class. This term reflects the key characteristics that identify tRG cells - translocation and radial glial identity. Furthermore, ‘tRG’ reflects the fact that these cells translocate through and are present in the VZ, the iSVZ, and the oSVZ. It is likely that subtypes of tRG cells exist within this class. Indeed, recent studies have advanced our understanding of the functions of tRG cells [10,15], and have also shown that the tRG cell class includes outer VZ progenitor cells [18], and basal radial glia [47,48]. In all cases the translocating cells share a core set of characteristics: maintenance of the pial process, translocation away from the ventricle toward the overlying cortical plate, loss of the ventricular contacting process, and asymmetric cell division that produces self renewed Pax6+ translocating radial glial cells. We suggest that these cells, meticulously described by different laboratories in several mammalian species, belong to a single cell class and we propose the simplified term ‘tRG’ to encompass members of this cell class. This term reflects the key characteristics that identify tRG cells - translocation and radial glial identity. Furthermore, ‘tRG’ reflects the fact that these cells translocate through and are present in the VZ, the iSVZ, and the oSVZ. It is likely that subtypes of tRG cells exist within this class. Indeed, recent studies have advanced our understanding of the functions of tRG cells [10,15], and have also shown that the tRG cell class includes diverse subtypes that can be identified based on marker expression [44]. In addition, our current results demonstrate that the transcription factor Olig2 can be used to distinguish two Pax6+ tRG cell subtypes (Fig. 19): those that presumably generate neurons and those that presumably generate astrocytes. Future studies should more closely examine the commonalities and differences between the various translocating cells that have been identified in the mammalian neocortex. Some recent studies have proposed that tRG cells are unique to the developing primate cortex, and are sparsely present in rodents. However, our analysis shows that tRG cells are also quite prominent in rodents. G cells are a common cell type in mammalian RG cells have long been known to translocate away from the ventricle and transform into astrocytes [12–15]. Recent work has shown translocating RG cells are present in humans [10,11], non- human primates including lissencephalic primates [45], carnivores such as ferrets [11,46], and rodents [17,18]. Furthermore, recent work shows that translocating radial glial cells in the oSVZ divide and produce neuronal and glial daughter cells. Multiple names have been applied to these cells including outer RG (oRG) cells [10], oSVZ progenitor cells [11], intermediate radial glia [42], January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 27 Mammalian Outer Subventricular Zone Figure 16. tRG cells express Pax6 during and after neurogenesis. (a–c) 4A4+ tRG cells (green) in the E17 rat somatosensory cortex that express Pax6 (red). Blue channel shows DAPI staining. (d–f) Examples of 4A4+ tRG cells (green) in post-neurogenic somatosensory cortex of macaque (d), ferret (e), and rat (f) that also express Pax6 (red). Blue channel shows DAPI staining. Scale bar in left panels applies to all panels in each set. doi:10.1371/journal.pone.0030178.g016 Figure 16. tRG cells express Pax6 during and after neurogenesis. (a–c) 4A4+ tRG cells (green) in the E17 rat somatosensory cortex th express Pax6 (red). Blue channel shows DAPI staining. (d–f) Examples of 4A4+ tRG cells (green) in post-neurogenic somatosensory cortex of macaq (d), ferret (e), and rat (f) that also express Pax6 (red). Blue channel shows DAPI staining. Scale bar in left panels applies to all panels in each set doi:10.1371/journal.pone.0030178.g016 Figure 16. tRG cells express Pax6 during and after neurogenesis. (a–c) 4A4+ tRG cells (green) in the E17 rat somatosensory cortex that express Pax6 (red). Blue channel shows DAPI staining. (d–f) Examples of 4A4+ tRG cells (green) in post-neurogenic somatosensory cortex of macaque (d), ferret (e), and rat (f) that also express Pax6 (red). Blue channel shows DAPI staining. Scale bar in left panels applies to all panels in each set. doi:10.1371/journal.pone.0030178.g016 outer VZ progenitor cells [18], and basal radial glia [47,48]. In all cases the translocating cells share a core set of characteristics: maintenance of the pial process, translocation away from the ventricle toward the overlying cortical plate, loss of the ventricular contacting process, and asymmetric cell division that produces self renewed Pax6+ translocating radial glial cells. January 2012 | Volume 7 | Issue 1 | e30178 Identifying the inner and outer SVZ Seminal work that first described the oSVZ was performed in the macaque visual cortex [9]. In the macaque visual cortex the inner fiber layer (IFL) divides the iSVZ and oSVZ by age E78, and the outer fiber layer (OFL) serves as the superficial boundary for the oSVZ [9]. We found that the IFL and OFL were not apparent in coronal sections of the somatosensory cortex or other cortical areas in the frontal lobe (Figs. 5, 19 and 20). Smart et al., (2002) reported that the IFL extends from the ganglionic eminence to the pole of the occipital lobe [9]. At E65 in macaque somatosensory cortex we observed a slight decrease in cell density between the iSVZ and oSVZ (Fig. 19), but the IFL was not present at E80 in somatosensory cortex (Fig. 20). The IFL and OFL were not apparent at any age in ferret or rat (Figs. 21 and 22). We developed a new approach for identifying the boundaries between the VZ, iSVZ and oSVZ in any cortical region of the rat, ferret and macaque based on DAPI, Tbr2/Pax6, and Tau-1 staining (Fig. 24). This method makes identification of the iSVZ and oSVZ an unambiguous task. G cells are a common cell type in mammalian Our current results match previous studies in which we (Noctor et al., 2004, 2008) and others (Miyata et al., 2004) demonstrated through detailed time lapse analysis the presence of mitotic translocating RG cells in the embryonic rodent neocortex that maintain their pial process, translocate away from PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org PLo January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 28 Mammalian Outer Subventricular Zone Figure 17. Most mitotic Pax6+ cells express Sox2 during and after neurogenesis. (a) Coronal section from E17 rat somatosensory cortex immunostained for Pax6 (red) and Sox2 (green) and counter- stained with DAPI (blue). Boxes (1 and 2) show examples of Pax6+ mitotic cells that also express Sox2. (b) Coronal section from P3 rat somatosensory cortex immunostained for Pax6 (red) and Sox2 (green) and counterstained with DAPI (blue). Boxes (1 and 2) show examples of Pax6+ mitotic cells that also express Sox2. There was greater overlap of Pax6 and Sox2 expression at E17 than at P3, but the extent of colabeling in mitotic cells was similar at each age (see Table 14). Scale bar in (a) applies to all images. doi:10.1371/journal.pone.0030178.g017 the ventricle and undergo asymmetric divisions that yield self- renewed RG cells that retain translocating morphology [4,5,16]. We invite interested parties to view detailed time-lapse movies of translocating RG cells in the embryonic rat in online supplemental information accompanying our previous publications [4,5]. In addition, we present new data showing that Pax6+ tRG cells are still present in the postnatal rat neocortex at P3, and are present after neurogenesis has ended in P10 ferret and E151 macaque somatosensory cortex (Fig. 16). The function of mitotic tRG cells in the post-neurogenic neocortex is presumably related to gliogenesis, but this remains to be confirmed. January 2012 | Volume 7 | Issue 1 | e30178 Conclusion Numbers in ber of mitotic cells of each type (Sox2 or Pax6) that coexpress both proteins. 014 Numbers represent the coexpression of Sox2 and Pax6 by mitotic cells in the developing rat somatosensory cortex. Cells were identified by Sox2 and/or Pax6 immunoreactivity in a minimum of three 200 mm radial unit sections at each age. Mitoses were identified by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the total number of mitotic cells of each type (Sox2 or Pax6) that coexpress both proteins. doi:10.1371/journal.pone.0030178.t014 at 48 hrs post transfection and virus was concentrated by centrifugation at 50,0006g for 1.5 hours at 4uC, resuspended in Opti-MEM (Invitrogen), and stored at 280uC until use. rodent and primate cortex is one of quantity, not quality. We conclude that the developing rodent cerebral cortex shares key features with that of primates, and therefore serves as a valid model for understanding human cortical development under normal and pathological conditions. Nissl Staining Cryostat-sectioned coronal sections (14 mm) from embryonic rats, ferrets and macaques were mounted on Superfrost Plus slides. Slides were hydrated in a series of two minute incubations as follows: 100% ethyl alcohol (EtOH), 96% EtOH, 70% EtOH, 50% EtOH, and two incubations in Milli-Q H2O. Slides were then incubated for two minutes in a 0.1% cresyl violet solution. Slides were dehydrated in a series of two minute incubations as follows: Milli-Q H2O, Milli-Q H2O, 50% EtOH, 70% EtOH, and 96% EtOH. Slides were then incubated in 100% chloroform for two minutes on a shaker. Cresyl Violet stain was differentiated in 95% EtOH+glacial acetic acid until nucleoli were clearly visible (2–10 minutes). Slides were placed in 100% EtOH for two minutes, incubated in Safeclear (Fisher) for five minutes and coverslipped in DPX mounting medium (EMS). Preparation of Retrovirus Replication-incompetent pantropic retrovirus encoding en- hanced green fluorescent protein (eGFP) was prepared as described previously [3]. 293gp NIT-GFP cells were grown to 70–80% confluency and transiently transfected with pVSV-G (Clontech, generous gift of Dr. James Angelastro) using Lipofecta- mine 2000 (Invitrogen) or CalPhos Mammalian Transfection Kit (Clontech) to pseudotype viral particles. Supernatant was collected Tissue Processing All animals were transcardially perfused with 4% paraformal- dehyde (PFA) with the exception of E14 rats and E23 ferrets, which were immersed in 4% PFA overnight. Brains were removed, post-fixed in 4% PFA for two hours, cryoprotected overnight in 30% sucrose, sectioned coronally on a cryostat, mounted on Superfrost Plus slides (Fisher) and stored at 220uC until use. In Utero Intracerebral Retroviral Infection Pregnant rats at gestation days E16 through E20 were anesthetized with 3–5% Isoflurane, a laparotomy was performed, and the uterine horns containing embryos were temporarily removed. A solution containing concentrated retrovirus was injected into the lateral ventricles of embryos and uterine horns were replaced in the peritoneal cavity. During injections the uterine horns were lavaged with warm artificial cerebral spinal fluid (aCSF) containing (in mM): NaCl 125, KCl 5, NaH2PO4 1.25, MgSO4 1, CaCl2 2, NaHCO3 25, and glucose 20, pH 7. The peritoneal cavity was lavaged with aCSF and the incision was sutured. The mother was allowed to recover from anesthesia and treated with Buprenorphine for analgesia. Embryos were allowed to develop for 1–16 days (E17 - P10), removed from the mother and some embryos were transcardially perfused with 4% PFA. Prior to perfusion postnatal animals were anesthetized using ice- cold anesthesia according to UC Davis IACUC guidelines. Brains were removed, cryoprotected in 30% sucrose, and sectioned on a cryostat. Cells with morphology of tRG cells were identified, and slices containing cells were processed for immunohistochemistry (see below). Animals Procedures on rodents (Sprague Dawley rats and Swiss Webster mice) were approved by the Institutional Animal Care and Use Committee of the University of California, Davis and were conducted in accordance with the National Institutes of Health guidelines for the use of animals in research (Protocol #16368). Ferret tissue (Mustela putorius) was collected by the laboratories of Drs. Francisco Clasca, Sharon Juliano and Barbara Chapman for other purposes and sections of brain tissue were provided for this study as a generous gift. Macaque tissue (Macaca mulatta) was collected by the laboratory of Dr. David Amaral for another purpose [49], and sections of brain tissue were provided for this study as a generous gift. Procedures on macaques were approved by the Institutional Animal Care and Use Committee of the University of California, Davis (protocol # 12139), and strictly adhered to National Institutes of Health policies on primate animal subjects. Rat brains that were used included embryonic day (E)13, E14, E17, E18, E20, E21 and postnatal day (P)1, P2, P3, P7, and P10. E18 mice brains were used. Ferret tissue was obtained from animals at E23, E28, E31, E34, E38, P2, and P10. Macaque tissue was obtained from animals at E50, E65, E80, E100 and E151. For rats, ferrets and macaques these ages encompassed the neurogenic and early gliogenic phase for somatosensory cortex in each species [19–21]. Conclusion Rodents and primates are both in the superorder Euarchonto- glires while ferrets are in the superorder Laurasiatheria. Therefore macaques are more closely related to lissencephalic rats than they are to gyrencephalic ferrets. This suggests that gyrencephalic cortex may have evolved independently in ferrets and macaques, or alternatively that a blueprint for cortical expansion was present in ancestral mammalian species. We prefer the second hypothesis and propose that this ‘blueprint’ is the oSVZ. We show that rats possess an oSVZ, albeit limited in size and only present during late stages of cortical neurogenesis. Understanding how expansion of this structure occurred, for example through integrin signaling [11], and how its neurogenic function is maintained over a longer developmental period in some species will shed light on mechanisms that drove evolution of the human cerebral cortex. The complexity of laminar structures in the developing rat neocortex has been described in detail by Bayer and Altman [21], and the functional significance of the rat proliferative zones has become clear during the past decade as precursor cell types have been identified. Perhaps one of the greatest differences between PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 29 Mammalian Outer Subventricular Zone Table 14. Coexpression of Sox2 and Pax6 in mitotic cells. Rat age Total Sox2+ mitoses Total Pax6+ mitoses Total Sox2+ Pax6+ mitoses Percentage of Sox+ mitoses that are Pax6+ Percentage of Pax6+ mitoses that are Sox2+ E14 26 26 26 100% (26/26) 100% (26/26) E17 81 83 79 98% (79/81) 95% (79/83) E20 76 89 75 99% (75/76) 84% (75/89) P3 32 32 31 97% (31/32) 97% (31/32) Numbers represent the coexpression of Sox2 and Pax6 by mitotic cells in the developing rat somatosensory cortex. Cells were identified by Sox2 and/or Pax6 immunoreactivity in a minimum of three 200 mm radial unit sections at each age. Mitoses were identified by condensed chromatin in DAPI stained tissue. Numbers in parentheses indicate the total number of mitotic cells of each type (Sox2 or Pax6) that coexpress both proteins. doi:10.1371/journal.pone.0030178.t014 on of Sox2 and Pax6 by mitotic cells in the developing rat somatosensory cortex. Cells were identified by Sox2 and/or Pax6 three 200 mm radial unit sections at each age. Mitoses were identified by condensed chromatin in DAPI stained tissue. Time-Lapse Recording of tRG Cells One to four days post injection the pregnant mother was anesthetized with Isoflurane, a laparotomy was performed and uterine horns exposed. Embryos were removed one at a time and decapitated on ice. Brains were removed, embedded in 4% Low Melting Point Agarose (Promega) and sectioned at 400 mm on a PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 30 Mammalian Outer Subventricular Zone vibratome (Ted Pella) in bubbling carbogenated (95% O2/5% Glutamine (Invitrogen) and (w/v) 0.66% D+ Glucose (Sigma). Figure 18. Olig2 expression distinguishes two subtypes of tRG cells in macaque, ferret and rat. (a–c) Examples of 4A4+ tRG cells (green) in the E80 macaque (a), P2 ferret (b), and E21 rat (c), that express Pax6 (red). Shown are examples of Pax6+ tRG cells that are Olig2-negative (top row of panels), or Olig2+ (lower row of panels, Olig2 immunostaining: blue). (d–f) Graphs showing changes in the proportion of Pax6+ tRG cells that express Olig2 during cortical development. During early stages of cortical development most Pax6+ cells were Olig2-negative in each species. The proportion of Pax6+ tRG cells that expressed Olig2 increased as development proceeded. Graphs also show Pax6(2)/Olig2+, or Pax6(2)/Olig2(2) 4A4+ cells with a pial fiber. Scale bar in (a) applies to (b). doi:10.1371/journal.pone.0030178.g018 Figure 18. Olig2 expression distinguishes two subtypes of tRG cells in macaque, ferret and rat. (a–c) Examples of 4A4+ tRG cells (green) in the E80 macaque (a), P2 ferret (b), and E21 rat (c), that express Pax6 (red). Shown are examples of Pax6+ tRG cells that are Olig2-negative (top row of panels), or Olig2+ (lower row of panels, Olig2 immunostaining: blue). (d–f) Graphs showing changes in the proportion of Pax6+ tRG cells that express Olig2 during cortical development. During early stages of cortical development most Pax6+ cells were Olig2-negative in each species. The proportion of Pax6+ tRG cells that expressed Olig2 increased as development proceeded. Graphs also show Pax6(2)/Olig2+, or Pax6(2)/Olig2(2) 4A4+ cells with a pial fiber. Scale bar in (a) applies to (b). doi:10.1371/journal.pone.0030178.g018 Figure 18. Olig2 expression distinguishes two subtypes of tRG cells in macaque, ferret and rat. (a–c) Examples of 4A4+ tRG cells (green) in the E80 macaque (a), P2 ferret (b), and E21 rat (c), that express Pax6 (red). Shown are examples of Pax6+ tRG cells that are Olig2-negative (top row of panels), or Olig2+ (lower row of panels, Olig2 immunostaining: blue). January 2012 | Volume 7 | Issue 1 | e30178 Time-Lapse Recording of tRG Cells (d–f) Graphs showing changes in the proportion of Pax6+ tRG cells that express Olig2 during cortical development. During early stages of cortical development most Pax6+ cells were Olig2-negative in each species. The proportion of Pax6+ tRG cells that expressed Olig2 increased as development proceeded. Graphs also show Pax6(2)/Olig2+, or Pax6(2)/Olig2(2) 4A4+ cells with a pial fiber. Scale bar in (a) applies to (b). doi:10.1371/journal.pone.0030178.g018 Glutamine (Invitrogen) and (w/v) 0.66% D+ Glucose (Sigma). Slices were placed in a humidified, 37uC incubator containing ambient oxygen and 5% CO2 and allowed to equilibrate for four hours. Six-well plates containing slices were removed from the incubator and cells were imaged on an Olympus FV1000 confocal microscope at 2–3 hour intervals for up to 84 hours. Projection vibratome (Ted Pella) in bubbling carbogenated (95% O2/5% CO2) aCSF. Slices were placed in bubbling carbogenated aCSF until all brains were sectioned. Sections were then transferred to six-well plates containing Millicell culture inserts (Millipore) and slice culture media containing (v/v): 66% BME, 25% HBSS, 5% Fetal Bovine Serum, 1% N-2 Supplement, 1% Pen/Strep/ PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 31 Mammalian Outer Subventricular Zone Table 15. Pax6 and Olig2 expression by 4A4+ cells with a pial fiber (tRG cells). Time-Lapse Recording of tRG Cells doi:10.1371/journal.pone.0030178.t015 Numbers represent the expression of Pax6 and Olig2 by 4A4+ mitotic cells with a pial fiber (tRG cells) in the developing somatosensory cortex in each species. Cells were identified by 4A4, Pax6 and Olig2 immunoreactivity in a minimum of three 200 mm radial units from each age in each species. Numbers in parentheses indicate the total number of tRG cells of each type (Pax6 or Olig2) that coexpress both proteins. doi:10.1371/journal.pone.0030178.t015 Figure 19. The outer fiber layer (OFL) is not present in cortical areas rostral to the occipital lobe. (a, c) Nissl-stained coronal sections of somatosensory and visual cortex from the E65 macaque. (b,d) Higher magnification images taken from the sections shown in (a) and (c). At E65 the OFL is visible in visual cortex superficial to the outer subventricular zone (oSVZ), but the inner fiber layer has not yet developed. The OFL is not apparent in somatosensory cortex. The oSVZ in somatosensory cortex is characterized by a striated appearance created by tangential streams of cells. Scale bar in (b) applies to (d). IC, internal capsule; AC, anterior commissure; VZ, ventricular zone; iSVZ, inner subventricular zone; SP, subplate, CP, cortical plate. doi:10.1371/journal.pone.0030178.g019 images were made from Z-stacks that included all visible processes of individual GFP+ cells, and all GFP+ cells in individual clones on a PC running Fluoview (Olympus). Laser power was monitored to prevent phototoxicity. Transmitted light images of the slices were taken periodically to track cell movements within the slices. Time- lapse sequences were reconstructed using Photoshop (Adobe). The position of labeled cells was registered at each timepoint using transmitted light images, ventricular surface, adjacent clones, and the pial surface. After time-lapse recording, slices were fixed in 4% PFA for 15 minutes at RT, cryoprotected in 30% sucrose, sectioned at 50 mm on a cryostat, mounted on slides and processed for immunohistochemistry (see below). In utero electroporation Timed pregnant mice at E16 were anesthetized as above. A laparotomy was performed and the uterine horns were removed from the peritoneal cavity. In utero intracerebral injection of pEGFP-N1 (1.0 mg/mL, Clontech) was performed into the lateral ventricles of the embryos. Tweezertrode electrodes (Harvard Apparatus) were positioned with the positive electrode over the dorsal cortex of the embryos. A square wave electroporator (Harvard Apparatus) was used to drive five pulses at 50 ms/pulse, 30 volts/pulse, with a 1 second interval between pulses. After electroporation, the uterine horns were replaced and lavaged as above. The incision was sutured, and the mother was given Buprenorphine for analgesia. The mother was allowed to recover from anesthesia and survived until E18, when the embryos were removed, perfused and cryosectioned as above. Figure 19. The outer fiber layer (OFL) is not present in cortical areas rostral to the occipital lobe. (a, c) Nissl-stained coronal sections of somatosensory and visual cortex from the E65 macaque. (b,d) Higher magnification images taken from the sections shown in (a) and (c). At E65 the OFL is visible in visual cortex superficial to the outer subventricular zone (oSVZ), but the inner fiber layer has not yet developed. The OFL is not apparent in somatosensory cortex. The oSVZ in somatosensory cortex is characterized by a striated appearance created by tangential streams of cells. Scale bar in (b) applies to (d). IC, internal capsule; AC, anterior commissure; VZ, ventricular zone; iSVZ, inner subventricular zone; SP, subplate, CP, cortical plate. doi:10.1371/journal.pone.0030178.g019 Time-Lapse Recording of tRG Cells Macaque age Total tRG cells Proportion of tRG cells that are Pax6+/Olig2(2) Proportion of tRG cells that are Pax6+/Olig2+ Proportion of tRG cells that are Pax6(2)/Olig2+ Proportion of tRG cells that are Pax6(2)/Olig2(2) E65 66 94% (62/66) 1% (1/66) 0% (0/66) 5% (3/66) E80 56 57% (32/56) 38% (21/56) 0% (0/56) 5% (3/56) E100 21 24% (5/21) 43% (9/21) 9% (2/21) 24% (5/21) Ferret age Total tRG cells Proportion of tRG cells that are Pax6+/Olig2(2) Proportion of tRG cells that are Pax6+/Olig2+ Proportion of tRG cells that are Pax6(2)/Olig2+ Proportion of tRG cells that are Pax6(2)/Olig2(2) E34 50 92% (46/50) 2% (1/50) 0% (0/50) 6% (3/50) P2 72 81% (58/72) 18% (13/72) 0% (0/72) 1% (1/72) P10 26 31% (8/26) 61% (16/26) 0% (0/26) 8% (2/26) Rat age Total tRG cells Proportion of tRG cells that are Pax6+/Olig2(2) Proportion of tRG cells that are Pax6+/Olig2+ Proportion of tRG cells that are Pax6(2)/Olig2+ Proportion of tRG cells that are Pax6(2)/Olig2(2) E17 18 100% (18/18) 0% (0/18) 0% (0/18) 0% (0/18) E19 54 78% (42/54) 22% (12/54) 0% (0/54) 0% (0/54) E21 31 33% (10/31) 55% (17/31) 6% (2/31) 6% (2/31) P2 30 3% (1/30) 77% (23/30) 7% (2/30) 13% (4/30) Numbers represent the expression of Pax6 and Olig2 by 4A4+ mitotic cells with a pial fiber (tRG cells) in the developing somatosensory cortex in each species. Cells were identified by 4A4, Pax6 and Olig2 immunoreactivity in a minimum of three 200 mm radial units from each age in each species. Numbers in parentheses indicate the total number of tRG cells of each type (Pax6 or Olig2) that coexpress both proteins. doi:10.1371/journal.pone.0030178.t015 of Pax6 and Olig2 by 4A4+ mitotic cells with a pial fiber (tRG cells) in the developing somatosensory cortex in each species. Cells were mmunoreactivity in a minimum of three 200 mm radial units from each age in each species. Numbers in parentheses indicate the total Pax6 or Olig2) that coexpress both proteins. 015 Numbers represent the expression of Pax6 and Olig2 by 4A4+ mitotic cells with a pial fiber (tRG cells) in the developing somatosensory cortex in each species. Cells were identified by 4A4, Pax6 and Olig2 immunoreactivity in a minimum of three 200 mm radial units from each age in each species. Numbers in parentheses indicate the total number of tRG cells of each type (Pax6 or Olig2) that coexpress both proteins. However, note that the OFL-like structure in ferret is located within the oSVZ, in contrast to the OFL in macaque, which is superficial to the oSVZ. In both somatosensory and visual cortex the boundary between the inner SVZ (iSVZ) and oSVZ can be visualized in Nissl stained tissue as a sharp border created by different cellular density. The superficial boundary of the oSVZ can also be visualized in Nissl stained tissue based on cell density (determined through Tbr2 immunostaining). In somatosensory cortex the oSVZ is characterized by a striated appearance of cells organized into clusters that appear to stream at an oblique or tangential angle through the oSVZ. (e–g) The OFL-like structure in ferret resembles the macaque OFL since radially oriented clusters of cells stream through this structure as they do in macaque (see Fig. 4). But the ferret OFL-like structure differs from the macaque OFL since it is within the oSVZ rather than superficial to the oSVZ and because Tbr2+ cells are located throughout the OFL- like structure in ferret but do not penetrate the macaque OFL (See Fig. 4). VZ, ventricular zone; CP, cortical plate. Scale bar in (b) applied to (d). Scale bar in (g) applied to (e, f). doi:10.1371/journal.pone.0030178.g021 Figure 20. The inner fiber layer (IFL) and outer fiber layer (OFL) are present in the E80 macaque visual cortex but are not apparent in other cortical areas. (a,c) Nissl-stained coronal sections of somatosensory and visual cortex taken from E80 macaque. (b,d) Higher magnification images taken from the sections shown in (a) and (c). Dotted line in (b) represents the outer boundary of the outer subventricular zone (oSVZ) in somatosensory cortex determined through Tbr2 immunostaining on adjacent sections (see Fig. 5). The outer fiber layer (OFL) and inner fiber layer (IFL) are visible in the visual cortex but are not apparent in cortical areas rostral to the occipital lobe. In somatosensory cortex the boundary between the inner SVZ (iSVZ) and oSVZ can be visualized in Nissl stained tissue as a sharp border created by differences in cell density. In the visual cortex the IFL divides the iSVZ from the oSVZ. In somatosensory cortex the oSVZ extends much farther from the ventricle than it does in visual cortex and is characterized by a striated appearance. Immunohistochemistry In both somatosensory and visual cortex the boundary between the inner SVZ (iSVZ) and oSVZ can be visualized in Nissl stained tissue as a sharp border created by different cellular density. The superficial boundary of the oSVZ can also be visualized in Nissl stained tissue based on cell density (determined through Tbr2 immunostaining). In somatosensory cortex the oSVZ is characterized by a striated appearance of cells organized into clusters that appear to stream at an oblique or tangential angle through the oSVZ. (e–g) The OFL-like structure in ferret resembles the macaque OFL since radially oriented clusters of cells stream through this structure as they do in macaque (see Fig. 4). But the ferret OFL-like structure differs from the macaque OFL since it is within the oSVZ rather than superficial to the oSVZ and because Tbr2+ cells are located throughout the OFL- like structure in ferret but do not penetrate the macaque OFL (See Fig. 4). VZ, ventricular zone; CP, cortical plate. Scale bar in (b) applied to (d). Scale bar in (g) applied to (e, f). doi:10.1371/journal.pone.0030178.g021 Fi 21 I d t SVZ i th d l i f t t Figure 20. The inner fiber layer (IFL) and outer fiber layer (OFL) are present in the E80 macaque visual cortex but are not apparent in other cortical areas. (a,c) Nissl-stained coronal sections of somatosensory and visual cortex taken from E80 macaque. (b,d) Higher magnification images taken from the sections shown in (a) and (c). Dotted line in (b) represents the outer boundary of the outer subventricular zone (oSVZ) in somatosensory cortex determined through Tbr2 immunostaining on adjacent sections (see Fig. 5). The outer fiber layer (OFL) and inner fiber layer (IFL) are visible in the visual cortex but are not apparent in cortical areas rostral to the occipital lobe. In somatosensory cortex the boundary between the inner SVZ (iSVZ) and oSVZ can be visualized in Nissl stained tissue as a sharp border created by differences in cell density. In the visual cortex the IFL divides the iSVZ from the oSVZ. In somatosensory cortex the oSVZ extends much farther from the ventricle than it does in visual cortex and is characterized by a striated appearance. In the E80 macaque the ventricular zone has become very thin and the cell dense proliferative zone that surrounds the lateral ventricle consists almost entirely of iSVZ. Figure 21. Inner and outer SVZ in the developing ferret cortex. (a,c) Nissl-stained coronal sections of somatosensory and visual cortex from P2 ferret. (b,d) Higher magnification images taken from the sections shown in (a) and (c). Dotted line in (b) represents the outer boundary of the outer subventricular zone (oSVZ) in somatosensory cortex determined through Tbr2 immunostaining. The outer fiber layer (OFL) and inner fiber layer are not present in somatosensory cortex, but a structure that resembles the OFL (OFL-like) is apparent in some areas of the ferret occipital lobe. However, note that the OFL-like structure in ferret is located within the oSVZ, in contrast to the OFL in macaque, which is superficial to the oSVZ. In both somatosensory and visual cortex the boundary between the inner SVZ (iSVZ) and oSVZ can be visualized in Nissl stained tissue as a sharp border created by different cellular density. The superficial boundary of the oSVZ can also be visualized in Nissl stained tissue based on cell density (determined through Tbr2 immunostaining). In somatosensory cortex the oSVZ is characterized by a striated appearance of cells organized into clusters that appear to stream at an oblique or tangential angle through the oSVZ. (e–g) The OFL-like structure in ferret resembles the macaque OFL since radially oriented clusters of cells stream through this structure as they do in macaque (see Fig. 4). But the ferret OFL-like structure differs from the macaque OFL since it is within the oSVZ rather than superficial to the oSVZ and because Tbr2+ cells are located throughout the OFL- like structure in ferret but do not penetrate the macaque OFL (See Fig. 4). VZ, ventricular zone; CP, cortical plate. Scale bar in (b) applied to (d). Scale bar in (g) applied to (e, f). doi:10.1371/journal.pone.0030178.g021 Figure 21. Inner and outer SVZ in the developing ferret cortex. l d l f d l Figure 21. Inner and outer SVZ in the developing ferret cortex. (a,c) Nissl-stained coronal sections of somatosensory and visual cortex from P2 ferret. (b,d) Higher magnification images taken from the sections shown in (a) and (c). Dotted line in (b) represents the outer boundary of the outer subventricular zone (oSVZ) in somatosensory cortex determined through Tbr2 immunostaining. The outer fiber layer (OFL) and inner fiber layer are not present in somatosensory cortex, but a structure that resembles the OFL (OFL-like) is apparent in some areas of the ferret occipital lobe. Immunohistochemistry The outer fiber layer (OFL) and inner fiber layer are not present in somatosensory cortex, but a structure that resembles the OFL (OFL-like) is apparent in some areas of the ferret occipital lobe. However, note that the OFL-like structure in ferret is located within the oSVZ, in contrast to the OFL in macaque, which is superficial to the oSVZ. In both somatosensory and visual cortex the boundary between the inner SVZ (iSVZ) and oSVZ can be visualized in Nissl stained tissue as a sharp border created by different cellular density. The superficial boundary of the oSVZ can also be visualized in Nissl stained tissue based on cell density (determined through Tbr2 immunostaining). In somatosensory cortex the oSVZ is characterized by a striated appearance of cells organized into clusters that appear to stream at an oblique or tangential angle through the oSVZ. (e–g) The OFL-like structure in ferret resembles the macaque OFL since radially oriented clusters of cells stream through this structure as they do in macaque (see Fig. 4). But the ferret OFL-like structure differs from the macaque OFL since it is within the oSVZ rather than superficial to the oSVZ and because Tbr2+ cells are located throughout the OFL- like structure in ferret but do not penetrate the macaque OFL (See Fig. 4). VZ, ventricular zone; CP, cortical plate. Scale bar in (b) applied to (d). Scale bar in (g) applied to (e, f). doi:10.1371/journal.pone.0030178.g021 anti-Phosphohistone H3 (PH3) 1:50 (Chemicon), goat anti-Olig2 1:500 (R&D Systems), chicken anti-GFP 1:500 (Abcam) and rat anti-BrdU-FITC 1:50 (Abcam). Sections were rinsed in 0.1 M PBS then incubated for one hour at RT in secondary antibody Figure 21. Inner and outer SVZ in the developing ferret cortex. (a,c) Nissl-stained coronal sections of somatosensory and visual cortex from P2 ferret. (b,d) Higher magnification images taken from the sections shown in (a) and (c). Dotted line in (b) represents the outer boundary of the outer subventricular zone (oSVZ) in somatosensory cortex determined through Tbr2 immunostaining. The outer fiber layer (OFL) and inner fiber layer are not present in somatosensory cortex, but a structure that resembles the OFL (OFL-like) is apparent in some areas of the ferret occipital lobe. However, note that the OFL-like structure in ferret is located within the oSVZ, in contrast to the OFL in macaque, which is superficial to the oSVZ. Immunohistochemistry The subplate (SP) was identified according to Smart et al 2002 [9]. Scale bar in (b) applies to (d). CC, corpus callosum; IC, internal capsule; AC, anterior commissure; CP, cortical plate. doi:10 1371/journal pone 0030178 g020 Immunohistochemistry Tissue was prepared and mounted on slides as above. Antigen retrieval was performed on slide-mounted tissue by boiling sections in 10 mM Citrate Buffer (pH 6.0) containing 10 mM Citric Acid (Fisher) and (v/v) 0.5% Tween-20 (Acros) for fifteen minutes. Sections were blocked in blocking buffer containing (v/v) 10% fetal donkey serum, 0.1% Triton X-100 (Acros), and (w/v) 0.2% January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 32 Mammalian Outer Subventricular Zone gelatin (Acros) for a minimum of one hour at room temperature Figure 20. The inner fiber layer (IFL) and outer fiber layer (OFL) are present in the E80 macaque visual cortex but are not apparent in other cortical areas. (a,c) Nissl-stained coronal sections of somatosensory and visual cortex taken from E80 macaque. (b,d) Higher magnification images taken from the sections shown in (a) and (c). Dotted line in (b) represents the outer boundary of the outer subventricular zone (oSVZ) in somatosensory cortex determined through Tbr2 immunostaining on adjacent sections (see Fig. 5). The outer fiber layer (OFL) and inner fiber layer (IFL) are visible in the visual cortex but are not apparent in cortical areas rostral to the occipital lobe. In somatosensory cortex the boundary between the inner SVZ (iSVZ) and oSVZ can be visualized in Nissl stained tissue as a sharp border created by differences in cell density. In the visual cortex the IFL divides the iSVZ from the oSVZ. In somatosensory cortex the oSVZ extends much farther from the ventricle than it does in visual cortex and is characterized by a striated appearance. In the E80 macaque the ventricular zone has become very thin and the cell dense proliferative zone that surrounds the lateral ventricle consists almost entirely of iSVZ. The subplate (SP) was identified according to Smart et al 2002 [9]. Scale bar in (b) applies to (d). CC, corpus callosum; IC, internal capsule; AC, anterior commissure; CP, cortical plate. doi:10.1371/journal.pone.0030178.g020 Figure 21. Inner and outer SVZ in the developing ferret cortex. (a,c) Nissl-stained coronal sections of somatosensory and visual cortex from P2 ferret. (b,d) Higher magnification images taken from the sections shown in (a) and (c). Dotted line in (b) represents the outer boundary of the outer subventricular zone (oSVZ) in somatosensory cortex determined through Tbr2 immunostaining. Scale bar below (f) applies to (b, d, f). doi:10.1371/journal.pone.0030178.g022 Figure 23. Tau-1 and Tbr2 staining distinguishes the inner subventricular zone (iSVZ) from the outer SVZ (oSVZ). (a) A coronal section from E80 macaque somatosensory cortex immuno- stained for Tau-1 (green), Tbr2 (red), and counterstained with DAPI (blue). Tau-1 staining produces dense labeling in the cortical plate, striated staining in the outer subventricular zone (oSVZ) and an absence of label in the inner SVZ (iSVZ). The striated appearance of Tau-1 staining in the oSVZ complements the striated pattern of the oSVZ that is seen in Nissl or DAPI stained tissue. (b) Inset from (a) showing the pattern of cell density (DAPI, blue), Tbr2 staining (red), and Tau-1 staining (green). The Tau-free zone corresponds to the cell dense iSVZ/ VZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where the diffuse outer band of Tbr2+ cells is located. (c) Tau-1 and Tbr2 immunostaining produces the same pattern in the E20 rat. The Tau-free zone corresponds to the cell dense VZ/iSVZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where diffuse Tbr2+ cells are located. (d) Tau-1 and Tbr2 immunostaining produces the same pattern in the P2 ferret. The Tau-free zone corresponds to the cell dense VZ/iSVZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where diffuse Tbr2+ cells are located. doi:10.1371/journal.pone.0030178.g023 Figure 23. Tau-1 and Tbr2 staining distinguishes the inner subventricular zone (iSVZ) from the outer SVZ (oSVZ). (a) A coronal section from E80 macaque somatosensory cortex immuno- stained for Tau-1 (green), Tbr2 (red), and counterstained with DAPI (blue). Tau-1 staining produces dense labeling in the cortical plate, striated staining in the outer subventricular zone (oSVZ) and an absence of label in the inner SVZ (iSVZ). The striated appearance of Tau-1 staining in the oSVZ complements the striated pattern of the oSVZ that is seen in Nissl or DAPI stained tissue. The striated appearance of Tau-1 staining in the oSVZ complements the striated pattern of the oSVZ that is seen in Nissl or DAPI stained tissue. (b) Inset from (a) showing the pattern of cell density (DAPI, blue), Tbr2 staining (red), and Tau-1 staining (green). The Tau-free zone corresponds to the cell dense iSVZ/ VZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where the diffuse outer band of Tbr2+ cells is located. (c) Tau-1 and Tbr2 immunostaining produces the same pattern in the E20 rat. The Tau-free zone corresponds to the cell dense VZ/iSVZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where diffuse Tbr2+ cells are located. (d) Tau-1 and Tbr2 immunostaining produces the same pattern in the P2 ferret. The Tau-free zone corresponds to the cell dense VZ/iSVZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where diffuse Tbr2+ cells are located. doi:10.1371/journal.pone.0030178.g023 Figure 22. Inner and outer SVZ in the developing rat cortex. (a, c, e) Nissl-stained sections of somatosensory or visual cortex from the E18 rat. (b, d, e) Higher magnification images taken from the sections shown in (a), (c) and (e). The inner fiber layer and outer fiber layer are not apparent in any cortical areas of rat cortex. At late stages of rat cortical development, such as E18 or E21, the boundary between the inner subventricular zone (iSVZ) and outer SVZ (oSVZ) can be discriminated based on differences in cell density. At E17 and younger ages Tbr2 immunostaining is required to visualize the location of dense inner band of Tbr2+ cells to localize the iSVZ and oSVZ. The oSVZ is characterized by a stippled or striated appearance created by oblique and tangential clusters of cells that appear to stream across the oSVZ. At E21, the oSVZ has expanded compared to earlier stages of cortical development. Dotted line indicates the upper boundary of the oSVZ. AC, anterior commissure; IC, internal capsule; VZ, ventricular zone; CP, cortical plate. Inner and outer SVZ in the developing rat cortex. (a, c, e) Nissl-stained sections of somatosensory or visual cortex from the E18 rat. (b, d, e) Higher magnification images taken from the sections shown in (a), (c) and (e). The inner fiber layer and outer fiber layer are not apparent in any cortical areas of rat cortex. At late stages of rat cortical development, such as E18 or E21, the boundary between the inner subventricular zone (iSVZ) and outer SVZ (oSVZ) can be discriminated based on differences in cell density. At E17 and younger ages Tbr2 immunostaining is required to visualize the location of dense inner band of Tbr2+ cells to localize the iSVZ and oSVZ. The oSVZ is characterized by a stippled or striated appearance created by oblique and tangential clusters of cells that appear to stream across the oSVZ. At E21, the oSVZ has expanded compared to earlier stages of cortical development. Dotted line indicates the upper boundary of the oSVZ. AC, anterior commissure; IC, internal capsule; VZ, ventricular zone; CP, cortical plate. Scale bar below (f) applies to (b, d, f). doi:10.1371/journal.pone.0030178.g022 or Cy5/Dylight 649 (Jackson Laboratories) and included donkey anti-mouse, donkey anti-rabbit, donkey anti-chicken, donkey anti-goat and donkey anti-rat. Slices were then rinsed and coverslipped with Mowiol. In the E80 macaque the ventricular zone has become very thin and the cell dense proliferative zone that surrounds the lateral ventricle consists almost entirely of iSVZ. The subplate (SP) was identified according to Smart et al 2002 [9]. Scale bar in (b) applies to (d). CC, corpus callosum; IC, internal capsule; AC, anterior commissure; CP, cortical plate. doi:10.1371/journal.pone.0030178.g020 ; , p doi:10.1371/journal.pone.0030178.g020 gelatin (Acros) for a minimum of one hour at room temperature (RT). Sections were incubated in primary antibody buffer containing primary antibodies (see below), (v/v) 2% fetal donkey serum, 0.02% Triton X-100, and (w/v) 0.04% gelatin overnight at RT. Primary antibodies included mouse anti-phosphorylated vimentin (4A4) 1:500 (MBL), mouse anti-Pax6 1:1 (Developmen- tal Studies Hybridoma Bank), mouse anti-Pax6 1:50 (Abcam), mouse anti-Sox2 1:50 (R&D Systems), mouse anti-Tau-1 1:200 (Millipore), mouse anti-NeuN 1:200 (Millipore), rabbit anti- Pax6 1:100 (Covance), rabbit anti-Tbr2 1:500 (Abcam), rabbit gelatin (Acros) for a minimum of one hour at room temperature (RT). Sections were incubated in primary antibody buffer containing primary antibodies (see below), (v/v) 2% fetal donkey serum, 0.02% Triton X-100, and (w/v) 0.04% gelatin overnight at RT. Primary antibodies included mouse anti-phosphorylated vimentin (4A4) 1:500 (MBL), mouse anti-Pax6 1:1 (Developmen- tal Studies Hybridoma Bank), mouse anti-Pax6 1:50 (Abcam), mouse anti-Sox2 1:50 (R&D Systems), mouse anti-Tau-1 1:200 (Millipore), mouse anti-NeuN 1:200 (Millipore), rabbit anti- Pax6 1:100 (Covance), rabbit anti-Tbr2 1:500 (Abcam), rabbit anti-Phosphohistone H3 (PH3) 1:50 (Chemicon), goat anti-Olig2 1:500 (R&D Systems), chicken anti-GFP 1:500 (Abcam) and rat anti-BrdU-FITC 1:50 (Abcam). Sections were rinsed in 0.1 M PBS, then incubated for one hour at RT in secondary antibody buffer, which contained secondary antibodies (see below), (v/v) 2% fetal donkey serum, 0.02% Triton X-100, (w/v) 0.04% gelatin, and DAPI 1:1000 (Roche). Secondary antibodies were conjugated to Dylight 405, Cy2/Dylight 488, Cy3/Dylight 549, January 2012 | Volume 7 | Issue 1 | e30178 33 PLoS ONE | www.plosone.org Mammalian Outer Subventricular Zone Figure 23. Tau-1 and Tbr2 staining distinguishes the inner subventricular zone (iSVZ) from the outer SVZ (oSVZ). (a) A coronal section from E80 macaque somatosensory cortex immuno- stained for Tau-1 (green), Tbr2 (red), and counterstained with DAPI (blue). Tau-1 staining produces dense labeling in the cortical plate, striated staining in the outer subventricular zone (oSVZ) and an absence of label in the inner SVZ (iSVZ). (b) Inset from (a) showing the pattern of cell density (DAPI, blue), Tbr2 staining (red), and Tau-1 staining (green). The Tau-free zone corresponds to the cell dense iSVZ/ VZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where the diffuse outer band of Tbr2+ cells is located. (c) Tau-1 and Tbr2 immunostaining produces the same pattern in the E20 rat. The Tau-free zone corresponds to the cell dense VZ/iSVZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where diffuse Tbr2+ cells are located. (d) Tau-1 and Tbr2 immunostaining produces the same pattern in the P2 ferret. The Tau-free zone corresponds to the cell dense VZ/iSVZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where diffuse Tbr2+ cells are located. doi:10.1371/journal.pone.0030178.g023 Figure 22. Inner and outer SVZ in the developing rat cortex. (a, Figure 22. Inner and outer SVZ in the developing rat cortex. (a, c, e) Nissl-stained sections of somatosensory or visual cortex from the E18 rat. (b, d, e) Higher magnification images taken from the sections shown in (a), (c) and (e). The inner fiber layer and outer fiber layer are not apparent in any cortical areas of rat cortex. At late stages of rat cortical development, such as E18 or E21, the boundary between the inner subventricular zone (iSVZ) and outer SVZ (oSVZ) can be discriminated based on differences in cell density. At E17 and younger ages Tbr2 immunostaining is required to visualize the location of dense inner band of Tbr2+ cells to localize the iSVZ and oSVZ. The oSVZ is characterized by a stippled or striated appearance created by oblique and tangential clusters of cells that appear to stream across the oSVZ. At E21, the oSVZ has expanded compared to earlier stages of cortical development. Dotted line indicates the upper boundary of the oSVZ. AC, anterior commissure; IC, internal capsule; VZ, ventricular zone; CP, cortical plate. Scale bar below (f) applies to (b, d, f). doi:10.1371/journal.pone.0030178.g022 Figure 22. Analysis of Number of Mitoses by Nissl staining, 4a4 Immunostaining and PH3 Immunostaining Analysis was performed in the somatosensory cortex of rats (E14, E17, E18, E20), ferret (E28, E31, E34, E38, P2) and macaque (E50, E65, E80, E100). zone (oSVZ), or cortical plate (CP)/preplate (PP)/subplate (SP)/ marginal zone (MZ)) based on cytoarchitecture of the region. age in each species. Analysis was performed in the somatosensory cortex of rats (E14, E17, E18, E20), ferret (E28, E31, E34, E38, P2) and macaque (E50, E65, E80, E100). Analysis of Number of Mitoses by Nissl staining, 4a4 Immunostaining and PH3 Immunostaining (a) Coronal sections of somatosensory cortex from E20 rat stained with (from left to right) DAPI (blue), and immunostained for Tbr2 (red) and Tau-1 (green). The right panel is an adjacent section that was immunostained for Pax6 (green). DAPI staining identifies the boundary between the iSVZ and oSVZ. The VZ and iSVZ have a near uniform cell density, while the oSVZ is striated and the cell density is lower. Tau-1 staining also identifies the boundary between the iSVZ and oSVZ in macaque, ferret and rat. The iSVZ is located in the Tau-free zone. The border between iSVZ and oSVZ is delineated by the border between the Tau-free zone and the Tau- striated zone. Tau-1+ fibers are largely absent from the VZ and iSVZ. Tbr2 and Pax6 can be used to discriminate the VZ and iSVZ. The dense inner band of Tbr2 expression corresponds to the iSVZ and the dense inner band of Pax6 expression corresponds to the VZ. The dashed lines indicate the Figure 24. The boundaries between the ventricular zone (VZ), inner subventricular zone (iSVZ) and outer SVZ (oSVZ) can be identified through a combination of DAPI, Tau-1, Tbr2 and Pax6 staining. (a) Coronal sections of somatosensory cortex from E20 rat stained with (from left to right) DAPI (blue), and immunostained for Tbr2 (red) and Tau-1 (green). The right panel is an adjacent section that was immunostained for Pax6 (green). DAPI staining identifies the boundary between the iSVZ and oSVZ. The VZ and iSVZ have a near uniform cell density, while the oSVZ is striated and the cell density is lower. Tau-1 staining also identifies the boundary between the iSVZ and oSVZ in macaque, ferret and rat. The iSVZ is located in the Tau-free zone. The border between iSVZ and oSVZ is delineated by the border between the Tau-free zone and the Tau- striated zone. Tau-1+ fibers are largely absent from the VZ and iSVZ. Tbr2 and Pax6 can be used to discriminate the VZ and iSVZ. The dense inner band of Tbr2 expression corresponds to the iSVZ and the dense inner band of Pax6 expression corresponds to the VZ. The dashed lines indicate the boundary between the iSVZ and oSVZ. doi:10.1371/journal.pone.0030178.g024 age in each species. Analysis was performed in the somatosensory cortex of rats (E14, E17, E18, E20), ferret (E28, E31, E34, E38, P2) and macaque (E50, E65, E80, E100). age in each species. Analysis of Number of Mitoses by Nissl staining, 4a4 Immunostaining and PH3 Immunostaining sections were anterior to the genu of the corpus callosum, middle sections were at the level of the anterior commissure and/or interventricular foramen, and caudal sections were located caudal to the hippocampal formation in the occipital lobe. Mitoses were quantified in the entire dorsal cortical wall from the dorsomedial boundary with cingulate cortex to the corticostriatal junction. Mitoses were allocated to discrete histological zones (ventricular zone (VZ), inner subventricular zone (iSVZ), outer subventricular Mitoses were identified in Nissl-stained tissue by identifying condensed chromatin. Alternatively, mitoses were identified in tissue immunostained for 4A4 or PH3 and confirmed by condensed chromatin with DAPI counterstaining. At least three adjacent sections were analyzed from three different locations in the rostrocaudal axis (nine sections from each brain). Rostral PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 34 Mammalian Outer Subventricular Zone Figure 24. The boundaries between the ventricular zone (VZ), inner subventricular zone (iSVZ) and outer SVZ (oSVZ) can be identified through a combination of DAPI, Tau-1, Tbr2 and Pax6 staining. (a) Coronal sections of somatosensory cortex from E20 rat stained with (from left to right) DAPI (blue), and immunostained for Tbr2 (red) and Tau-1 (green). The right panel is an adjacent section that was immunostained for Pax6 (green). DAPI staining identifies the boundary between the iSVZ and oSVZ. The VZ and iSVZ have a near uniform cell density, while the oSVZ is striated and the cell density is lower. Tau-1 staining also identifies the boundary between the iSVZ and oSVZ in macaque, ferret and rat. The iSVZ is located in the Tau-free zone. The border between iSVZ and oSVZ is delineated by the border between the Tau-free zone and the Tau- striated zone. Tau-1+ fibers are largely absent from the VZ and iSVZ. Tbr2 and Pax6 can be used to discriminate the VZ and iSVZ. The dense inner band of Tbr2 expression corresponds to the iSVZ and the dense inner band of Pax6 expression corresponds to the VZ. The dashed lines indicate the boundary between the iSVZ and oSVZ. doi:10 1371/journal pone 0030178 g024 Figure 24. The boundaries between the ventricular zone (VZ), inner subventricular zone (iSVZ) and outer SVZ (oSVZ) can be identified through a combination of DAPI, Tau-1, Tbr2 and Pax6 staining. References 12. Schmechel DE, Rakic P (1979) A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes. Anatomy & Embryology 156: 115–152. 1. Samuelsen GB, Larsen KB, Bogdanovic N, Laursen H, Graem N, et al. (2003) The changing number of cells in the human fetal forebrain and its subdivisions: a stereological analysis. Cereb Cortex 13: 115–122. 13. Voigt T (1989) Development of glial cells in the cerebral wall of ferrets: direct tracing of their transformation from radial glia into astrocytes. J Comp Neurol 289: 74–88. 2. Bentivoglio M, Mazzarello P (1999) The history of radial glia. Brain Res Bull 49: 305–315. 3. Noctor SC, Flint AC, Weissman TA, Dammerman RS, Kriegstein AR (2001) Neurons derived from radial glial cells establish radial units in neocortex. Nature 409: 714–720. 14. Takahashi T, Misson JP, Caviness VS, Jr. (1990) Glial process elongation and branching in the developing murine neocortex: a qualitative and quantitative immunohistochemical analysis. J Comp Neurol 302: 15–28. 4. Noctor SC, Martı´nez-Cerden˜o V, Ivic L, Kriegstein AR (2004) Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat Neurosci 7: 136–144. 15. deAzevedo LC, Fallet C, Moura-Neto V, Daumas-Duport C, Hedin-Pereira C, et al. (2003) Cortical radial glial cells in human fetuses: depth-correlated transformation into astrocytes. J Neurobiol 55: 288–298. 5. Noctor SC, Martinez-Cerden˜o V, Kriegstein AR (2008) Distinct behaviors of neural stem and progenitor cells underlie cortical neurogenesis. J Comp Neurol 508: 28–44. 16. Miyata T, Kawaguchi A, Saito K, Kawano M, Muto T, et al. (2004) Asymmetric production of surface-dividing and non-surface-dividing cortical progenitor cells. Development 131: 3133–3145. 6. Gotz M, Stoykova A, Gruss P (1998) Pax6 controls radial glia differentiation in the cerebral cortex. Neuron 21: 1031–1044. 17. Wang X, Tsai JW, LaMonica B, Kriegstein AR (2011) A new subtype of progenitor cell in the mouse embryonic neocortex. Nat Neurosci 14: 555–561. 7. Englund C, Fink A, Lau C, Pham D, Daza RA, et al. (2005) Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex. J Neurosci 25: 247–251. 18. Shitamukai A, Konno D, Matsuzaki F (2011) Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors. J Neurosci 31: 3683–3695. 8. BrdU Birthdating Postnatal rats received two intraperitoneal injections of BrdU (80 mg/kg, Sigma) in the morning and evening on P1, P3 or P7 to label cells born on that day. Injected pups survived until P10. Animals were perfused transcardially with PFA, cryoprotected and cryostat sectioned as described above. Cryostat sections were washed in 0.1 M PBS, incubated in 2 N HCL for 30 minutes, and then processed for immunohistochemistry as above. To determine if neurogenesis occurred on any postnatal days (P1, P3, or P7), P10 sections were co-immunostained for BrdU and NeuN as above except using pretreatment with 2 N HCl at 37uC for 30 minutes in Quantification of Double Immunolabeling in Mitoses Quantification of Double Immunolabeling in Mitoses Rat, ferret and macaque sections that were double-immuno- stained using a variety of antibodies and counterstained with DAPI were imaged on a confocal microscope (Olympus). Z-stacks were created from images constructed from optical sections in which penetration of all antibodies and DAPI was complete. All mitoses, identified by condensed chromatin (DAPI) and 4A4/PH3 immunostaining, were identified in Z-stacks and the extent of costaining with different antibodies was assessed. Mitoses were allocated to discrete histological zones using DAPI staining as described above. The proportions of mitoses that immunostained positive or negative with specific antibodies were quantified for each area. The distribution of cells expressing 4A4/Pax6 and 4A4/Tbr2 was analyzed by quantifying the proportion of 4A4+/ Tbr2+ and 4A4+/Pax6+ cells in each histological zone in a 300 mm wide radial unit of cortex that stretched from the ventricle to the pial surface. Quantification of Total Pax6+ and Tbr2+ cells Coronal sections of rat, ferret, and macaque tissue immuno- stained for Pax6 or Tbr2 were imaged on a confocal microscope (Olympus). A montage of a cortical radial unit was created in Photoshop (Adobe) by combining individual optical sections from a series of overlapping high magnification (406) Z-stacks. A 200 mm wide bin was created that stretched from the ventricular The dense inner band and diffuse outer band of Tbr2- expressing cells were identified in combination with DAPI staining. All Tbr2+ cells in a 2500 mm2 region of interest (ROI) located in the dense inner band and the diffuse outer band were quantified. A minimum of nine separate ROIs from the Tbr2+ dense inner band and diffuse outer band were averaged at each January 2012 | Volume 7 | Issue 1 | e30178 PLoS ONE | www.plosone.org 35 Mammalian Outer Subventricular Zone lieu of antigen retrieval. Sections were imaged on a confocal and the pattern of BrdU and NeuN immunolabeling was analyzed. surface to the pial surface in the dorsal somatosensory cortex. All Pax6+ or Tbr2+ cells in the bin were counted, and allocated into discrete histological zones (VZ, iSVZ, oSVZ or CP/PP/SP/MZ) that were identified by DAPI staining. The number of positive cells/bin and the relative distribution of Pax6+ and Tbr2+ cells were compared across ages and species. Acknowledgments We are grateful to Dr. David Amaral for macaque tissue; and Drs Francisco Clasca, Barbara Chapman, and Sharon Juliano for ferret tissue. We thank Chavis Jackson, Derik Neilson, Laura Bomze, Joseph Hamera, David Wong, Annette Masterson, Adrienne Ng, Mark Ensign, Roxanne Parker, Katherine Hanel, Kyle Spaulding and Joseph Elsbernd for assistance with tissue processing and analysis. We are indebted to Chavis Jackson for technical assistance with the confocal microscope. Retroviral packaging cell stocks were the kind gift of Drs. Fred Gage and Theo Palmer, pVSV-G plasmid was the kind gift of Dr. James Angelastro. Author Contributions Conceived and designed the experiments: SCN VMC CLC. Performed the experiments: VMC CLC JC JLA ANP MEC AIW. Analyzed the data: SCN VMC CLC. Contributed reagents/materials/analysis tools: SCN VMC. Wrote the paper: SCN VMC CLC. Supporting Information Figure S1 4A4+/Pax6+ mitotic cells with ventricular and tangential oriented processes are present in macaque cortex. (a) Coronal section from E80 macaque immunostained for 4A4 (green), Pax6 (red) and counterstained with DAPI (blue). The cells indicated by boxes (1 and 2) show Pax6+ mitotic cells with a single process oriented toward the ventricular surface. These cells appear to be unipolar. (b) Coronal section from E80 macaque immunostained for 4A4 (green), Pax6 (red) and counterstained with DAPI (blue). The cell highlighted with the box shows a 4A4+ mitotic cell that expresses Pax6 and possesses a short tangential process. VZ, ventricular zone; iSVZ, inner subventricular zone; oSVZ, outer subventricular zone. (TIF) Mammalian Outer Subventricular Zone Bani-Yaghoub M, Tremblay RG, Lei JX, Zhang D, Zurakowski B, et al. (2006) Role of Sox2 in the development of the mouse neocortex. Dev Biol 295: 52–66. 43. Rakic P (1988) Specification of cerebral cortical areas. Science 241: 170–176. p 30. Marshall CA, Novitch BG, Goldman JE (2005) Olig2 directs astrocyte and oligodendrocyte formation in postnatal subventricular zone cells. J Neurosci 25: 7289–7298. 44. Van Essen DC (1997) A tension-based theory of morphogenesis and compact wiring in the central nervous system. Nature 385: 313–318. 31. Mikula S, Stone JM, Berman AL, Jones EG (2011) A digital stereotaxic atlas of the brain of the monkey, Macaca mulatta. 45. Garcia-Moreno F, Vasistha NA, Trevia N, Bourne JA, Molnar Z (2011) Compartmentalization of Cerebral Cortical Germinal Zones in a Lissencephalic Primate and Gyrencephalic Rodent. Cereb Cortex. y 32. Blinkov SM, Glezer II (1968) The human quantitative book. New York: Plenum Press. 32. Blinkov SM, Glezer II (1968) The human brain in figures and tables, A quantitative book. New York: Plenum Press. 46. Reillo I, Borrell V (2011) Germinal Zones in the Developing Cerebral Cortex of Ferret: Ontogeny, Cell Cycle Kinetics, and Diversity of Progenitors. Cereb Cortex. q 33. Herculano-Houzel S, Collins CE, Wong P, Kaas JH (2007) Cellular scaling rules for primate brains. Proc Natl Acad Sci U S A 104: 3562–3567. 47. Kelava I, Reillo I, Murayama AY, Kalinka AT, Stenzel D, et al. (2011) Abundant Occurrence of Basal Radial Glia in the Subventricular Zone of Embryonic Neocortex of a Lissencephalic Primate, the Common Marmoset Callithrix jacchus. Cereb Cortex. 34. Christensen JR, Larsen KB, Lisanby SH, Scalia J, Arango V, et al. (2007) Neocortical and hippocampal neuron and glial cell numbers in the rhesus monkey. Anat Rec (Hoboken) 290: 330–340. y ( ) 35. Korbo L, Pakkenberg B, Ladefoged O, Gundersen HJ, Arlien-Soborg P, et al. (1990) An efficient method for estimating the total number of neurons in rat brain cortex. J Neurosci Methods 31: 93–100. 48. Hevner RF, Haydar TF (2011) The (Not Necessarily) Convoluted Role of Basal Radial Glia in Cortical Neurogenesis. Cereb Cortex. J 36. Herculano-Houzel S, Lent R (2005) Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain. J Neurosci 25: 2518–2521. 49. Judson MC, Amaral DG, Levitt P (2011) Conserved subcortical and divergent cortical expression of proteins encoded by orthologs of the autism risk gene MET. Mammalian Outer Subventricular Zone Mammalian Outer Subventricular Zone 24. Kamei Y, Inagaki N, Nishizawa M, Tsutsumi O, Taketani Y, et al. (1998) Visualization of mitotic radial glial lineage cells in the developing rat brain by Cdc2 kinase-phosphorylated vimentin. Glia 23: 191–199. 37. Mooney SM, Napper RM (2005) Early postnatal exposure to alcohol reduces the number of neurons in the occipital but not the parietal cortex of the rat. Alcohol Clin Exp Res 29: 683–691. 37. Mooney SM, Napper RM (2005) Early postnatal exposure to alcohol reduces the number of neurons in the occipital but not the parietal cortex of the rat. Alcohol Clin Exp Res 29: 683–691. p 38. Haubensak W, Attardo A, Denk W, Huttner WB (2004) Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis. Proc Natl Acad Sci U S A 101: 3196–3201. p p y 25. Noctor SC, Flint AC, Weissman TA, Wong WS, Clinton BK, et al. (2002) Dividing precursor cells of the embryonic cortical ventricular zone have morphological and molecular characteristics of radial glia. J Neurosci 22: 3161–3173. 39. Tarabykin V, Stoykova A, Usman N, Gruss P (2001) Cortical upper layer neurons derive from the subventricular zone as indicated by Svet1 gene expression. Development 128: 1983–1993. 26. Weissman T, Noctor SC, Clinton BK, Honig LS, Kriegstein AR (2003) Neurogenic radial glial cells in reptile, rodent and human: from mitosis to migration. Cereb Cortex 13: 550–559. p p 40. Nieto M, Monuki ES, Tang H, Imitola J, Haubst N, et al. (2004) Expression of Cux-1 and Cux-2 in the subventricular zone and upper layers II–IV of the cerebral cortex. J Comp Neurol 479: 168–180. 27. Cunningham CL, Martinez Cerdeno V, Navarro Porras E, Prakash AN, Angelastro JM, et al. (2011) Premutation CGG-repeat expansion of the Fmr1 gene impairs mouse neocortical development. Hum Mol Genet 20: 64–79. 41. Kriegstein A, Noctor S, Martı´nez-Cerden˜o V (2006) Patterns of neural stem and progenitor cell division may underlie evolutionary cortical expansion. Nat Rev Neurosci 7: 883–890. 28. Arai Y, Pulvers JN, Haffner C, Schilling B, Nusslein I, et al. (2011) Neural stem and progenitor cells shorten S-phase on commitment to neuron production. Nat Commun 2: 154. 42. Reillo I, de Juan Romero C, Garcia-Cabezas MA, Borrell V (2011) A role for intermediate radial glia in the tangential expansion of the mammalian cerebral cortex. Cereb Cortex 21: 1674–1694. 29. References Smart IH (1973) Proliferative characteristics of the ependymal layer during the early development of the mouse neocortex: a pilot study based on recording the number, location and plane of cleavage of mitotic figures. J Anat 116: 67–91. 19. Noctor SC, Scholnicoff NJ, Juliano SL (1997) Histogenesis of ferret somatosensory cortex. J Comp Neurol 387: 179–193. 9. Smart IH, Dehay C, Giroud P, Berland M, Kennedy H (2002) Unique morphological features of the proliferative zones and postmitotic compartments of the neural epithelium giving rise to striate and extrastriate cortex in the monkey. Cereb Cortex 12: 37–53. 20. Rakic P (1974) Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition. Science 183: 425–427. . Bayer SA, Altman J (1991) Neocortical Development. New York: R 10. Hansen DV, Lui JH, Parker PR, Kriegstein AR (2010) Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature 464: 554–561. 22. Carney RS, Bystron I, Lopez-Bendito G, Molnar Z (2007) Comparative analysis of extra-ventricular mitoses at early stages of cortical development in rat and human. Brain Struct Funct 212: 37–54. 11. Fietz SA, Kelava I, Vogt J, Wilsch-Brauninger M, Stenzel D, et al. (2010) OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling. Nat Neurosci 13: 690–699. 23. Boulder_Committee (1970) Embryonic vertebrate central nervous system: revised terminology. Anatomical Record 166: 257–261. PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 January 2012 | Volume 7 | Issue 1 | e30178 36 Mammalian Outer Subventricular Zone Cereb Cortex 21: 1613–1626. PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30178 37
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Supraglottic Cancer pT4a TNM Finding v8
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Supraglottic Cancer pT4a TNM Finding v8 National Cancer Institute National Cancer Institute Qeios ID: J66GB8 · https://doi.org/10.32388/J66GB8 Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Source National Cancer Institute. Supraglottic Cancer pT4a TNM Finding v8. NCI Thesaurus. Code C133106. Code C133106. Supraglottic cancer with moderately advanced local disease. Tumor invades through the outer cortex of the thyroid cartilage and/or invades tissues beyond the larynx (e.g., trachea, soft tissues of neck including deep extrinsic muscle of the tongue, strap muscles, thyroid, or esophagus). (from AJCC 8th Ed.) 1/1
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New records of insect pests infesting Dalbergia latifolia (Roxb.) from Uttarakhand, India
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Abstract The present study conducted in Uttarakhand, India aimed to investigate the insect pests associated with Dalbergia latifolia, commonly known as rosewood. Rosewood is an economically important timber-producing tree species indigenous to South and Southeast Asia. Although insect pests have been reported to attack rosewood trees, their impact and threat to nurseries and plantations in India have been insignificant thus far. The periodic survey was done and insect pest were recorded infesting D. latifolia nursery and plantation. The study recorded ten different insect species on D. latifolia in the region, with eight of them being reported for the first time on this particular species. The newly recorded insect species included Gastrophysa viridula, Chrysochus cobaltinus, Apoderus crenatus, Hyposidra talaca, Ricania speculum, Leptocorisa acuta, Leptocentrus taurus, and Dorsicha stebbingi. Further, nature of damage caused by these insect pests, and seasonal incidence was also studied. This investigation aimed to gather valuable information on the insect pest fauna associated with rosewood in Uttarakhand, India, and assess their potential effects on this valuable timber species. By studying the insect pests and their damage patterns, researchers can develop effective pest management strategies to mitigate any potential economic losses caused by these pests. Such information is crucial for the sustainable management and conservation of Dalbergia latifolia in the region. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/18 Page 1/18 INTRODUCTION Dalbergia latifolia (Roxb.) commonly known as Indian rosewood, Bombay blackwood, Indian palisandre and Java palisandre. D. latifolia is a native species of India, Nepal and Indonesia and exotic species of other countries like Kenya, Malaysia, Myanmar, Nigeria, Philippines, Sri Lanka, Tanzania Vietnam and many other parts of South East Asia (Sujatha, 2008; Orwa et al., 2009).The distribution of this species is very scattered and occurs in mixed deciduous forest throughout the Indian peninsula (Sasidharan et al., 2020). D. latifolia is widely distributed to low-elevation tropical monsoon forests of Central and southern parts of India at altitudes from 900 to 1350 MSL. D. latifolia is one of the important quality timber yielding species and its timber is internationally known for its grain quality, strength, natural dark colour, and its timber is widely used for making furniture, panel, decorative articles, musical instrument industry and other multiple products. Additionally, its bark is used for source of tannin, which is utilised in medicine industry (Anon., 2018; Damaiyani and Prabowo, 2019; Chaterjee et al., 2020). Plantation of D. latifolia is widely done due to its ecological services as CO2 sequester, nitrogen fixation and soil repairing quality (Soerianegara & Lemmens, 1994; Sujatha, 2008; Lahiri et al., 2019). The species regenerates normally by seeds and occasionally by root suckers, but it is categorized as threatened species under IUCN red data list due to its poor regeneration, slow growth and long rotation period. Subsequently, rapid increase in demand resulted in its over and illegal exploitation in India (Orwa et al., 2009; Treanor, 2015). It has been recorded that more than forty insect pests pose threat to D. latifolia and quality, growth and productivity of this species are adversely affected by frequent outbreaks of insect pests. Some important groups of insects belonging to orders Coleoptera, Hymenoptera, Lepidoptera and Isoptera were recorded as major pests that cause severe damage and high economic loss to the nursery and plantations. Among these, most important pests are cutworms, termites and cockchafers, besides some defoliators, sapsuckers and shoot borers as major pests. In northern India D. latifolia was introduced in the year 2003 onwards and many Page 2/18 plantations under introduction trails were established in Uttarakhand, but insect pest infestation study of D. latifolia from Northern region of India is available. Hence, the survey was undertaken to find out the insect pests fauna associated with D. INTRODUCTION latifolia and their nature of damage in Uttarakhand, India. Gastrophysa viridula (De Geer) Adult beetle was oval in shape of about 5.02 mm long and dark metallic green in colour, with reddish brown legs (Fig. 1A & D). Infestation of this beetle was found at New Forest Campus nursery on young leaves and FRC Lalkuan Uttarakhand plantation. Both male and female beetle was recorded feeding on the leaves. Adult beetle feed on young leaves from margin under field and laboratory condition. RESULTS The result presented in table-1 revealed that D. latifolia was found to be infested with ten insect pests belonging to three different insect orders viz. Coleoptera, Lepidoptera and Hemiptera and their nature of damage was also recorded. Chrysochus cobaltinus Leconte Adult beetle was oval in shape of about 7.35–7.75 mm long with dark metallic iridescent cobalt-blue exoskeleton and brown legs. The beetle was collected from New Forest Campus nursery found to be infested on young leaves. Both adult male and female specimens have been recovered from young leaves (Fig. 1B & E). Feeding pattern of adult beetle has been monitored under field and laboratory conditions. Through regular observation it was found that the adult beetle feeds gregariously on leaves, tender shoots and cut the leaves from margin. MATERIALS AND METHOD The present survey of insect pest of D. latifolia was done at New Forest campus, Forest Research Institute, Dehradun lies at lat 30◦ 20’ 31.56’’ N and long 77◦ 59’ 50.28’’ E and Forest Research Centre Lalkuan, Silva Sal unit Haldwani, lies at lat 29◦03’50.5’’N to long 79◦ 30’55.3’’ E in the state of Uttarakhand, India (Fig. 3). The investigation was done over nursery at New Forest Campus (Fig. 4A) and eleven year old plantation at Lalkuan site (Fig. 4B) of D. latifolia during round the year from 2021–2023. Weekly field visit was done for collection of insect pests and observation of their nature of damage. Insect pests associated with root, bark, shoot and leaves were observed, collected and their nature of damage was recorded simultaneously. Seasonality of the insect pest infestation was recorded on monthly bais. Collected insect pests were brought to the laboratory for further studies viz. nature of feeding, adult emergence and identification. Adult stages were preserved for further record. Field photo of insect pest and their nature of damage was captured using DSLR Canon 7D camera and some of the pictures were captured under steriozoom microscope Leica M205 fitted with camera MC190. Hyposidra talaca Walker This insect is also known as black inch looper or tea looper size about 5.0-5.5 cm. Only larval stage of this pest was found to be damaging on leaves. Different larval stages of black looper (Fig. 1H) were studied in the laboratory and it was observed that initially larvae were black coloured and creamy brown coloured at maturity. The infestation of this larva was from New Forest Campus nursery. The semi-looper larva was found to damage tender leaf by feeding from margin of the leaf. The study conducted under laboratory condition revealed that the 4th and 5th instar caterpillar stage are most destructive to young leaves. Plecoptera reflexa Guenee The insect adult stage was nocturnal in nature and only larval stage was found to be damaging to the plant. Caterpillar was light green, smooth, and cylindrical in shape (Fig. 1G). ). Infestation of this bug was recorded from New Forest Campus and FRC Lalkuan under plantation. The young caterpillar was found to be damaging on the lower surface of leaf. Mature larvae of size about 4.0-4.5 cm. Larvae of this species consume whole leaf including petiole and the green shoot. Full grown larvae consume three to four leaves in a day. Ectropis bhurmitra Hubner The larva (Fig. 1I) was light grey- brown in colour, body with swollen darkened diagonal ridge that ends at dorsal tubercle. Head light grey with darker brown herringbone pattern on lobes and horizontal streak at top of front diamond on mid-abdominal segments. Infestation of this larva was recorded from New Forest Campus and FRC Lalkuan under plantation. The larva of this species was found damaging the tender leaves of the plant under both nursery and plantation. Apoderus crenatus Voss Page 3/18 Page 3/18 The weevil (Fig. 1C) was small about 4.75 mm long, orange red in colour with row of elytra; glabrous rostrum, subconical prothorax, semicircular scutellum and densely pubescent meso and meta sternal epimera. The weevil was different from the A. sissu. Infestation of this weevil was recorded from New Forest Campus and FRC Lalkuan under plantation. Adult was mainly found infesting on soft tender leaves making irregular scraping pattern (Fig. 1C). Additionally, female cut the young leaf from the base edge and roll the leaf into a compact cylindrical shape capsule (Fig. 1F). Female lay eggs one to three in each rolled leaf, and larva feed internally inside the leaf roll till pupation. The weevil (Fig. 1C) was small about 4.75 mm long, orange red in colour with row of elytra; glabrous rostrum, subconical prothorax, semicircular scutellum and densely pubescent meso and meta sternal epimera. The weevil was different from the A. sissu. Infestation of this weevil was recorded from New Forest Campus and FRC Lalkuan under plantation. Adult was mainly found infesting on soft tender leaves making irregular scraping pattern (Fig. 1C). Additionally, female cut the young leaf from the base edge and roll the leaf into a compact cylindrical shape capsule (Fig. 1F). Female lay eggs one to three in each rolled leaf, and larva feed internally inside the leaf roll till pupation. Ricania speculum Walker The adult has dark brown wings with central wavy horizontal bands and irregular transparent patches of different sizes. The precostal area of the forewings showed dense transverse veinlets and the costal margin was distinctly convex near the base (Fig. 2C & D). Infestation of this bug was recorded from New Forest Campus and FRC Lalkuan under plantation. Both the nymph and adult were found feeding on the tender parts of the plants. Leptocentrus taurus Fabrichus The insect commonly known as tree hopper, triangular shaped body measured 4–7 mm long, yellow eyes, winged with three pairs of legs, two curved horns like projections on the thorax (Fig. 2A). Infestation of this hopper was recorded from aerial apical parts of D. latifolia from FRC Lalkuan plantation. Both nymph and adult suck the plant sap from tender parts of the plant. The hopper fed on apical portions of the shoot apices, making them rough and woody in appearance, brown in colour that gradually dried and apical leaves shed off. Leptocorisa acuta Thunberg Leptocorisa acuta Thunberg Leptocorisa acuta Thunberg Page 4/18 Page 4/18 The bug was greenish yellow to yellow-brown in colour (Fig. 2E & F). Head was longer than wide, rostrum relatively shorter; the scutellum (triangular shaped plate) was found on the thorax, posterior to the pronotum. The fourth antennal segment was curved. Infestation of this bug was recorded from FRC Lalkuan under plantation. Both nymph and adults were found to be feeding on leaves and tender shoots of the plant. Nymph feed gregariously on the tender shoots, resulted in wilting and drying of tender shoots. Table 2: Seasonal incidence of Insect pest infesting to D. latifolia Drosicha stebbingi Green Insect species January February March April May June July August September October 1 Gastrophysa viridula (De Geer)                   2 Chrysocus cobaltinus Leconte                 3 Apoderus crenatus Voos                     4 Plecoptera reflexa Guenee                     5 Ectropis bhurmitra Hubner                     6 Hyposidra talaca Walker                     7 Ricania speculum  Walker                     8 Leptocorisa     acuta Thunberg                     9 Leptocentrus taurus Fabrichus                     10 Drosicha stebbingi Green Drosicha stebbingi Green Commonly known as mango mealy bug of about 1.0-1.5 cm long, oval white body (Fig. 2B). ). Infestation of this bug was recorded from FRC Lalkuan under plantation only. The nymph and adult the stages were observed to be infesting on tender parts of the plant. The late instar nymph and adult female was flat, oval and waxy white. They remained stationary and adhered to the total length on shoots. Infested shoot part was covered by the sooty mould. The seasonal incidence (Table 2) of leaf cutting beetle i.e. G.viridula and C. cobaltinus was recorded infesting new from April to June. The infestation of A. crenatus was recorded to be during April to July, while leaf defoliator larvae of P. reflecta was appeared in the month of April and remain infesting to the D. latifolia plantation up to August. The larvae of E. burmitra were recorded infesting the leaves of the plant during July to September; and H. talaca were recorded feeding on D. latifolia leaves during the month of May to August. The infestation of R. speculum was recorded during hot months of May to July, while the infestation of L. acuta was recorded during April to August. The infestation of L. Taurus was recorded during March to October, while infestation of D. stebbingi was recorded to be during premonsoon to monsoon period from February to August. Page 5/18 Page 5/18 Page 5/18 Table 1 Insect pests infesting D. latifolia in Uttarakhand and their nature of damage. Sr. no. Species Order: Family Nature of damage 1. Gastrophysa viridula (De Geer) Coleoptera: Chrysomelidae Defoliator 2. Chrysocus cobaltinus Leconte Coleoptera : Chrysomelidae Defoliator 3. Apoderus crenatus Voos Coleoptera: Attelabidae Defoliator 4. Plecoptera reflexa Guenee Lepidoptera: Noctuidae Defoliator 5. Ectropis bhurmitra Hubner Lepidoptera.: Geomatridae Defoliator 6. Hyposidra talaca Walker Lepidoptera: Geomatridae Defoliator 7. Ricania speculum Walker Hemiptera: Ricanidae Sap sucker 8. Leptocorisa acuta Thunberg Hemiptera: Alydidae Sap sucker 9. Leptocentrus taurus Fabrichus Hemiptera: Membracidae Sap sucker 10. Drosicha stebbingi Green Hemiptera : Margarodidae Sap sucker Table 1 Table 1 Table 2: Seasonal incidence of Insect pest infesting to D. latifolia Table 2: Seasonal incidence of Insect pest infesting to D. latifolia Page 6/18 SL. DISCUSSION viridula was recorded as an effective biocontrol agent due to its high food consumption efficiency and more abundance which decreases the dock population in growing season (Renner, 1970; Brooks and Whittaker 1998). This is the first record of feeding on D. latifolia, though Engel (1956) has reported about 38 species of ten plant families as occasional or regular hosts, but there was no record of Dalbergia sp. This insect was also recorded from Rheum palmatum (Balachowsky & Mensil, 1936), Begoniax tuberhybrida in UK (Salisbury and Platoni, 2013). This species was also reported as a biological control agent for Rumex species (Engel, 1956; Chevin, 1968; Martinková & Honěk, 2004). In the present study we have recorded ten insect pests from Uttarakhand province of Northern, India, among Gastrophysa viridula leaf beetle, first time recorded feeding on D. latifolia. Beetle feed on the young leaves by cutting the leaf from its margin to make a circular pattern in the centre of the leaf. G. viridula has been recorded as a pest of Begonia (Begoniaceae) (Salisbury & Platoni, 2013), though, it has been considered an important biocontrol agent of dock weed (Swatonek 1972, Barbattini et al., 1986, Hatcher et al., 1997). G. viridula was recorded as an effective biocontrol agent due to its high food consumption efficiency and more abundance which decreases the dock population in growing season (Renner, 1970; Brooks and Whittaker 1998). This is the first record of feeding on D. latifolia, though Engel (1956) has reported about 38 species of ten plant families as occasional or regular hosts, but there was no record of Dalbergia sp. This insect was also recorded from Rheum palmatum (Balachowsky & Mensil, 1936), Begoniax tuberhybrida in UK (Salisbury and Platoni, 2013). This species was also reported as a biological control agent for Rumex species (Engel, 1956; Chevin, 1968; Martinková & Honěk, 2004). The leaf beetle, Chrysochus cobaltinus native to North American (Arnett, 1968; Lopatin, 1984) have been recorded for the first time in this study. Adults emerge in early summer and adults feed on leaves of Apocynum cannabinum (Apocynaceae) and Asclepias spp. (Asclepiadaceae) (Dickinson, 1995; Sennblad & Bremer, 1996); Asclepias speciosa and A. eriocarpa (Sady, 1994; Dickinson, 1995; Dobler & Farrell, 1999). Larvae of this species are obligate root feeders, single generation per year and persist for approximately six weeks (Williams, 1992, Dickinson, 1995). C. DISCUSSION The expansion of the ecological niche and host range of insect pests may be influenced by global climatic changes, and this phenomenon is considered part of a dynamic evolutionary process. Studies conducted by Bernays and Chapman (1994), Janz et al. (2006), Williams and Liebhold (1995), and Van et al. (2004) have highlighted the potential impact of climate change on the behavior and distribution of herbivorous insects. In order to successfully expand their range to new habitats or hosts, herbivorous insects often require behavioral adaptations (Henniges-Janssen et al., 2011). However, the regular assessment of geographical and host range extensions of insect pests in forestry is often lacking. This knowledge gap makes it challenging to predict and manage potential pest outbreaks effectively. In the case of Dalbergia latifolia, the quality, growth, and productivity of this tree species are negatively affected by frequent insect pest outbreaks. There are many insect pests were previously recorded from D. latifolia viz. Karria lacca Kerr a sap feeder, Lecanium hesperidum Linn, Gragara sordid Fun., Oxyrachis mangiferana Dis., Oxyrachis trandus Fun., (Hemipteran sap suckers), Aulacophora fovecollis Lucans beetle, Anomala dalbergiae Arrow (leaf feeding beetle), Argyroploce aprobola Meyrick (Eucosmidae); Anisodes obrinaria Guenee, Ectropis bhurmitra Walker (Geometridae); Tapena thwaitesi Moore, Tapena thwaitesi Moore (Hesperiidae); Cosmotriche laeta Walker (Lasiocampidae); Page 7/18 Page 7/18 Dasychira dalbergiae Moore (Lymantriidae); Achaea janata Linnaeus, Anoba polyspila Walker, Ericeia inangulata Guenee, Hamodes aurantiaca Guenee, Metachrostis trigona Hampson, Midea rectalis Walker, Mocis undata Fabricius, Plecoptera quaesita Guenee, Plecoptera reflexa Guenee, Rhesala imparata Walker (Noctuidae), Spataloides costalis Moore (Notodontidae); Charaxes polyxena Moore, Neptis viraja Moore (Nymphalidae); Bocchoris onychinalis Guenee, Lamprosema imphealis Walker, Maruca testulalis Geyer, Nephopteryx sp. (Pyralidae); Clanis titan titan Rothschild & Jordan (Sphingidae); Striglina scitaria Walker (Thyrididae) (lepidopteran defoliators); and bark feeding lepidopteran insect pests: Labdia molybdaula Meyrick (Cosmopterygidae); Opogona xanthocrita Meyrick (Lyonetiidae); Hapsifera rugosella Stainton (Family: Tineidae) (Beeson, 1941; Mathur and Singh 1959, Chatopadhyay, 2021). In the present study we have recorded ten insect pests from Uttarakhand province of Northern, India, among Gastrophysa viridula leaf beetle, first time recorded feeding on D. latifolia. Beetle feed on the young leaves by cutting the leaf from its margin to make a circular pattern in the centre of the leaf. G. viridula has been recorded as a pest of Begonia (Begoniaceae) (Salisbury & Platoni, 2013), though, it has been considered an important biocontrol agent of dock weed (Swatonek 1972, Barbattini et al., 1986, Hatcher et al., 1997). G. DISCUSSION talaca was also reported from cinchona, tea, coffee, cocoa and fruit trees in tropics (Entwistle, 1972), Shorea robusta and Cinchona (Shankar et al., 1998), new pest of forest tree Quercus incana (Singh & Singh, 2004), on Perilla frutescens (Uniyal & Singh, 2010). It has a short life cycle with multiple overlapping generations. Apart from that, factors like faster multiplication, lack of efficient natural enemies cause high density of larvae and notices more active in winter months (Das et al., 2010). Ectropis bhurmitra has a broad distribution in Asian-Pacific regions and well recorded from Borneo, Buru, Java, India, New Guinea, Peninsular Malaysia, Philippines, Solomon Islands Sumatra, Sri Lanka, Sulawesi, Taiwan, and Thailand (Sato, 2007). We have record of E. bhurmitra on D. latifoilia, though this species recorded on this species by Mathur and Singh, (1959). Additionally, on a wide range of host plants, e.g. Aleurites montana, Artemisia vulgaris, Bombax malabaricum, Lantana aculeata, Phoebe lanceolata, Shorea robusta and Tectona grandis (Beeson 1941). The presence of large number of alternative host plants has induced the looper problem in tea garden in most of the areas of Assam and West Bengal of India (Antony, 2012). Leptocentrus taurus was found to be feed on apical part of D. latifolia first time, though this insect pest was recorded on Dalbergia sissu, Zizyphus jujuba and on Solanum melongena, Parthenium hysterophorusin Tamilnadu, India from India (Thangavelu, 1980; McKamey, 2017; Biwas et al., 1994, Kumar, 2017). We have recorded that female of this insect lay egg in the tender shoots and both nymph and adult feed gregariously on tender shoot. The insect species was found everywhere in the province and found very common. Kumar (2017) have also observed that both the adults and nymphs were found to suck the sap from the tender parts particularly from the shoot during March to June and about 91% plants of D. sissoo were infested in Jharkhand. Drosicha stebbingi both nymph and adult suck the plant sap from stem of the plant. D. DISCUSSION Cobaltinus range in British Columbia south through Washington, Idaho, Montana, Oregon, California, Nevada, Utahand Colorado to Arizona and New Mexico (Peterson et al., 2001). We have recorded Apoderus crenatus Voss species feeding on the leaf of D. latifolia for the first time from India. The adult beetle makes leaf roll similar as done by A. sissu in D. sissoo. We have identified this species with morphological characters of type of this species at ICAR-Indian Agricultural Research Institute, New Delhi, and the species is entirely different from A. sissu Marshall (type collection (NFIC-FRI, Dehradun India). The nature of damage was similar to the Apoderus sissu, as its adult stage was found to cause damage to new flush either by rolling them off or by cutting through the mid rib and defoliation (Kumar, 2017). Plecoptera reflexa a well-known defoliator and considered as serious pest of D. sissoo in India and poor quality plantations have often been derelict, it was first reported in 1899 and regular annual defoliation was recorded in Pakistan and northern India in the month of April (Beeson, 1941; Rawat & Singh, 2003). We have Page 8/18 Page 8/18 recorded this species first time from Uttarakhand, Northern India. Roychoudhury & Mishra, 2021 have recorded this species as a major pest of D. latifolia Central from India. recorded this species first time from Uttarakhand, Northern India. Roychoudhury & Mishra, 2021 have recorded this species as a major pest of D. latifolia Central from India. Hyposidra talaca is distributed throughout the oriental region and it was first reported as a tea pest from Indonesia and later was reported as a destructive tea pest from West Bengal (Biswas et al., 2004).. H. talaca is a dominantly prefer forest host plants to feed (Shankar et al., 1998), such as Bombax ceiba, Cassia sp., Cedrela toona, Ficus glomerata, Shorea robusta, Syzygium cumini, Tectona grandis, but we have recorded this species for the first time from D. latifolia in India. Majorly it is a defoliating tea pest, it creates a periodical and regular problem in north-eastern tea plantations of India. It was also reported as weed control against Chromolena odorata, as a major biocontrol agent (Muniappan & Viraktamath, 1986). H. CONCLUSION It is noteworthy that some of these insect pests were documented for the first time on D. latifolia, indicating the need for further research and monitoring to understand their impact and develop appropriate pest management strategies. The information gathered from such studies is valuable for assessing the potential threats posed by insect pests, improving pest management practices, and safeguarding the health and productivity of D. latifolia plantations. The study has given the current status of insect pest of D. latifolia of northern India, with some new insect pest records. The infestation of insect pest shows that the introduction of D. latifolia in new environment of northern India making the species susceptible and favourable to insect pest to host upon. After observing the insect pests on D. latifolia it may also be concluded that in near future these pest may become a serious threat to high valued plantation of D. lattifolia, therefore, more emphasis will be required for selection of superior / resistant progeny for successful introduction and sustainable growth of D. latifolia in Northern India. Though, continued research in this area will contribute to a better understanding of the ecological dynamics and potential impacts of insect pests on this economically important timber species. DISCUSSION stebbingi is a major polyphagus sap sucking pest of forestry, horticulture and agriculture species as it has recorded to be infesting on Shorea robusta later from Mangifera indica, Carica papaya, Zizyphus jujuba, Prunus persica, Prunus domestica, Artocarpus heterophyllus, Bauhinia variegata, Helianthus annuus, Rosa indica, Althaea rosea, Citrus sp., Nerium odorum, Eugenia jambolana, Eriobotrya japonica, Vitisvini fera, Jasminum sambac, Aloizzia lebbek, Hibiscus sp., Pyrus malus, Juglans regia, and Pyruscom munis, Litsaea polyantha, Butea frondosa, Holarrhena antidysenterica and Mallotus philippinensis (Dutt, 1925; Latif, 1949; Beeson, 1941; Rahman, 1944). Page 9/18 Leptocorisa acuta, commonly known as the rice ear bug, is a significant pest of rice in India. Both the nymphs (immature stages) and adult bugs have been observed feeding on the young tender parts of plants in various locations. We have recorded both nymph and adult of L. acuta feeding on young tender part of the plant at FRI nursery and at Lalkuan plantation. L. acuta was also recorded infesting to Dalbergia sissoo and causing considerable damage by sucking the sap of tender shoot and leaves in nursery and young plantations in Jharkhand (Chattopadhyay, 2021), on Calotropis procera in Madhya Pradesh (Chandra et al., 2011) and on nutmeg trees (Abraham & Mony, 1977). In our study Ricania speculum recorded to be the new insect pest of D. latifolia in India. This hopper commonly known as black plant hopper and it is distributed mainly in Asia, Australia and tropical Africa (Mazza et al., 2014; Rossi & Lucchi, 2015). It has also been recorded in Genoa and La Spezia, China, Indonesia, Japan, Korea, Philippines, Taiwan, and Vietnam (Bourgoin, 2016). R. speculum broadly polyphagous pest, females lay eggs into the woody twigs of many host plants, and subsequently nymphs feed the sap of the plant and develop (Mazza et al., 2014; Rossi & Lucchi, 2015; Rossi et al., 2015). References 1. Abraham, C.C., & Mony, K.S.R. (1977). Occurrence of Leptocorisa acuta Fabr. (Coreidae, Hemiptera) as a pest of nutmeg trees. Journal of the Bombay Natural History Society. 74(3): 553. 2. Anonymous (2018). “Dalbergia latifolia – The High-Valued Indian Rosewood,” https://www.winrock.org/factnet-a-lasting-impact/fact-sheets/dalbergia-latifolia-the-high-valued-indian- rosewood. 2. Anonymous (2018). “Dalbergia latifolia – The High-Valued Indian Rosewood,” https://www.winrock.org/factnet-a-lasting-impact/fact-sheets/dalbergia-latifolia-the-high-valued-indian- rosewood. 3. Antony, B. (2012). Looper caterpillar invasion in north east Indian tea agro ecosystem: change of weather and habitat loss may be possible causes. 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Chaterjee, M., Soumyashree, Manohara, T.N., & Shettannavara, V. (2020). Population structure, regeneration status and carbon sequestration potential of Dalbergia latifolia Roxb. (Rosewood) in Rajeev Gandhi National Park, Karnataka. My Forest, 56(1&2): 141–155. 12. Chaterjee, M., Soumyashree, Manohara, T.N., & Shettannavara, V. (2020). ACKNOWLEDGEMENT The authors are greatly indebted to the National CAMPA authority, Government of India for providing funding support for this study. We are also grateful to the Director General, Indian Council of Forestry Research and Education (ICFRE), Uttarakhand, India for providing necessary research facilities for this study. FUNDING: First author is a team member of fund receiving team from CAMPA, Ministry of environment, Forest and climate change, Government of India. Author second and thirds are part of the project, experimentation of the study and manuscript writing. 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(2017). References Species 2000 & ITIS Catalogue of Life, 30th June 2017. Digital resource at www.catalogueoflife.org/col. Species 2000: Naturalis, Leiden, the Netherlands. 34. Muniappan, R., & Viraktamath, C.A. (1986). Insects and mites associated with Chromolaenaodorata (L.) R.M. King and H. Robinson (Asteraceae) in Karnataka and Tamil Nadu. Entomon. 11:285–287. 35. Orwa, C., Mutua, A., Kindt, R., Jamnadass, R., & Simons, A. (2009). Agroforest tree Database: a tree reference and selection guide version 4.0. 36. Peterson, M.A., Dobler, S., Holland, J.T.L., & Locke, S. (2001). "Behavioral, Molecular, and Morphological Evidence for a Hybrid Zone Between Chrysochus auratus and C. cobaltinus (Coleoptera: Chrysomelidae). Annals of the Entomological Society of America. 94 (1):1–10. https://doi:10.1603/0013- 8746(2001)094[0001:BMAMEF]2.0.CO;2. 37. Rawat, V., & Singh, S. (2003). Early epidemic of Plecoptera reflexa on Dalbergia sissoo an indication of Doon valley climate change. Indian Forester, 129(4): 539–540. 37. Rawat, V., & Singh, S. (2003). Early epidemic of Plecoptera reflexa on Dalbergia sissoo an indication of Doon valley climate change. Indian Forester, 129(4): 539–540. 38. Renner, K. (1970). Beziehungen zwischen Nahrungs-menge, Körpergewicht und Entwicklungsalter bei Gastroidea viridula Deg. (Col. Chrysomelidae). Anz. Schädl.-Kde Pfl.-Schutz, 43: 119–122. 38. Renner, K. (1970). Beziehungen zwischen Nahrungs-menge, Körpergewicht und Entwicklungsalter bei Gastroidea viridula Deg. (Col. Chrysomelidae). Anz. Schädl.-Kde Pfl.-Schutz, 43: 119–122. 39. Rossi, E., & Lucchi, A. (2015). The Asian planthopperRicania speculum (Walker) (Homoptera: Ricaniidae) on several crops in Italy: a potential threat to the EPPO region? OEPP/EPPO Bulletin, 45: 119–122. 39. Rossi, E., & Lucchi, A. (2015). The Asian planthopperRicania speculum (Walker) (Homoptera: Ricaniidae) on several crops in Italy: a potential threat to the EPPO region? OEPP/EPPO Bulletin, 45: 119–122. 40. Rossi, E., Stroinski, A., & Lucchi, A. (2015). Egg morphology, laying behaviour and record of the host plants of Ricania speculum (Walker, 1851), a new alien species for Europe (Hemiptera: Ricaniidae). Zootaxa, 4044 (1): 93–104. 41. Roychoudhury, N., & Mishra, R.K. (2021). Shisham defoliator Plecoptera reflexa and its control measures. Van Sangyan, 8 (3): 30–32. 42. Sady, M.B. (1994). Survey of the blue milkweed beetle, Chrysochus cobaltinus LeConte (Coleoptera: Chrysomelidae), in western Nevada. Coleop. Bull. 48: 299. 43. Salisbury, A., & Platoni, A. (2013). The green dock beetle, Gastrophysa viridula (De Geer) (Chrysomelidae) - a pest on Begonia (Begoniaceae), a new host record. The Coleopterist 22(3): 124–125. 44. Sato, R. (2007). The genus Ectropis (Geometridae, Ennominae) in Indonesia, with descriptions of two new species. Transactions of the Lepidopterological Society of Japan, 58(2): 205–214. 45. References Sennblad, B., & Bremer, B. (1996). The familial and subfamilial relationships of Apocynaceae and Asclepiadaceae evaluated with rbcl data. Plant Syst. Evol. 202: 153–175 45. Sennblad, B., & Bremer, B. (1996). The familial and subfamilial relationships of Apocynaceae and Asclepiadaceae evaluated with rbcl data. Plant Syst. Evol. 202: 153–175 46. Shankar, U., Lama, S.D., & Bawa, K.S. (1998). Ecosystem reconstruction through taungya agrosystems following commercial logging in dry mixed deciduous forest in Darjeeling Himalaya. Forest Ecology and Management, 102, 131–142. 46. Shankar, U., Lama, S.D., & Bawa, K.S. (1998). Ecosystem reconstruction through taungya agrosystems following commercial logging in dry mixed deciduous forest in Darjeeling Himalaya. Forest Ecology and Management, 102, 131–142. 47. Singh, K.P., & Singh, S. (2004) Hyposidra talaca walk. (Lepidoptera: Geometridae) a new pest of Quercus incana Roxb. Indian Forester, 130:231–232 47. Singh, K.P., & Singh, S. (2004) Hyposidra talaca walk. (Lepidoptera: Geometridae) a new pest of Quercus incana Roxb. Indian Forester, 130:231–232 48. Soerianegara, I., & Lemmens, R.H.M.J. (1994). ”Plant Resources of South-East Asia No 1(5). Timber trees: Major commercial timbers,” Bogor, Indonesia, pp.160. 48. Soerianegara, I., & Lemmens, R.H.M.J. (1994). ”Plant Resources of South-East Asia No 1(5). Timber trees: Major commercial timbers,” Bogor, Indonesia, pp.160. Page 13/18 49. Sujatha, M.P., Thomas, T.P., & Florance, E.J.M. (2008). Grwoth and enhancement of Dalbergia latifolia through soil management techniques. KFRI Research Report No 303. KFRI370/01 50. Swatonek, F., (1972). Ein Beitrag zur Biologie des Ampferbl attkäfers (Gastroidea viridula Deg.). Anz. Schädl.-Kde Pfl.-Schutz, 45: 116–119. 51. Thangavelu K (1980) Report of Leptocentrus taurus fabricius (Membracidae: Homoptera) feeding on Partheniumhysterophorus Linn. Entomon. 5 (4), 357. 52. Treanor, N.B. (2015). China’s Hongmu consumption Boom: Analysis of the Chinese rosewood Trade and Link to illegal activity of Tropical forsted countries. Pp 1–48. Forest Trend, Washinton D.C., USA. 53. Uniyal, N., & Singh, S. (2010). First record of Hyposidra talaca Walker (Lepidoptera: Geometridae) on Perilla frutescens Linn. Indian Forester, 136(3): 400–401. 54. Van, B.S.A., Aiello, A., Valderrama, A., Medianero, E., Samaniego, M., & Wright, S.J. (2004). General herbivore outbreak following an El-Nino related drought in a lowland Panamanian forest. Journal of Tropical Ecology 20, 625–633. 55. Williams, C.E. (1992). Movement of the dogbane beetle, Chrysochus auratus (Coleoptera: Chrysomelidae) in a patchy environment. Banisteria 1: 8–10. 56. Williams, D.W., & Liebhold, A.M. (1995). Herbivorous insects and global change: potential changes in the spatial distribution of forest defoliator outbreaks. Journal of Biogeography, 22, 665–671. Figures Page 14/18 Figure 1 (A-I): Leaf defoliators insects feeding on Dalbergia latifolia. A- Gastroph C- Adult Apoderus crenatus weevil & feeding symptoms; D-  leaf feeding leaf feeding symptoms of Chrysocus cobaltinus; F- leaf rolling symptom Plecoptera reflexa; H- Ectropis bhurmitra ; I-Larva of Hyposidra talaca. Figure 1 (A-I): Leaf defoliators insects feeding on Dalbergia latifolia. A- Gastrophysa viridula; B-Chrysocus cobaltinus; C- Adult Apoderus crenatus weevil & feeding symptoms; D-  leaf feeding symptoms of Gastrophysa viridula; E- leaf feeding symptoms of Chrysocus cobaltinus; F- leaf rolling symptoms of Apoderus crenatus; G- Larva of Plecoptera reflexa; H- Ectropis bhurmitra ; I-Larva of Hyposidra talaca. Page 15/18 Page 15/18 Figure 2 (A-F): Sap sucking insects feeding on Dalbergia latifolia. A- Leptocentrus taurus ; B- Drosicha stebbingi; C&D Nymph & Adult of Ricania speculum;  E&F- Nymphs & Adult of Leptocorisa acuta . Figure 2 (A-F): Sap sucking insects feeding on Dalbergia latifolia Nymph & Adult of Ricania speculum;  E&F- Nymphs & A Figure 2 (A-F): Sap sucking insects feeding on Dalbergia latifolia. A- Leptocentrus taurus ; B- Drosicha stebbingi; C&D Nymph & Adult of Ricania speculum;  E&F- Nymphs & Adult of Leptocorisa acuta . Page 16/18 Figure 3 Insect pest survey and study area of D. latifolia Figure 3 Insect pest survey and study area of D. latifolia Insect pest survey and study area of D. latifolia Page 17/18 Figure 4 (A) Nursery of D. latifolia at New Forest Campus; (B) plantation of D. latifolia at L Figure 4 (A) Nursery of D. latifolia at New Forest Campus; (B) plantation of D. latifolia at Lalkuan Page 18/18
https://openalex.org/W2046736974
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The School Question in Belgium
˜The œelementary school journal/˜The œElementary school journal
1,921
public-domain
3,849
This content downloaded from 080.082.077.083 on January 11, 2018 20:29:04 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and- JULIAN PIERCE Washington, D.C. The solution of the school question in Belgium is the subject of a recent comprehensive address by M. Jules Destr6e, Belgian Minister of Sciences and Arts. The Belgian primary schools are administered by the authorities of the communes. The central government, however, contributes large subsidies to the support of the primary schools and establishes certain requirements to which the primary schools must conform. The administration of the subsidies, including the national public- school inspection service, falls to the Minister of Sciences and Arts. p , For fifty years or more the so-called school war has been waged in Belgium over religious courses in the public schools and state control of private religious schools. Religious instruction in the public schools was established by the law of 1895 and has been in operation since that date. Parents who do not wish their children to take the religious courses may have them exempted. g y p Political control of the public schools has also been a major issue for many years in Belgium. In politics the school war is reflected in the activities of the Catholic or Clerical party and of the Liberal or anti-clerical party. Each of these parties has undertaken to use the public schools to impress the minds of the children with its political principles. p p p The present political situation in Belgium gives added interest to M. Destr6e's address. The Belgium government is of the parliamentary form, the cabinet being chosen from the members of the Chamber of Deputies. The present chamber is composed of seventy-one Catholics, seventy Socialists, thirty-four Liberals, and ten non-party members. The parliamentary elections occur on November 20. There is a possibil- ity that the Socialist party may supersede the Catholic as the largest party in the chamber. Should this happen, the Socialist party 290 This content downloaded from 080.082.077.083 on January 11, 2018 20:29:04 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c THE SCHOOL QUESTION IN BELGIUM 291 will take the prime ministership and the general administration of the government, including its educational system. Consequently M. Destr6e's analysis of the school question and its solution from the Socialist viewpoint is of more than passing interest. p p g M. JULIAN PIERCE Washington, D.C. Destr6e is one of the four Socialist members of the present Belgian cabinet, having held the portfolio of Minister of Sciences and Arts since the organization of the "government of democratic union" in November, 1918. , An understanding of the dispute and the importance which the disputants attach to it is necessary in the search for the solution of the school question. Therefore M. Destree gives this historical summary of the matter: During the first revision of the Belgian constitution in 1830 the rulers, recruited solely from the taxpaying electors of the bourgeoisie, experienced a temporary period of relative harmony. They were all preoccupied in assuring the fruits of the Revolution. Moreover, they were united by a community of interests. Then divergencies of opinion accentuated themselves and gave birth to two organized parties, the Liberal party and the Clerical party. g p , p y p y The school question was the basic reason for the manifestation of this distinction and its persistence. All the other differences were unimportant; the incomprehension of the needs and rights of the people was about equal in the two bourgeois parties. p The Liberals, however, saw the importance of primary education and wished to separate it from religious influences. The Clericals, less convinced of the benefits of the wide diffusion of instruction, insisted that it should be strongly stamped with the impression of the confessional. Since 1842 law has followed law, unceasingly renewing the dispute and always exhibiting the divergence of opinion. p Primary education was a communal institution. The Liberals were in control of the large cities. Consequently schools multiplied there and always sought to bring victory for Liberal ideas in spite of legal obstacles. On the other hand, the Catholics, who were reactionary, developed and multiplied the schools over which they were the masters. Thus, thanks to constitutional liberty, there arose two opposing school systems, the so-called "free" schools and the "official" schools,' with the strange consequence that when the Catholics were in power and had control of public instruction they exerted themselves to impede and curtail it to the profit of the competing schools. p p p g This struggle has endured for more than half a century and is not yet over. JULIAN PIERCE Washington, D.C. The claims it has made on both parties for zeal, energy, and sacrifice are I The primary schools of Belgium under the administration of the local authorities and subsidized by the central government are called "official" schools. The private Catholic parochial schools are called "free" or "Catholic" or "confessional" schools. This content downloaded from 080.082.077.083 on January 11, 2018 20:29:04 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-an THE ELEMENTARY SCHOOL JOURNAL 292 [Decembe [Decembe incalculable. It has poisoned all our public life. It has engendered, even in the villages, lamentable civil discords, suffering, and persecutions of all sorts. It has made irreducible enemies out of citizens who otherwise would have understood, esteemed, and assisted each other. After pointing out that the contest over the school question is responsible for the slow progress of the democratic ideas in the Catholic party, and that it is the desire to retain control of the public schools which maintain the Catholic party in Belgium, although that party has disappeared from other European countries, M. Destree declares that a real solution of the problem cannot be effected until the school question divests itself of certain illusions which have caused the continued quarrels. The first illusion is the idea that the public school is neutral, tolerant, and free from religious influence. Do not forget that public instruction is under the administration of the commune. In cities with a Liberal administration it occasionally has neutral qualities. It is these occasional instances which its enthusiastic supporters have in mind. But let them enlarge the field of their observation; let them include the innumerable communes where the administration is Catholic, and there they will find different conditions. y Quite recently my attention was directed to a public school in which an altar to Saint Joseph was set up and where they recited prayers for souls in purgatory! What more could they do in a Catholic school? I could multiply these examples. p I am convinced that, taken as a whole, public instruction does not possess sufficient merit to warrant the feverish solicitude of certain people. The public school does not escape the influence of local environment, and certain public schools in the Flemish region are more religious than are certain Catholic schools in the industrial communes of the Walloon provinces. Another illusion is the much praised quality of the public schools. This content downloaded from 080.082.077.083 on January 11, 2018 20:29:04 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and- JULIAN PIERCE Washington, D.C. While there are some public schools as well as some Catholic schools that are excellent, M. Destree is disposed to believe that "taken as a whole, the two educational systems are about equivalent- equivalent in mediocrity." q y But the Belgian Minister of Education positively declares that both the Catholic and Liberal parties have sought to use the primary schools to make voters for their respective political parties. This partisan policy, however, is based upon another illusion which must be cast aside in the Socialist solution of the school question. This content downloaded from 080.082.077.083 on January 11, 2018 20:29:04 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and- THE SCHOOL QUESTION IN BELGIUM 19211 293 Those who control the schools control the nation, is a saying that is often repeated. To my mind this is an illusion which close observation of the facts will demonstrate as such. I know this belief is widespread. Liberals as well as Catholics share in the opinion. But I am convinced that it does not cor- respond with reality and that it has been considerably overvalued. p y y To attribute to me the thought that primary education is unimportant would be to misunderstand me completely. I simply say that it has not the decisive importance which is too often accorded to it. Even in the bourgeois class we see former students of the Jesuits among the well-known anti-clericals; this clearly shows that in their intellectual development other elements than the lessons of their professors have intervened. It is still more evident in the working class, where school impressions are quickly effaced by the impressions of the workshop and the factory and where the life of the adult singularly transforms the recollections of the child. It was for the most part generations who graduated from the schools of the Petits Freres that made the French Revolution and pushed official impiety to its last limits. For the past, this example is sufficiently instructive. p , p y As for the present, if the school controls the nation, how explain the fact that although nine-tenths of the scholars take the school courses in religious instruction their religious training does not reflect itself in the electoral body, for only four-tenths of the electors vote for the Catholic party ? JULIAN PIERCE Washington, D.C. To be exact, in the last general election held in November, 1919, 36.64 per cent of the votes were cast for the candidates of the Catholic party. The discrepancy occurs because other influences than those of the school come into action in determining the views of the electors. Born of the desire of the opposing bourgeois parties to assure themselves a voting constituency, the school question, therefore, has its origin in an error of judgment as to the political importance of primary education in social life. It is an illusion-a bourgeois illusion. M. Destree finds his keynote for Labor's solution of the school question in the fact that the working people of Belgium take but little interest in religious instruction in the schools. "It is always the bourgeois elements who carry on the propaganda to increase the exemptions." In spite of this propaganda the number of exemp- tions reaches but 33,432 out of 960,818 scholars. This emphatically demonstrates that the peasants and industrial workers have other preoccupations than the consideration of religious courses in the schools. How right the workers are! How many other questions affect them more directly! One thing is certain. The Clerical-Liberal question has too long masked the social question; and for the proletariat the latter is much more important than the former. This content downloaded from 080.082.077.083 on January 11, 2018 20:29:04 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c THE ELEMENTARY SCHOOL JOURNAL [Decem THE ELEMENTARY SCHOOL JOURNAL 294 [Decembe [Decembe [Decembe At the same time, the Socialist party must guard against the illusions which are at the base of the school war. We must resolutely renounce making use of the schools for political purposes. p p Both Liberals and Clericals have undertaken to use the primary schools to make Liberals and Clericals. We, on the other hand, undertake to make Socialists; but not by means of the primary school. An educational institution should have an educational and not a political object, either open or dis- simulated. We shall endeavor to use the public school to make well-informed citizens, equipped for the struggles of life. It is these citizens who will later, and outside of the schools, charge themselves with the function of showing the voters the political party which they should support. JULIAN PIERCE Washington, D.C. p p y y pp Here is what the working people expect of the school: instruction, educa- tion, preparation for life. This and nothing more. Some say the child belongs to the parents; others affirm that he belongs to the state. These views are equally false. The child cannot be the property of anyone. He is a being with respect to whom both the parents and the state have rights; but above all, and because of the child's weakness, they have duties. It is in the order of nature that the parents fulfill these duties. It may happen, however, that, unconsciously or through inability, the parents neglect to perform their duties. Then the care of the state intervenes. Among its necessary attributes the state includes protection for the weak as well as the development of productive energies. "It cannot abandon to parents the right to determine whether their children shall be instructed; social interest rises above individual caprice. Primary instruction must therefore be made legally obligatory. This was done in Belgium by the law of May 18, 1914, and the principle is no longer opposed. Belgium y y , , p p g pp Rights of the state, the obligation for instruction on the one side, but liberty of instruction, free choice of the school by the parents on the other side -this is the conciliation which has inspired the legislator. It seems to me that this policy should be approved. Suppression of the private religious schools, advocated by certain ultra anti-clericals, should have no place in Belgium's public policy. Above all, such a policy is foreign to the underlying principles of the Socialist party of Belgium and consequently cannot figure in the Socialist solution of the school question. I have never been one of those who exaggerate the rights of the state to the point of monopoly. That is a sectarian pretension which leads to insupport- able tyrannies. It has never had more than a few supporters in Belgium, a country attached by its Constitution as well as its traditions to freedom of thought and liberty of education. This content downloaded from 080.082.077.083 on January 11, 2018 20:29:04 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and- THE SCHOOL QUESTION IN BELGIUM Z921] 295 There are Socialists who sometimes have a tendency to exaggerate the importance of power. JULIAN PIERCE Washington, D.C. Desirous of hastening the progress of the ideas which are dear to them, they believe in the possibility of accomplishing it by compul- sion-a dangerous course, which quickly ends in tyranny. Human happiness is not brought about against the will of mankind. In certain cases the law should impose restraint; it becomes inefficacious as soon as it passes beyond what the nation is capable of accepting. As for me, I cannot separate socialism from liberty. Especially in the domains which touch conscience and thought, liberty should be scrupulously respected. y p y p If it is proper, therefore, to consider education as a public service, the same as justice, the army, the police, and the railroads are public services, it is also proper not to demand the authority of the state except within the limits compatible with liberty of opinion. Moreover, suppression of the "free" schools is unthinkable from the point of view of the children themselves. Compulsory educa- tion implies enough schools to accommodate all the scholars. As a matter of fact, the number of schools is scarcely sufficient now, even when the confessional schools are included. If there were enough intolerants to desire the suppression of the Catholic schools with enough power to realize their desire, they would thereby make the application of the compulsory school attendance law impracticable and throw thousands of children into the streets and its hazards; moreover, the children would be mostly poor children. A Socialist, free thinker though he may be, cannot consent to such an enormity. He must therefore tolerate the confessional school. I go still further. To my mind "tolerate" is an improper term. "Accept" is the word to use. Accept the confessional school without preconceived hostility. Accept it as a fact which it is vain to wish does not exist. Accept it as worthy of respect since its existence and prosperity result from the desires of thousands of compatriots. Accept it as useful, because it supplements the numerical insufficiency of public education. y p We Socialists have other things to do than to take up the traditions and rancors of the Liberal party. On the question of private religious schools we should declare that we shall neither persecute nor annoy them. JULIAN PIERCE Washington, D.C. Why not hope that by this attitude we shall inspire as a consequence a less hostile attitude and that one day, instead of being the malignant competitor of public school educa- tion, the parochial schools will become the cordial collaborator ? , p If we become permeated with these hopes we begin to perceive the solution sought, the bridge across the abyss. Education, regardless of whether it is parochial or public, must be made a great national enterprise. This nationali- zation can be brought about without any serious attack on either the autonomy of the communes or the desires of the leaders of parochial education with respect This content downloaded from 080.082.077.083 on January 11, 2018 20:29:04 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and- THE ELEMENTARY SCHOOL JOURNAL 296 [Decembe [Decembe to the religious character of their schools. But it should increase the rights of the state. In 1919 the Belgian Parliament enacted a law giving the same subsidies to private religious schools as to public schools, provided the former conformed with the minimum standards laid down by the state for the latter. The bill was supported by M. Destr6e and most of the other Socialist members. Their action caused con- siderable criticism, it being charged, among other things, that M. Destree had made concessions to the Catholics. In reply to this criticism M. Destree insists that his general conception of equal subsidies for the private religious schools is the same conception that the Socialist councilors instinctively affirmed before the war when the establishment of school canteens was under consideration. In certain municipal councils the Liberals undertook to limit the advantages of the canteens to the public scholars. The Socialist councilors took a more generous position and insisted that the lunches should be given to the school children regardless of the religious beliefs of their parents. "Today we are doing for the sustenance of the brain what we formerly did for the sustenance of the body." y M. Destree has always been classed as an anti-clerical. Has he given up his anti-clerical views? He has never been an ultra anti-clerical. He is still a conservative anti-clerical. Understand me thoroughly. I am not abandoning anti-clericalism. I am simply abandoning its illiberal and sectarian aspects which, moreover, I have never supported. JULIAN PIERCE Washington, D.C. But I remain detached from every creed whatsoever, and am firmly determined to defend the liberty of the people without religious belief against the encroachments, always to be feared, of the religionists. In short, I propose to substitute for negative tactics, which are likely to create opposition, an actual constructive doctrine which is necessaryfor a government. pp , necessary government. "What! Are you going to give public money to priests and nuns?" I am asked. I reply, "Why not? I subsidize them not because they are priests and nuns but because they perform to my satisfaction a work specified by law and recognized as beneficial. That is sufficient; and, unless it is desired to give an exhibition of a deplorable Jacobinistic spirit, that is as much as can reasonably be demanded." Other critics say that these people whom I endow with public funds are our enemies and that they will use against us the money with which you subsidize them. This content downloaded from 080.082.077.083 on January 11, 2018 20:29:04 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.ed THE SCHOOL QUESTION IN BELGIUM 1921] 297 I reply, "Set yourself at ease with respect to that. I do not consent to a subsidy except on condition that it is accompanied by control. Naturally the one does not take place without the other. From the time that private parochial education accepts money from the state it is not free from state control; it is subject to the elementary rule of probity that he who receives money for a definite purpose is not free to expend that money for anything he sees fit. He is required to show that the money has been expended for the prescribed purposes. JULIAN PIERCE Washington, D.C. From that point there may be differences of a legitimate character between the public and the parochial schools, since they are the result of liberty of education and are desired by the children's parents; but for the rest, both of them should conform to the law which assists them equally--both of them are educational institutions subsidized by the state and are to a certain extent public." p If the future should show that my attempt to realize school peace is vain through the fault of those who oppose it, if the parochial schools wish to remain parochial schools simply for the purpose of securing subsidies, but should refuse state control, then it would be necessary to return to the principles of rigorous secular public education, apply those principles more energetically than did the Liberals, and leave the Catholics merely the liberty to have their own schools at their own expense, risks, and perils. Does the Socialist solution of the school question in Belgium offer the only hope for school peace ? M. Destree believes it does. Under the school laws of 1919 and 1920o the discretionary power of persecution and favoritism by the Minister of Education is con- siderably enlarged. "The anti-clericals will never consent to have this power given to a Catholic. On the other hand, and for similar reasons, a Liberal will be suspected by the Clericals, and even though he desired school peace he would also fall quickly under the suspicion of his friends." There remains but the solution offered by M. Destree as the spokesman of the Socialist party: In school matters a Socialist minister should develop and attempt to realize a solution different from the solutions brought forward by the old parties. Other ministers than I have undertaken to solve the school question; Georges Lorand and Leon Furnemont among others. But the time had not come. g Has the time come now ? Has the catastrophe of the war with all its misfortunes brought us this benefit ? g I cannot tell. So many dangerous germs are still fermenting, so many incomprehensions and so much ill will still occasionally appear that I sometimes doubt it. But the time will come. And those who succeed me will achieve the work of national peace which the first Socialist Minister of Sciences and Arts outlined. JULIAN PIERCE Washington, D.C. This content downloaded from 080.082.077.083 on January 11, 2018 20:29:04 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-
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Different warm-ups on the maximum repetition performance in resistance training
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p Paulo Henrique Marchetti Programa de Pós Graduação em Ciências do Movimento Humano, Faculdade de Ciências da Saúde, Universidade Metodista de Piraci- caba (UNIMEP). Rodovia do Açúcar Km 156, Bloco 7, Sala 39, Taquaral. 13400-911. Piracicaba, SP, Brasil. pmarchetti@unimep.br Correspondência: p Paulo Henrique Marchetti Programa de Pós Graduação em Ciências do Movimento Humano, Faculdade de Ciências da Saúde, Universidade Metodista de Piraci- caba (UNIMEP). Rodovia do Açúcar Km 156, Bloco 7, Sala 39, Taquaral. 13400-911. Piracicaba, SP, Brasil. pmarchetti@unimep.br Keywords: athletic performance, resistance training, force. Palavras-chave: desempenho esportivo, treinamento de resistência, força. Palavras-chave: desempenho esportivo, treinamento de resistência, força. DIFERENTES AQUECIMENTOS NO DESEMPENHO DE REPETIÇÕES MÁXIMAS NA MUSCULAÇÃO Artigo Original Artigo Original RESUMO Universidade São Judas Tadeu, São Paulo, SP, Brasil. 4. Faculdade de Educação Física da UNIFIEO, Osasco, SP, Brasil. 2. Instituto de Ortopedia e Traumatologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brasil. 3. Universidade São Judas Tadeu, São Paulo, SP, Brasil. 4. Faculdade de Educação Física da UNIFIEO, Osasco, SP, Brasil. RESUMO Danilo Atanázio da Luz Junior3 (Educador Físico) Aylton Figueira Junor3 (Educador Físico) Érica Paes Serpa1 (Educador Físico ) Willy Andrade Gomes1 (Educador Físico) Enrico Gori Soares1 (Educador Físico) Charles Ricardo Lopes2 (Educador Físico) Luis Felipe Milano Teixeira4 (Educador Físico ) Paulo Henrique Marchetti1,2 (Educador Físico) Introdução: Apesar do aquecimento ser considerado essencial na prática dos esportes, pouco se conhece sobre seu efeito no treinamento de força. Objetivo: Avaliar o efeito de diferentes estratégias de aquecimento no desempenho neuromuscular em adultos jovens submetidos a testes de repetições máximas de membro superior e inferior. Métodos: Dois experimentos foram realizados separadamente. Para o primeiro experimento, 16 adultos jovens hígidos realizaram um teste de repetições máximas a 70% de 1RM no supino reto (previa- mente determinado). Quatro condições de aquecimento foram testadas: Controle (CON) sem aquecimento prévio, esteira (EST) 5’ a 60% VO2max, resistência de força (REF) 15 repetições com 40% de 1RM e força máxima (FM) duas séries de duas repetições com 90% de 1RM. Para o segundo experimento, 14 adultos jovens hígidos, realizaram um teste de repetições máximas a 70% de 1RM no leg press unilateral (previamente determinado). Cinco condições de aquecimento foram testadas: controle (CON) sem aquecimento prévio, esteira (EST) 5’ a 60% VO2max, bicicleta (BIC) 5’ a 60% VO2max, resistência de força (REF) 15 repetições com 40% de 1RM e força máxima (FM) duas séries de duas repetições com 90% de 1RM. Para ambos os experimentos o número de repetições realizadas e a percepção subjetiva de esforço foram avaliadas. Resultados: Para o primeiro experi- mento o protocolo FM foi superior a todos os outros protocolos. Não foram observadas diferenças entre as outras condições. Para o segundo experimento, foi observado diferenças entre todos os protocolos, exceto CON e EST. Sendo FM>BIC>REF>EST=CON. Conclusão: Os resultados do presente estudo sugerem que um aquecimento de força máxima levou a uma maior produção de força para os membros superiores e inferiores. Adicionalmente, aquecimentos aeróbios e de resistência de força apresentaram melhora no desempenho de força para os membros inferiores em uma menor magnitude. 1. Departamento de Ciências do Movimento Humano, Universidade Metodista de Piracicaba, Piracicaba, SP, Brasil. 1. Departamento de Ciências do Movimento Humano, Universidade Metodista de Piracicaba, Piracicaba, SP, Brasil. 1. Departamento de Ciências do Movimento Humano, Universidade Metodista de Piracicaba, Piracicaba, SP, Brasil. 2. Instituto de Ortopedia e Traumatologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brasil. 3. ABSTRACT Introduction: Although the warm-up is considered essential for sports, little is known about its effect on strength training. Objective: To evaluate the effect of different warm-up strategies in neuromuscular performance in young adults undergoing maximum repetitions of upper and lower limb exercises. Methods: Two experiments were conducted separa- tely. For the first experiment, 16 healthy young adults performed a test of maximal repetitions at 70% 1RM in the bench press (previously determined). Four warm-up conditions were tested: control (CON) without prior warm-up, 5 min on the treadmill at 60% VO2max (EST), strength endurance (REF) with 1 set x 15 repetitions with 40% 1RM and maximal strength (MS) with two sets of two reps with 90% 1RM. For the second experiment, 14 healthy young adults performed a maximal number of repetitions at 70% 1RM in the unilateral leg press (previously determined). Five warm-up conditions were tested: control (CON) without prior warm-up, 5 min of cycling at 60% VO2max (BIC), 5 min on the treadmill to 60% VO2max (EST), strength endurance (REF) with 1 set vs. 15 repetitions with 40% 1RM and maximal strength (MS) with two sets of two reps with 90% 1RM. For both experiments the number of repetitions performed and perceived exertion were evaluated. Results: For the first experiment, the MS condition was superior when compared to other protocols. No differences between the other conditions were observed. For the second experiment differences between all protocols were observed, except CON and EST, being MS> BIC> REF> = CON. Conclusion: The results of this study suggest that a warming-up of maximum strength may produce higher force production for the upper and lower limbs. Additionally, aerobic and strength resistance warm-up showed an improvement in strength performance for the lower limbs in a smaller magnitude. DIFFERENT WARM-UPS ON THE MAXIMUM REPETITION PERFORMANCE IN RESISTANCE TRAINING Artigo Original DIFERENTES CALENTAMIENTOS EN EL DESEMPEÑO DE REPETICIONES MÁXIMAS EN LA MUSCULACIÓN Artigo recebido em 19/02/2014, aprovado em 22/09/2014 Artigo recebido em 19/02/2014, aprovado em 22/09/2014 DOI: http://dx.doi.org/10.1590/1517-86922014200602012 Procedimentos Experimento 1: Os sujeitos compareceram ao laboratório em quatro sessões distintas, com um intervalo de uma semana entre sessões. As coletas em cada uma das sessões foram realizadas sempre no mesmo dia da semana e horários, e pelo mesmo avaliador. Na primeira sessão foram coletados os dados antropométricos (estatura, massa, distância biacromial) e dados pessoais (nome, idade, tempo de prática na musculação). A dis- tância biacromial foi utilizada para normatizar a distância entre as mãos de cada sujeito posicionadas (empunhadura) na barra do supino, sendo que todos realizaram a empunhadura com uma distância de 2 vezes a distância biacromial. Após a avaliação inicial, os sujeitos realizaram dois testes máxi- mos de força: teste de 1RM no exercício supino reto8 e após 30 minutos de recuperação, os sujeitos realizaram um teste controle (CON), que consistiu na realização do número máximo de repetições sem aquecimento prévio contra uma sobrecarga externa de 70%1RM (CON) até a falha mecânica concêntrica. Foram consideradas válidas apenas as repetições completas, caracterizadas pelo toque da barra na região torácica (fase excêntrica) e extensão completa dos cotovelos (fase concêntrica)9 A segunda, terceira INTRODUÇÃO Schar- mann et al.,5 investigaram os efeitos do aquecimento no teste de 1RM no leg press e supino e não verificaram aumento na produção de força, entretanto estudo de Barroso et al.,6 observou que o aquecimento geral de longa du- ração e baixa intensidade resultou em melhora no desempenho durante o teste de 1RM. Em outro estudo, Serra et al.,7 avaliou o efeito de alongamento estático na força máxima em diferente exercícios em indivíduos treinados e destreinados demonstrando que o alongamento promoveu efeito ne- gativo de 17% na força máxima membros superiores e 8% em membros inferiores. Ainda demonstraram que indivíduos destreinados apresentaram os menores valores de força máxima em relação aos treinados. Na prática, diferentes estratégias de aquecimento específico são realizadas previamente ao treinamento de força, com a manipulação do número de repetições e intensidade (ex. alto número de repetições e baixa sobrecarga). Entretanto, não se conhece o real efeito destas estratégias no desempenho do treino. O bj i d d f i li f i d dif Os voluntários assinaram o Termo de Consentimento Livre Esclarecido, respeitando as Diretrizes do CNS 196/96, sendo que todos os procedimen- tos foram aprovados pelo Comitê de Ética em Pesquisa da Universidade Metodista de Piracicaba, Piracicaba, SP, Brasil (Protocolo #97/12). Rev Bras Med Esporte – Vol. 20, No 6 – Nov/Dez, 2014 INTRODUÇÃO organizado com diferentes estratégias de aquecimento analisando o número máximo de repetições no exercício supino reto, e o experimen- to dois seguiu as mesmas estratégias, mas avaliando o desempenho no exercício leg press unilateral. O aquecimento é uma prática altamente difundida entre atletas, técnicos e praticantes de atividade física visando diminuir lesões ou preparar-se para a atividade subsequente1. Tradicionalmente, pode-se definir o aquecimento como um breve período onde exercícios aeróbios são seguidos por alongamentos e/ou exercícios específicos para determi- nada prática esportiva. Em relação ao desempenho, o aquecimento visa preparar o organismo para a melhor função durante a prática específica subsequente2,3. Diversos são os mecanismos responsáveis associados ao efeito do aquecimento na melhora do desempenho, como aumento da temperatura muscular, redução da rigidez muscular e articular, maior ca- pacidade circulatória pulmonar, resultando na oferta de oxigênio nas vias aéreas e corrente sanguínea, aumento da sensibilidade da hemoglobina e mioglobina, elevação do consumo de oxigênio de base e aumento do fluxo sanguíneo muscular, ativação da atividade enzimática, facilitando a velocidade das reações metabólicas, aumento da taxa de condução nervosa, aumento da tensão de termorregulação, efeito de potenciali- zação pós-ativação (PPA) e efeitos psicológicos1,4. A amostra foi composta de adultos jovens, hígidos, do sexo masculino, aptos fisicamente para a realização do estudo e com experiência mínima em treinamento de força de 3 anos, utilizando os exercícios de supino reto e/ou leg press nos respectivos programas de treinamento. O experimento 1 utilizou o exercício supino reto; n=16, média de idade: 24 ± 4 anos; massa corporal: 75 ± 7 kg; estatura: 176 ± 6 cm; distância bi-acromial: 37 ± 2 cm; carga máxima no teste de 1RM: 89 ± 17 kg. O experimento 2 foi realizado com o exercício de leg press unilateral; n=14, média de idade: 27 ± 6 anos; massa corporal: 76 ± 7 kg; estatura: 176 ± 8 cm; carga máxima no teste de 1RM: 149 ± 26 kg. Os critérios de inclusão na composição amostral em ambos experimentos foram: (1) sem histórico prévio de cirurgia, lesão ou qualquer acometimento osteomioarticular em membros superiores e inferiores; (2) não terem treinado a área muscular envolvida nas 48 horas precedentes ao protocolo experimental; (3) não apresentar desordens neurológicas periféricas e/ou centrais. Apesar de o aquecimento ser considerado essencial na prática dos es- portes, pouco se conhece sobre seu efeito no treinamento de força. RESUMEN Introducción: A pesar de que el calentamiento sea considerado esencial en la práctica de los deportes, poco se conoce sobre su efecto en el entrenamiento de fuerza. Objetivo: Evaluar el efecto de diferentes estrategias de calentamiento en el desempeño neuromuscular en adultos jóvenes sometidos a tests de repeticiones máximas de miembro superior e inferior. Métodos: Dos experimentos fueron realizados separadamente. Para el primer experimento, 16 adultos jóvenes 461 Rev Bras Med Esporte – Vol. 20, No 6 – Nov/Dez, 2014 Rev Bras Med Esporte – Vol. 20, No 6 – Nov/Dez, 2014 hígidos realizaron un test de repeticiones máximas a 70% de 1RM en el supino recto (previamente determinado). Cuatro condiciones de calentamiento fueron probadas: Control (CON) sin calentamiento previo, cinta ergonométrica (EST) 5’ a 60% VO2max, resistencia de fuerza (REF) 15 repeticiones con 40% de 1RM y fuerza máxima (FM) dos series de dos repe- ticiones con 90% de 1RM. Para el segundo experimento, 14 adultos jóvenes hígidos realizaron un test de repeticiones máximas a 70% de 1RM en el leg press unilateral (previamente determinado). Cinco condiciones de calentamiento fueron probadas: control (CON) sin calentamiento previo, cinta ergonométrica (EST) 5’ a 60% VO2max, bicicleta (BIC) 5’ a 60% VO2max, resistencia de fuerza (REF) 15 repeticiones con 40% de 1RM y fuerza máxima (FM) dos series de dos repeticiones con 90% de 1RM. Para ambos experimentos el número de repeticiones realizadas y la percepción subjetiva de esfuerzo fueron evaluadas. Resultados: Para el primer experimento el protocolo FM fue superior a todos los otros protocolos. No fueron observadas diferencias entre las otras condiciones. Para el segundo experimento, fueron observadas diferencias entre todos los protocolos, excepto CON y EST, siendo FM>BIC>REF>EST=CON. Conclusión: Los resultados del presente estudio sugieren que un calentamiento de fuerza máxima llevó a una mayor producción de fuerza para los miembros superiores e inferiores. Adicionalmente, los calentamientos aeróbicos y de resistencia de fuerza presentaron mejora en el desempeño de fuerza para los miembros inferiores en una menor magnitud. Palabras clave: desempeño deportivo, entrenamiento de resistencia, fuerza. MATERIAIS E MÉTODOS Visando entender os efeitos do aquecimento no desempenho neu- romuscular em membros superiores e inferiores, o presente estudo foi realizado como dois experimentos independentes. O experimento 1 foi 462 na tabela 1. Não foram observadas diferenças significantes na PSE em nenhuma das condições experimentais (EST: 8 ± 1; RF: 8 ± 1; FM: 9 ± 1). na tabela 1. Não foram observadas diferenças significantes na PSE em nenhuma das condições experimentais (EST: 8 ± 1; RF: 8 ± 1; FM: 9 ± 1). O exercício leg press unilateral demonstrou diferenças significativas entre as seguintes estratégias: CON x FM (P=0,001), BIC x FM (P=0,008), EST x FM (P=0,001), RF x FM (P=0,001) e BIC x EST (P=0,005) (figura 2). As comparações do tamanho do efeito são apresentadas na tabela 2. Não foram observadas diferenças significativas na PSE entre os protocolos de aquecimento (EST: 9 ± 1; BIC: 9 ± 1; RF : 9 ± 1; FM: 9 ± 1). e quarta sessões foram constituídas por três modelos distintos de aqueci- mento, aleatorizados, sendo: (1) esteira (EST): O indivíduo caminhou 5 min à 50%VO2máx controlado por meio de um frequencímetro (Marca Oregon®); (2) resistência de força (RF): 15 repetições à 40%1RM e (3) força máxima (FM): duas séries de duas repetições com 90%1RM. Após o aquecimento específico foi dado 1 min de intervalo de recuperação e um novo teste de repetições máximas com 70%1RM foi realizado. Após o teste 70%1RM, os sujeitos foram questionados sobre a percepção subjetiva de esforço (PSE) em uma escala que variou entre um e 10 (sendo, um muito fácil e 10 esforço máximo)10. O exercício leg press unilateral demonstrou diferenças significativas entre as seguintes estratégias: CON x FM (P=0,001), BIC x FM (P=0,008), EST x FM (P=0,001), RF x FM (P=0,001) e BIC x EST (P=0,005) (figura 2). As comparações do tamanho do efeito são apresentadas na tabela 2. Não foram observadas diferenças significativas na PSE entre os protocolos de aquecimento (EST: 9 ± 1; BIC: 9 ± 1; RF : 9 ± 1; FM: 9 ± 1). Figura 1. Média e desvio padrão do número máximo de repetições para 70%1RM após os protocolos de aquecimento para o exercício supino reto. *P<0,05; CON: controle; EST: esteira; RF: resistência de força; FM: força máxima. Análise estatística A normalidade e homogeneidade das variâncias foram verificadas utilizando o teste de Shapiro-Wilk e de Levene, respectivamente. Os dados analisados em valores médios e desvio padrão (DP). A ANOVA de medidas repetidas foi utilizada na comparação das diferenças entre as variáveis dependentes (Percepção subjetiva de esforço e o número máximo de repetições) nas diferentes condições experimentais para cada exercício. Um post hoc de Bonferroni (com correção) foi utilizado para verificar as diferenças entre estratégias. O cálculo do tamanho do efeito (software EffectSizeGenerator 2.3®) foi realizado através da formula de Cohen (ES) e os resultados se basearam nos seguintes critérios: <0,35 sem expressão; 0,35-0,80 pequeno; 0,80-1,50 moderado; e >1,5 grande, para sujeitos treinados de forma recreacional baseado em Rhea12.Uma significância de 5% foi utilizada em todos os testes estatísticos. *Diferença entre FM e todos os outros protocolos, P<0,01. +P<0,05. CON:controle; EST: esteira; BIC: bicicleta; RF: resistência de força; FM: força máxima. Figura 2. Média e desvio padrão do número máximo de repetições para 70%1RM após os protocolos de aquecimento para o exercício leg press unilateral. Tabela 2. Magnitude do efeito entre protocolos de aquecimento no exercício leg press unilateral. Comparação entre condições Tamanho do Efeito CON x EST 0,16 (trivial) CON x BIC 0,50 (pequeno) CON x RF 0,30 (trivial) CON x FM 0,97 (moderado) EST x BIC 0,46 (pequeno) EST x RF 0,14 (trivial) EST x FM 0,83 (moderado) BIC x RF 0,15 (trivial) BIC x FM 0,50 (pequeno) RF x FM 0,61 (pequeno) CON: controle; EST: esteira; BIC: bicicleta; RF: resistência de força; FM: força máxima MATERIAIS E MÉTODOS Comparação entre condições Tamanho do efeito CON x EST 0,035 (trivial) CON x RF 0,78 (pequeno) CON x FM 0,78 (pequeno) EST x RF 0,66 (pequeno) EST x FM 0,66 (pequeno) RF x FM 1,33 (moderado) CON:controle; EST: esteira; RF: resistência de força; FM: força máxima. Tabela 1. Tamanho do efeito entre protocolos de aquecimento no exercício supino reto. Tabela 1. Tamanho do efeito entre protocolos de aquecimento no exercício supino reto. Comparação entre condições Tamanho do efeito CON x EST 0,035 (trivial) CON x RF 0,78 (pequeno) CON x FM 0,78 (pequeno) EST x RF 0,66 (pequeno) EST x FM 0,66 (pequeno) RF x FM 1,33 (moderado) CON:controle; EST: esteira; RF: resistência de força; FM: força máxima. Figura 2. Média e desvio padrão do número máximo de repetições para 70%1RM após os protocolos de aquecimento para o exercício leg press unilateral. *Diferença entre FM e todos os outros protocolos, P<0,01. +P<0,05. CON:controle; EST: esteira; BIC: bicicleta; RF: resistência de força; FM: força máxima. Teste de repetições máximas (70% 1RM) CON EST BIC RF FM 40 30 20 10 0 Teste de repetições máximas (70% 1RM) CON EST BIC RF FM 40 30 20 10 0 O número de repetições máximas atingidas no teste T70 e a PSE após cada condição de aquecimento foi quantificado e utilizado para verificar o efeito dos diferentes protocolos de aquecimento no desempenho neu- romuscular em ambos os exercícios (supino reto e leg press unilateral). Rev Bras Med Esporte – Vol. 20, No 6 – Nov/Dez, 2014 MATERIAIS E MÉTODOS Teste de repetições máximas (70% 1RM) CON EST RF FM 30 20 10 0 Experimento 2: Os sujeitos compareceram ao laboratório em cinco sessões distintas, com um intervalo de uma semana entre sessões. As coletas foram realizadas no mesmo dia e horários, pelo mesmo avaliador. Na primeira sessão foram coletados os dados antropométricos (estatura, massa) e dados pessoais (nome, idade, tempo de prática na musculação e dominância podal). Após a avaliação inicial, os sujeitos realizaram dois testes máximos de força: teste de 1RM no exercício leg press unilateral8 e após 30 minutos de recuperação os sujeitos realizaram o teste con- trole (CON) sendo o número máximo de repetições sem aquecimento contra uma sobrecarga externa de 70%1RM (CON) até a falha mecânica concêntrica. Foi realizada uma marcação no equipamento garantindo que todos os sujeitos atingissem uma flexão máxima de joelhos de 90º, de forma unilateral com o membro dominante. Foram consideradas válidas, apenas as repetições completas pelos critérios de alinhamento da marca adicionada ao trilho e a plataforma do equipamento levando a um ângulo de 90º de flexão do joelho (fase excêntrica) e extensão completa do joelho (fase concêntrica)11. A segunda, terceira, quarta e quinta sessões foram constituídas por quatro modelos distintos de aquecimento, aleatorizados entre as sessões, sendo: (1) os indivíduos caminharam em uma esteira (EST) por 5 min à 50%VO2máx controlado por meio de um frequencímetro (Marca Oregon®); (2) os indivíduos pedalaram um cicloergômetro (BIC) por 5 min à 50%VO2máx controlado por meio de frequencímetro (Marca Oregon®); (3) resistência de força (REF): 15 repetições com 40%1RM e (4) força máxima (FM): duas séries de duas repetições com 90%1RM. Após o aquecimento específico foi dado 1 min de intervalo e um novo teste com 70% 1RM foi realizado, seguido por um questionamento oral sobre a PSE10. *P<0,05; CON: controle; EST: esteira; RF: resistência de força; FM: força máxima. Figura 1. Média e desvio padrão do número máximo de repetições para 70%1RM após os protocolos de aquecimento para o exercício supino reto. Figura 1. Média e desvio padrão do número máximo de repetições para 70%1RM após os protocolos de aquecimento para o exercício supino reto. Tabela 1. Tamanho do efeito entre protocolos de aquecimento no exercício supino reto. DISCUSSÃO Diversos mecanismos são responsáveis pelo desempenho físico, sendo que as atividades específicas realizadas na rotina de aqueci- mento poderiam contribuir com o desempenho em um exercício, em função do aumento da temperatura corporal, aumento da viscosidade muscular, aumento ou melhora da taxa de condução nervosa, altera- ção para direita da curva de força-velocidade, possível aumento na provisão de energia anaeróbia, melhora na termorregulação, elevação do consumo inicial de oxigênio, acidose, potencialização pós-ativação (PPA), bem como efeitos psicológicos (estado de prontidão/atenção)4,13. ( ), p g ( p ç ) Os resultados encontrados no experimento 1 demonstraram que o aquecimento realizado pelo protocolo de força máxima e resistência de força apresentaram maior rendimento quando comparados ao controle. Entretanto, o aquecimento de FM contribuiu com maior número de repe- tições (13%) em relação ao RF e 27% em relação à esteira. Considerando que em um programa de exercícios, não é comum utilizar a FM como uma estratégia de aquecimento, os dados encontrados sugerem que esta estratégia parece ser a mais eficaz em indivíduos experientes, embora muitos praticantes apontem que cargas menores sejam utilizadas como estratégia de aquecimento quando se objetiva a redução de lesões14. Hipotetizamos que a melhora no desempenho após o aquecimento em FM tenha ocorrido em função ao aumento da ativação dos precursores da capacidade de fosforilação das cadeias leves de miosina (aumentando sua sensibilidade ao Ca2+) e pela maior excitabilidade dos motoneurônios alfa15-17. De acordo com Ament e Verkerke18 a capacidade de contração muscular é diretamente afetada por seu histórico contrátil, considerado como os efeitos residuais de atividades pregressas. A análise do número de repetições máximas no supino reto, não foi alterado quando precedi- do do aquecimento em esteira. Resultados similares foram encontrados por Raddi et al19 no qual não foram apresentadas diferenças na produção de força e número de repetições máximas no exercício supino reto, após 45 minutos em esteira rolante a 70% da FCMax. Não foram observadas diferenças significativas em nenhum dos experimentos e das condições experimentais de aquecimento na PSE, sugerindo que os sujeitos não apresentaram diferentes percepções em relação às estratégias de aquecimento, possivelmente em função da relação volume e intensidade serem muito diferentes. RESULTADOS O exercício supino reto apresentou diferenças significativas entre as seguintes estratégias de aquecimento: CON x RF (P=0,007), CON x FM (P=0,014), FM x RF (P=0,001), EST x RF (P=0,001), EST x FM (P=0,021) (figura 1). As comparações da magnitude de mudança são apresentadas 463 associados à fadiga neuromuscular. Quanto aos protocolos de aqueci- mento aeróbio, os resultados no exercício leg press unilateral demons- tram que somente o protocolo na bicicleta ergométrica apresentou efeito trivial na melhora do desempenho em relação ao CON. Barroso et al.,22 analisaram o efeito do aquecimento em duas intensidades (40 e 70% VO2max) e duração (5’ e 15’) no desempenho de força máxima (1RM) no exercício leg press. Dentre as combinações testadas, apenas a condição de intensidade moderada (70% VO2max) e longa duração (15’) promoveram queda no desempenho, sendo que na condição de baixa intensidade (40% VO2max) e longa duração (15’) foi observada melho- ra na força máxima do exercício leg press. O presente estudo utilizou duração (5’) e intensidade (50% VO2max.) similar ao estudo de Barroso et al.,22, embora melhora no desempenho na força tenha sido encontrado apenas no protocolo de bicicleta quando comparado a esteira. Isto pode estar relacionado à maior amplitude de movimento e ações contráteis da musculatura envolvida na caminhada em esteira assim como equilí- brio nesta atividade. Por outro lado, sabe-se que intensidades mais altas promovem aumentos significativos na temperatura muscular, embora a maior depleção das reservas de fosfato de alta energia seja conhecida3, caso a intensidade esteja acima de 60% do VO2max, fato que pode res- ponder as diferenças encontradas entre protocolos. Todos os autores declararam não haver qualquer potencial conflito de interesses referente a este artigo. Todos os autores declararam não haver qualquer potencial conflito de interesses referente a este artigo. DISCUSSÃO Embora o presente estudo apresente limitações como a ausência de controle da tempera- tura muscular em relação às estratégias de aquecimento, avaliação das respostas mioelétricas e metabólicas, potencialmente influenciadas pelas diferentes estratégias de aquecimento, apresenta indicador aplicado em programas de exercícios, como o número de repetições, pois representa a relação colume-intensidade de treinamentos com pesos. Os resultados do experimento 2, demonstraram um aumento sig- nificativo no número de repetições máximas comparando a estratégia de força máxima e as outras condições, fato similar ao encontrado no experimento 1. Adicionalmente, Weber, Brown et al.20 e McCann e Flanagan21 verificaram melhora do desempenho de saltos quando precedidos de exercícios de alta intensidade. Porém, no referido estu- do não foram observadas diferenças entre a estratégia de resistência de força e a condição controle. O número total de repetições após o aquecimento de resistência de força foi 24% menor que o de FM, pelo fato da coexistência de mecanismos de melhora do desempenho, REFERÊNCIAS 11. Marchetti PH, Gomes WA, Da Luz Junior DA, Giampaoli B, Amorim MA, Bastos HL, et al. Aspectos neuromecânicos do exercício agachamento. Revista CPAQV. 2013;5(2):1-16. 1. Fradkin AJ, Zazryn TR, Smoliga JM. Effects of warming-up on physical performance: a systematic review with metaanalysis. J Strength Cond Res. 2010;24(1):140-8. 1. Fradkin AJ, Zazryn TR, Smoliga JM. Effects of warming-up on physical performance: a systematic review with metaanalysis. J Strength Cond Res. 2010;24(1):140-8. 12. Rhea MR. Determining the magnitude of treatment effects in strength training research through the use of the effect size. J Strength Cond Res. 2004;18(4):918-20. 12. Rhea MR. Determining the magnitude of treatment effects in strength training research through the use of the effect size. J Strength Cond Res. 2004;18(4):918-20. 2. Fradkin AJ, Zazryn TR, Smoliga JM. Effects of warming-up on physical performance: a systematic review with meta-analysis. J Strength Cond Res. 2010;24(1):140-8. 2. Fradkin AJ, Zazryn TR, Smoliga JM. Effects of warming-up on physical performance: a systematic review with meta-analysis. J Strength Cond Res. 2010;24(1):140-8. 13. Di Alencar TAM, Matias KFS. Princípios Fisiológicos do Aquecimento e Alongamento Muscular na Atividade Esortiva. Rev Bras Med Esporte. 2010;16(3):230-4. 13. Di Alencar TAM, Matias KFS. Princípios Fisiológicos do Aquecimento e Alongamento Muscular na Atividade Esortiva. Rev Bras Med Esporte. 2010;16(3):230-4. 3. Bishop D. Warm up II: performance changes following active warm up and how to structure the warm up. Sports Med. 2003;33(7):483-98. 3. Bishop D. Warm up II: performance changes following active warm up and how to structure the warm up. Sports Med. 2003;33(7):483-98. 14. Lin J, Chen T. Diversity of strength training methods: A theoretical approach. Strength Cond. J. 2012;34(2):42-9. 4. Bishop D. Warm up I: potential mechanisms and the effects of passive warm up on exercise perfor- mance. Sports Med. 2003;33(6):439-54. 4. Bishop D. Warm up I: potential mechanisms and the effects of passive warm up on exercise perfor- mance. Sports Med. 2003;33(6):439-54. 15. Hodgson M, Docherty D, Robbins D. Post-activation potentiation: underlying physiology and implica- tions for motor performance. Sports Med. 2005;35(7):585-95. Epub 2005/07/20. 5. Scharmann A, Eves S, Kimpel S. The effects of warm-up duration on 1-repetition maximum leg press/ bench press and push-ups to axhaustion. J Strength Cond Res. 2011;25(1):S79. 16. Ide BN, Lopes CR, Sarraipa MF. Fisiologia do treinamento esportivo: Força, potência, velocidade, resis- tência, periodização e habilidades psicológicas. São Paulo: Phorte Editora; 2010. bench press and push-ups to axhaustion. J Strength Cond Res. CONCLUSÃO O aquecimento de força máxima para ambos os membros (superior e inferior) se apresentou efetivo na melhora do desempenho. Entre- tanto, a utilização da bicicleta apresentou melhora no desempenho de membros inferiores durante o leg press unilateral. Todos os autores declararam não haver qualquer potencial conflito de interesses referente a este artigo. Rev Bras Med Esporte – Vol. 20, No 6 – Nov/Dez, 2014 REFERÊNCIAS 2011;25(1):S79 6. Barroso R, Silva-Batista C, Tricoli V, Roschel H, Ugrinowitsch C. The effects of different intensities and durations of the general warm-up on leg press 1RM. J Strength Cond Res. 2013;27(4):1009–13. 17. Lorenz D. Postactivation potentiation: an introduction. Int J Sports Phys Ther. 2011;6(3):234-40. 8. Ament W, Verkerke GJ. Exercise and fatigue. Sports Med. 2009;39(5):389-422. 7. Serra AJ, Silva Jr JA, Marcolongo AA, Manchini MT, Oliveira JVA, Santos LFN, et al. Experience in resis- tance training does not prevent reduction in muscle strength evoked by passive static stretching. J Strength Cond Res. 2013;27(8):2304–8. 19. Raddi LLO, Gomes RV, Charro MA, Bacurau RFP, Aoki MS. Treino de corrida não interfere no desempenho de força de membros superiores. Rev Bras Med Esporte. 2008;14(6):544-7. 8. Baechle TR, Earl RW. Fundamentos do Treinamento de Força e do Condicionamento Físico. São Paulo: Manole; 2010. 8. Baechle TR, Earl RW. Fundamentos do Treinamento de Força e do Condicionamento Físico. São Paulo: Manole; 2010. 20. Weber, KR; Brown,LE, et al. Acute Effects of Heavy-Load Squats on Consecutive Squat Jump Performance. J Strength Cond Res. 2008;22(3):726-30. 9. Marchetti PH, Arruda CC, Segamarchi LF, Soares EG, Ito DT, Da Luz Junior DA, et al. Exercício supino: uma breve revisão sobre os aspectos biomecânicos. Braz. J. Sports and Exerc. Res. 2010;1(2):135-42. 21. McCann, MR; Flanagan,SP. The Effects of Exercise Selection and Rest Interval on Postactivation Potentia- tion of Vertical Jump Performance. J Strength Cond Res. 2010;24(5):1285-91. 22. Barroso R, Silva-Batista C, Tricoli V, Roschel H, Ugrinowitsch C. The effects of different intensities and durations of the general warm-up on leg press 1RM. J Strength Cond Res. 2013;27(4):1009-13. 10. Borg G, Ljunggren G, Ceci R. The increase of perceived exertion, aches and pain in the legs, heart rate and blood lactate during exercise on a bicycle ergometer. Eur J. Appl Physiol. 1985;54(4):343-9. 10. Borg G, Ljunggren G, Ceci R. The increase of perceived exertion, aches and pain in the legs, heart rate and blood lactate during exercise on a bicycle ergometer. Eur J. 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Assessment of phenotypic variability among EEA INTA Pergamino sunflower lines: Its relationship with the grain yield and oil content
Oilseeds and fats, crops and lipids/OCL. Oilseeds & fats crops and lipids
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☆Contribution to the Topical Issue “Sunflower / Tournesol”. *Correspondence: dominguez.matias@inta.gob.ar Mots clés : tournesol / sélection / variabilité génétique / hétérosis ☆Contribution to the Topical Issue “Sunflower / Tournesol”. *C d d i ti @i t b This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Assessment of phenotypic variability among EEA INTA Pergamino sunflower lines: Its relationship with the grain yield and oil t t☆ Matías Domínguez1,2,*, Stefanía Herrera3 and Julio Horacio González1 1 Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria, Pergamino, Buenos Aires, Argentina 2 Consejo Nacional de Investigaciones Científicas y Técnicas, Pergamino, Buenos Aires, Argentina 3 Universidad Nacional del Noroeste de Buenos Aires, Pergamino, Buenos Aires, Argentina 1 Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria, Pergamino, Buenos Aires, Argentina 2 Consejo Nacional de Investigaciones Científicas y Técnicas, Pergamino, Buenos Aires, Argentina 3 Universidad Nacional del Noroeste de Buenos Aires, Pergamino, Buenos Aires, Argentina Received 11 February 2021 – Accepted 11 May 2021 Abstract – The aims of the present study were to assess the phenotypic diversity among 221 sunflower accessions of INTA Pergamino Sunflower Breeding Program, to obtain discriminant functions that allow the classification of new accessions in similar groups and to evaluate the relationship between genetic distance pairwise accessions and hybrid performance for grain yield and oil content. We used 19 quantitative descriptors to evaluate phenotypic and morphological variability. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were used to evaluate simultaneously all the variables and to describe phenotypic variation patterns of the germplasm. The distribution of germplasm in the dendrogram did not follow a clear pattern with regard to the predefined groups. This study revealed the variability observed among the lines that form the INTA Pergamino breeding program despite the highly selective forces applied to obtain inbred lines that produce superior hybrids for the Argentinean sunflower area. This work demonstrates the need for more in-depth study of genetic variability to be used as a predictor of heterosis in sunflower. Keywords: sunflower / breeding / genetic variability / heterosis This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommon unrestricted use, distribution, and reproduction in any medium, provided the original work is prop OCL 2021, 28, 33 © M. Domínguez et al., Published by EDP Sciences, 2021 https://doi.org/10.1051/ocl/2021021 Oilseeds & fats Crops and Lipid OCL OCL 2021, 28, 33 © M. Domínguez et al., Published by EDP Sciences, 2021 https://doi.org/10.1051/ocl/2021021 O f C OCL Available online at: www.ocl-journal.org Available online at: www.ocl-journal.org Available online at: www.ocl-journal.org *Correspondence: dominguez.matias@inta.gob.ar Keywords: sunflower / breeding / genetic variability / heterosis Résumé – Évaluation de la variabilité phénotypique parmi les lignées de tournesol EEA INTA Pergamino : relation avec le rendement en grains et la teneur en huile. Les objectifs de la présente étude étaient d’évaluer la diversité phénotypique parmi 221 lignées de tournesol issues du programme de sélection de tournesol argentin à l’INTA Pergamino, afin d’identifier des fonctions discriminantes permettant de classer les nouvelles accessions dans des groupes similaires et d’évaluer la relation entre la distance génétique entre couples d’accessions et la performance des hybrides pour le rendement en grain et la teneur en huile. Nous avons utilisé 19 descripteurs quantitatifs pour évaluer la variabilité phénotypique et morphologique. Des analyses en Composantes Principales (ACP) et en Clusters Hiérarchiques (ACH) ont permis d’évaluer simultanément toutes les variables et de décrire les modèles de variation phénotypique du germplasme. La distribution du germplasme dans le dendrogramme n’a pas suivi un schéma clair par rapport aux groupes prédéfinis. Cette étude a démontré la variabilité observée parmi les lignées issues du programme de sélection de l’INTA Pergamino malgré les fortes pressions de sélection appliquées pour obtenir des parents d’hybrides performants et bien adaptés aux conditions argentines. Les résultats indiquent le besoin d’études plus approfondies de la variabilité génétique afin de l’utiliser pour prédire l’hétérosis chez le tournesol. Mots clés : tournesol / sélection / variabilité génétique / hétérosis s article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. M. Domínguez et al.: OCL 2021, 28, 33 lines of domesticated sunflowers, a grouping of Argentine germplasm and lastly a group of Romanian and South African female lines. The US restorer lines tend to be good sources for disease resistance and fertility restorer genes and the Argentine germplasm provides female parental lines with disease resistance and high grain yield. Also, French and Serbian inbred lines are important and widely used in sunflower seed industry. 2 Materials and methods A set of 221 inbred lines (maintainer and restorer) with desirable characteristics for subsequent use in hybrids and bred by the Sunflower Breeding Program of the Instituto Nacional de Tecnología Agropecuaria (INTA) in the Estación Experi- mental Agropecuaria (EEA) in Pergamino (Buenos Aires, Argentina) were included in this study. The only line that was included in this study and was not developed by INTA is the public line RHA 278. Five groups of accessions were previously determined according to the origin of the germplasm, the agronomic characteristics and if there are maintainer or restorer lines. The lines that form each group are shown in Supplementary Table 1. The characteristics of each group of lines and their origin are detailed in Table 1. For more information on the development of the lines released by INTA, their characteristics and selection strategies used, can be found in the work previously presented by González et al. (2015). , ; , ) Knowledge of the amount and distribution of genetic diversity within sunflower breeding germplasm is essential to ensure the maintenance of the genetic variability of breeding pools, to make crop improvement more efficient through the directed accumulation of desirable alleles and to select the most diverse parental lines in crosses intended to generate breeding populations (Hladni et al., 2017). For studies of genetic diversity in sunflower, morphological, physiological, biochemical, pedigree and molecular data have been used (Rama Subrahmanyam et al., 2003; Dudhe et al., 2019; Filippi et al., 2015, 2020). Melchinger (1999) concluded that quantitative traits such as yield and heterotic response are expected to increase with parental genetic distance. Melchinger and Gumber (2015) defined a heterotic group as a group of related or unrelated genotypes from the same or different populations that exhibit similar combining ability when crossed with genotypes from other germplasm groups. According to Miller (1999) and Vear and Miller (1993) four heterotic groups are being utilized worldwide in sunflower breeding: a group of inbred female maintainer lines derived from the Russian open-pollinated varieties, a US restoration group, formed by crossing wild annual sunflower species with 1 Introduction Therefore, it is used routinely to assess genetic diversity in germplasm collections (Peeters and Martinelli, 1989). DA maximizes between-group variability, minimizes within-group variability and provides a visual assessment of their relatedness. It also works as a predictive tool, enabling the classification of new accessions to previously characterized groups (Hernández et al., 2019; Palacio et al., 2020). y p ( g ) Since 1931, Argentina has been working on sunflower breeding by exploiting the diversity of a broad range of international genetic resources in combination with intro- gressions of wild Helianthus species. At its Experimental Stations in Manfredi (Córdoba, Argentina) and Pergamino (Buenos Aires, Argentina), the Instituto Nacional de Tecno- logía Agropecuaria (INTA), has pioneered Argentinean sun- flower breeding and has become one of the most prolific sunflower breeding groups in the country (Filippi et al., 2020). g g p y ( pp , ) Multivariate analysis refers to a statistical technique that is widely used to analyze data which arise from more than one variable. Principal component analysis (PCA), hierarchical cluster analysis (HCA) and discriminant analysis (DA) are commonly employed multivariate techniques. PCA is one of the most useful methods employed in diversity studies. It reduces the dimensionality of the data while retaining most of the variation in the data set, and it is used to order genotypes according to their relationships while identifying the traits which explain most of the variability in the data set (Ringnér, 2008; Dudhe et al., 2019). HCA is a multivariate analysis widely used to assess relatedness and distance of any type of samples characterized by any type of descriptors. Therefore, it is used routinely to assess genetic diversity in germplasm collections (Peeters and Martinelli, 1989). DA maximizes between-group variability, minimizes within-group variability and provides a visual assessment of their relatedness. It also works as a predictive tool, enabling the classification of new accessions to previously characterized groups (Hernández et al., 2019; Palacio et al., 2020). The aims of the present study were (i) to assess the diversity among a set of INTA Pergamino sunflower accessions using multivariate techniques, (ii) to compare the patterns of phenotypic variation obtained according to the different predefined groups of accessions, (iii) to obtain discriminant functions that allow the classification of new accessions and (iv) to evaluate the relationship between the genetic distance among accessions and the hybrid performance for grain yield and oil content. 1 Introduction Sunflower (Helianthus annuus) is produced in more than fifty countries, and two thirds of its production is concentrated in Europe, including Ukraine, Russia and Turkey. Other major producing countries are Argentina, China, United States, and the South-Eastern parts of Africa. Sunflower oilseed came in fourth position on vegetable oils market in 2017/18 after palm, soybean, and rapeseed (Pilorgé, 2020). The proposed heterotic groups in sunflower are less rigid than in other hybrid crops. The within group genetic distances appear to be greater and between group distances smaller than those observed in maize. Hongtrakul et al. (1997) also emphasized the need for further study of heterotic groups in sunflower, in their work on a group of public maintainer and restorer lines and using AFLP markers they found up to 4 subgroups within the restorer lines and 2 subgroups within the maintainer lines, suggesting that these could be defined as distinct heterotic groups. The heterotic groups are very useful for sunflower to catalog diversity and direct the process to introgress new alleles and create new heterotic groups (Cheres and Knapp, 1998; Cheres et al., 2000; Lochner, 2011). y , p ( g , ) Since 1931, Argentina has been working on sunflower breeding by exploiting the diversity of a broad range of international genetic resources in combination with intro- gressions of wild Helianthus species. At its Experimental Stations in Manfredi (Córdoba, Argentina) and Pergamino (Buenos Aires, Argentina), the Instituto Nacional de Tecno- logía Agropecuaria (INTA), has pioneered Argentinean sun- flower breeding and has become one of the most prolific sunflower breeding groups in the country (Filippi et al., 2020). Multivariate analysis refers to a statistical technique that is widely used to analyze data which arise from more than one variable. Principal component analysis (PCA), hierarchical cluster analysis (HCA) and discriminant analysis (DA) are commonly employed multivariate techniques. PCA is one of the most useful methods employed in diversity studies. It reduces the dimensionality of the data while retaining most of the variation in the data set, and it is used to order genotypes according to their relationships while identifying the traits which explain most of the variability in the data set (Ringnér, 2008; Dudhe et al., 2019). HCA is a multivariate analysis widely used to assess relatedness and distance of any type of samples characterized by any type of descriptors. 2.2 Field experiments Restorer of fertility lines were obtained from PRIII; Sel.105; Sel.105G; Sel.3; Sel.3107; Sel.34507; Sel.36506; Sel.379; Sel.70; Sel.731; Sel.860; Sel.871; Sel.90014 and Sel.9021. 5 Imidazolinone resistant lines were obtained from IMI-Sun N.Dakota; B10/B83/HA425 and A83-3/RfI. From Sel.2329 IMI-Sun N.Dakota and Sel.2330 IMI-Sun N.Dakota were originated Verticillium resistant lines. Imidazolinone resistant lines were obtained from IMI-Sun N.Dakota; B10/B83/HA425 and A83-3/RfI. From Sel.2329 IMI-Sun N.Dakota and Sel.2330 IMI-Sun N.Dakota were originated Verticillium resistant lines. 2.3 Quantitative descriptors consisted of one row 6m long by 0.52m wide, with a planting distance of 0.25m, resulting in 24plants per row. The dates of sowing in each season were in the first week of October. We measured phenotypic and morphological variability in four randomly selected plants from each row using 19 descriptors of the International Board for Plant Genetic Resources list (IBPGR, 1985) (Tab. 3). g A group of 70 A  R hybrids obtained from parental lines included in the collection of lines studied were evaluated in multi-environment trials (MET) in five locations of the Argentine sunflower region during the 2014–2015, 2015–2016, 2016–2017, 2017–2018 and 2018–2019 growing seasons (austral summer). The locations were Pergamino (33° 56’ S, 60° 33’ W), Bellocq (35° 54’ S, 61° 32’ W), San Carlos de Bolívar (36° 13’ S, 61° 09’ W), Coronel Suárez (37° 25’ S, 61° 51’ W) and Asturias (36° 10’ S, 61° 52’ W). The parental lines that form each hybrid included in the analysis are presented inTable2.TheAlinesusedtodevelopthehybridswereobtained by incorporating a unique source of cytoplasmic male sterility Helianthus petiolaris (CMS) PET1 gene (Leclercq, 1969) to the maintainer lines by backcrossing. DTF, PH, NL, SD, LL, LW, PL, CD, BL, NRF, RFW and RFL were measured in R5.5 (50% of the disk flowers have completed or are in anthesis) (Schneiter and Miller, 1981). The % Oil was determined by Nuclear Magnetic Resonance (NMR) with an Oxford MQ5 equipment calibrated by solvent extraction (Grandlund and Zimmerman, 1975). 2.2 Field experiments The germplasm characterization was conducted in the EEA INTA Pergamino (Buenos Aires, Argentina, 33° 56’ S, 60° 33’W) during the 2014–2015, 2015–2016, 2016–2017, 2017–2018, 2018–2019 and 2019–2020 growing seasons (austral summer). All sunflower accessions were sown in three replicate plots per accessioninfullyrandomizeddesignsineachtrialseason.Eachplot Page 2 of 11 M. Domínguez et al.: OCL 2021, 28, 33 Table 1. Characteristics and origin of each group of INTA Pergamino sunflower’s lines. Group Characteristics Origin 1 High content of oleic acid (maintainer lines) High content of oleic acid lines were obtained from ND 01; VNNIMK 1646/ND 01; Sel HA 343; Luch/AO 01/03 and ND 01/BXC. 2 Maintainer lines with desirable traits such as grain yield, disease resistance and bird resistance Verticillium resistance was originated from VNIIMK 1646; VNIIMK 1646/ND 01; SB/BZ BXC/KLM, BXC/KLM/HA 300, E/DXT; Local/ Silvestre; P4 and HA 335/HA 412/ND 01/BZ. Downy mildew resistance was originated from E.75x MP 557/Negro Bellocq/KLM/HA 300; HA 335/HA412/71/538 /BXC; MP557/Negro Bellocq/KLM/HA 300/V112/E/BXC and MP557/Negro. From Bellocq/ KLM/HA 300/V112/E/BXC were also obtained lines that were moderately resistant to Verticillium. Sclerotinia resistance was originated from MP557/Negro Bellocq/HA 89; ND 01; PGRK, V-13/BxC and AXB/BXC. From ND 01 was originated also high oleic lines and from AXB/BXC moderately Verticillium resistant lines. Resistance to rust was obtained from BXC and Pehuén/Caburé/Charata- SelRN160. Resistance to birds was originated from PGRK and sintética Os2. 3 Confectionary use lines Confectionery lines were obtained from ND-NONOIL composite. 4 Restorer of fertility branched lines (oleic and linoleic) Restorer of fertility lines were obtained from PRIII; Sel.105; Sel.105G; Sel.3; Sel.3107; Sel.34507; Sel.36506; Sel.379; Sel.70; Sel.731; Sel.860; Sel.871; Sel.90014 and Sel.9021. 5 Resistance to Imidazolinone Imidazolinone resistant lines were obtained from IMI-Sun N.Dakota; B10/B83/HA425 and A83-3/RfI. From Sel.2329 IMI-Sun N.Dakota and Sel.2330 IMI-Sun N.Dakota were originated Verticillium resistant lines. Table 1. Characteristics and origin of each group of INTA Pergamino sunflower’s lines. G Ch i i O i i Table 1. Characteristics and origin of each group of INTA Pergamino sunflower’s lines. High content of oleic acid lines were obtained from ND 01; VNNIMK 1646/ND 01; Sel HA 343; Luch/AO 01/03 and ND 01/BXC. Resistance to rust was obtained from BXC and Pehuén/Caburé/Charata- SelRN160. Resistance to birds was originated from PGRK and sintética Os2. Resistance to birds was originated from PGRK and sintética Os2. Confectionery lines were obtained from ND-NONOIL composite. Confectionery lines were obtained from ND-NONOIL composite. 2.4 Statistical analysis Descriptive and multivariate analyses were carried out to characterize the phenotypic diversity of sunflower accessions. The mean, range, standard deviation and coefficient of variation (CV) were obtained. Both statistical analyses were conducted using the statistical software R (R core Team, 2020). In each environment, a randomized incomplete block design with three replicates was used to test the 70 A  R hybrids. Environments were considered as random and hybrids as fixed. The trials were planted at 45000 plants/ha. A plot size of 2 rows  6 m and inter-row spacing of 0.70 m was used in all the trials. Planting took place within the normal sowing window (mid-October) at each location. Grain yield was determined by hand harvesting of 3.92 m2 (both rows discarding border plants) and is presentedat 11% moisture. Oil content wasdetermined on a 10 g oven-dried achene sample Nuclear Magnetic Resonance (NMR) with an Oxford MQ5 equipment calibrated by solvent extraction (Grandlund and Zimmerman, 1975). PCA and HCA were used to simultaneously examine several variables for each accession and to describe phenotypic variation patterns in the germplasm characterized (Franco and Hidalgo, 2003). Principal component analysis was performed with standardized Euclidean distance obtained by Vegan package in R (Oksanen et al., 2018) and depicted in a two-dimensional scatter plot using FactoMineR and factoextra R packages (Lê et al., 2008; Kassambara and Mundt, 2020). For classification (HCA), the hierarchical agglomerative method with the average as fusion criteria was chosen using Page 3 of 11 Page 3 of 11 M. Domínguez et al.: OCL 2021, 28, 33 Table 2. Parental lines of 70 hybrids tested in MET. 2.4 Statistical analysis A-Line/ R-Line IMI_05/04 IMI_13/03 IMI_13/04 Rf_00/03 Rf_00/04 Rf_00/07 Rf_00/08 Rf_00/09 Rf_00/10 Rf_00/11 Rf_00/13 Rf_00/17 Rf_00/18 Rf_15/03 Rf_17/01 AO_01/01 X X X X AO_01/02 X X AO_01/03 X X X X X X AO_01/04 X X X X X X X X AO_03/03 X X X X X AO_03/05 X AO_03/14 X X X X X X AO_03/15 X X X X AO_04/01 X X AO_04/02 X X X X X GP_02/04 X X GP_02/05 X X X X X GP_03/02 X X X X X X X X X GP_06/04 X X X GP_07/14 X X X X SB_04/01 X X X SB_04/02 X Page 4 of 11 A-Line/ R-Line IMI_05/04 IMI_13/03 IMI_13/04 Rf_00/03 Rf_00/04 Rf_00/07 Rf_00/08 Rf_00/09 Rf_00/10 Rf_00/11 Rf_00/13 Rf_00/17 Rf_00/18 Rf_15/03 Rf_17/01 AO_01/01 X X X X AO_01/02 X X AO_01/03 X X X X X X AO_01/04 X X X X X X X X AO_03/03 X X X X X AO_03/05 X AO_03/14 X X X X X X AO_03/15 X X X X AO_04/01 X X AO_04/02 X X X X X GP_02/04 X X GP_02/05 X X X X X GP_03/02 X X X X X X X X X GP_06/04 X X X GP_07/14 X X X X SB_04/01 X X X SB_04/02 X Page 4 of 11 Page 4 of 11 Page 4 of 11 M. Domínguez et al.: OCL 2021, 28, 33 Table 3. Quantitative descriptors used. Reference Quantitative descriptor DTF Days to flowering from sowing NAC Number of achenes per capitulum W100 Weight of 100 achenes (g) Kernel (%) Percentage of seed kernel Oil (%) % of oil content AW Achene width (mm) AL Achene length (mm) AT Achene thickness (mm) PH Plant height (cm) NL Number of leaves per plant SD Stem diameter (mm) LL Leave length (cm) LW Leave width (cm) PL Petiole length (cm) CD Capitulum diameter (central capitulum in branched lines) (cm) BL Bract length (cm) NRF Number of ray flowers RFW Ray flower width (mm) RFL Ray flower lenght (mm) The PCA results showed that the first three components concentrate 43.7% of total variation. The first component(PC1) explained most of this variation (21.8%) and ordered the accessions according to a gradient of achene size, plant height, number of rayflowers, size of leaves (leave length and width) and weight of 100 achenes. 2.4 Statistical analysis In the opposite direction it ordered the accessions according to a gradient of oil content and kernel. As indicated in Figure 1, accessions with taller plants, wider capitulum, longer and wider achenes, longer petioles, numerous ray flowers and bigger leaves were placed in quadrants 1(upper right) and 4 (lower right). Most of the confectionary lines were placed inthesequadrants. Accessionswithhigher % of kerneland oil content were located in quadrants 2(upper left) and 3(lower left). The majority of restorer lines were in the quadrant 3. The second component (PC2) explained 11.3% of variation and ordered the accessions according to the size of ray flowers, locatingtheaccessionswiththelargestrayflowersinquadrants1 and 2. In the same direction, it ordered the accessions with the most days to flowering, the highest number of leaves per plant and the number of achenes per capitulum. The accessions with the longer time to flowering, the largest number of achenes per capitulum and the number of leaves per plant were located in quadrants 1 and 2 (Fig. 1). All other components PC3 (10.6%), PC4 (9.8%) and PC5 (6.7%) explained less variation and did not differentiate between accessions. The biplot also showed that group 2 is the most diverse phenotypically and could discrimi- nate between maintainer and restorer lines. Besides, it showed the positive associations between DTF and NAC, also positive associations were noticed between the variables of dimension of achenes and between the variables obtained on ray flowers. The vector of Oil showed an opposite direction to the vectors of the achene dimension variables. Euclidean distance derived from the principal components 1, 2 and 3 (PC1, PC2, PC3). The dendrogram was visualized and edited with ape and factoextra R packages (Paradis and Schliep, 2019; Kassambara and Mundt, 2020). The optimal number of clusters was determined using the NbCLust R package (Charrad et al., 2014). Euclidean distance derived from the principal components 1, 2 and 3 (PC1, PC2, PC3). The dendrogram was visualized and edited with ape and factoextra R packages (Paradis and Schliep, 2019; Kassambara and Mundt, 2020). The optimal number of clusters was determined using the NbCLust R package (Charrad et al., 2014). Based on the groups obtained from the cluster analysis, a Linear Discriminant Analysis (LDA) was performed to define functions to discriminate between groups and to classify new accessions. 2.4 Statistical analysis LDA1 ¼ 0:068 DTF ð Þ  0:002 NAC ð Þ  0:099 W100 ð Þ þ 0:065 Kernel ð Þ þ 0:001 Oil ð Þ  0:401 AL ð Þ  0:012 PH ð Þ  0:002 NL ð Þ þ 0:051 SD ð Þ  0:016 LL ð Þ  0:076 PL ð Þ þ 0:024 CD ð Þ  0:034 BL ð Þ  0:035 NRF ð Þ  0:048 RFL ð Þ; LDA2 ¼ 0:022 DTF ð Þ þ 0:002 NAC ð Þ  0:340 W100 ð Þ þ 0:059 Kernel ð Þ þ 0:128 Oil ð Þ  0:321 AL ð Þ þ 0:005 PH ð Þ þ 0:041 NL ð Þ  0:011 SD ð Þ þ 0:050 LL ð Þ þ 0:005 PL ð Þ þ 0:115 CD ð Þ  0:164 BL ð Þ  0:013 NRF ð Þ  0:011 RFL ð Þ: LDA2 ¼ 0:022 DTF ð Þ þ 0:002 NAC ð Þ  0:340 W100 ð Þ þ 0:059 Kernel ð Þ þ 0:128 Oil ð Þ  0:321 AL ð Þ þ 0:005 PH ð Þ þ 0:041 NL ð Þ  0:011 SD ð Þ þ 0:050 LL ð Þ þ 0:005 PL ð Þ þ 0:115 CD ð Þ  0:164 BL ð Þ  0:013 NRF ð Þ  0:011 RFL ð Þ: 2.4 Statistical analysis Discriminant functions were obtained by splitting the original database in two subsets: the training data set (80%) and the testing data set (20%). Also, the variables that were highly correlated and provided redundant information were removed (AW, AT, LW and RFW). The testing data set was used to evaluate the accuracy of the LDA functions to classify new accessions into the groups. The LDAwas performed using MASS R package (Venables and Ripley, 2002). The LD functions were graphed in a two-dimensional space using ggplot2 R package (Wickham, 2016). The quality of the discriminant analysis was evaluated using the Wilks’ Lambda statistic and was done using the basic R “manova” function. The dendrogram resulting from HCA formed at a cut-off of 5.0 Euclidean distance showed three groups (S1, S2 and S3) and two un-grouped accessions (Fig. 2). The smallest group (S1) contained 23 accessions, the second (S2) has 45 and the third one (S3) 151 accessions. The main cluster was formed primarily by maintainer lines from group 1 and 2 and restorer lines from group 4. The second cluster was formed mainly by accessions derived from group 2 and in minor proportion by lines derived from group 5 and restorer lines. Most of the confectionary lines were concentrated in the smallest cluster. The HCA showed that group 2 was the predominant group in all clusters. g p p g p The LDA performed on the three clusters defined in the HCA showed that the discriminant functions obtained were significant in the Wilks test (p-value < 0.001) (Fig. 3). The two LDA functions obtained were: g The adjusted means for variables grain yield (GY) and Oil derived from multi-environment trials (MET) of the 70 A  R hybrids were regressed on Euclidean distance estimates obtained from the PC1, PC2 and PC3 to evaluate their association (Supplementary Table 2). The performance of these hybrids was also evaluated in relation to the group that was classified as the parental lines in the HCA. The relationship between the adjusted means of GY and Oil and the groups of classification of the parental lines were graphed in a violin plot using ggplot2 R package (Wickham, 2016). 3 Results Summary statistics for each quantitative descriptor are presented in (Tab. 4). Page 5 of 11 The variables that differentiated best between accession groups were achene length and weight of 100 achenes, because they obtained the highest coefficients in both functions. The discriminant function LDA1 explained 78.8% of the variability while the function LDA2 accounted for the rest (Fig. 3). The predictive capacity of the LDA functions obtained was evaluated through the testing data set by using a confusion matrix, showing an accuracy of 89%. The associations between the adjusted means of GY and Oil and Euclidean distance were not significant. The simple Table 4. Summary statistics for quantitative descriptors included in this study. Descriptor Mean SD CV Range Days to flowering from sowing 71.83 7.90 11.00 57–99.5 Number of achenes per capitulum 581.59 197.83 34.01 135–1650 Weight of 100 achenes (g) 5.29 1.27 23.92 2.2–10 Percentage of seed kernel 73.18 5.35 7.31 29.6–84.6 % of oil content 46.53 5.42 11.64 29–57.5 Achene width (mm) 4.76 0.89 18.69 2.64–9.1 Achene length (mm) 9.57 1.26 13.19 5.44–13.26 Achene thickness (mm) 2.86 0.72 25.03 0.5–5.38 Plant height (cm) 131.75 24.17 18.35 71.25–185 Number of leaves per plant 26.98 6.67 24.73 15.33–72 Stem diameter (mm) 20.89 3.88 18.54 12–32.23 Leave length (cm) 18.79 3.45 18.35 9.63–28.63 Leave width (cm) 17.64 3.80 21.52 8.7–33.71 Petiole length (cm) 10.02 2.78 27.75 3.63–18.35 Capitulum diameter (cm) 13.57 3.41 25.12 6–24.25 Bract length (cm) 4.70 1.12 23.93 1.1–8.07 Number of ray flowers 42.94 8.91 20.76 10.67–72.5 Ray flower width (mm) 13.25 3.96 29.90 6–25.67 Ray flower lenght (mm) 57.62 13.81 29.89 23.4–89.75 Fig. 1. Biplot of EEA INTA Pergamino sunflower lines. M. Domínguez et al.: OCL 2021, 28, 33 M. Domínguez et al.: OCL 2021, 28, 33 Table 4. Summary statistics for quantitative descriptors included in this study. 3 Results Descriptor Mean SD CV Range Days to flowering from sowing 71.83 7.90 11.00 57–99.5 Number of achenes per capitulum 581.59 197.83 34.01 135–1650 Weight of 100 achenes (g) 5.29 1.27 23.92 2.2–10 Percentage of seed kernel 73.18 5.35 7.31 29.6–84.6 % of oil content 46.53 5.42 11.64 29–57.5 Achene width (mm) 4.76 0.89 18.69 2.64–9.1 Achene length (mm) 9.57 1.26 13.19 5.44–13.26 Achene thickness (mm) 2.86 0.72 25.03 0.5–5.38 Plant height (cm) 131.75 24.17 18.35 71.25–185 Number of leaves per plant 26.98 6.67 24.73 15.33–72 Stem diameter (mm) 20.89 3.88 18.54 12–32.23 Leave length (cm) 18.79 3.45 18.35 9.63–28.63 Leave width (cm) 17.64 3.80 21.52 8.7–33.71 Petiole length (cm) 10.02 2.78 27.75 3.63–18.35 Capitulum diameter (cm) 13.57 3.41 25.12 6–24.25 Bract length (cm) 4.70 1.12 23.93 1.1–8.07 Number of ray flowers 42.94 8.91 20.76 10.67–72.5 Ray flower width (mm) 13.25 3.96 29.90 6–25.67 Ray flower lenght (mm) 57.62 13.81 29.89 23.4–89.75 Table 4. Summary statistics for quantitative descriptors included in this study. Fig. 1. Biplot of EEA INTA Pergamino sunflower lines. Fig. 1. Biplot of EEA INTA Pergamino sunflower lines. Fig. 1. Biplot of EEA INTA Pergamino sunflower lines. The predictive capacity of the LDA functions obtained was evaluated through the testing data set by using a confusion matrix, showing an accuracy of 89%. The variables that differentiated best between accession groups were achene length and weight of 100 achenes, because they obtained the highest coefficients in both functions. The discriminant function LDA1 explained 78.8% of the variability while the function LDA2 accounted for the rest (Fig. 3). The associations between the adjusted means of GY and Oil and Euclidean distance were not significant. The simple Page 6 of 11 Page 6 of 11 M. Domínguez et al.: OCL 2021, 28, 33 Fig. 2. Hierarchical cluster for EEA INTA Pergamino sunflower lines. Fig. 2. Hierarchical cluster for EEA INTA Pergamino sunflower lines. themphenotypicallyandgenotypicallytounderstandthegenetic diversity available in order to maximize the genetic gain in the breeding process. linear regression coefficient of Oil on Euclidean distance was 0.14 and the regression coefficient of GY on Euclidean distance was 16.27. Neither regression models explained the variability of the data, both multiple R2 were near to 0. g p PCA identified the traits which contributed most to explanation of the variability in the data. 3 Results In this study, the variables related to achene size play an important role in the arrangement of accessions, with the greatest capacity to discriminate between them. Terzić et al. (2020) in a phenotypic study of the UGA-SAM1 population also reported achene size among the descriptors with the greatest power of genotype discrimination. Dudhe et al. (2019), in a diversity study on a germplasm bank collection, reported that oil content was one of the traits that contributed most to the variability of the data, whereas in our work the results were not similar. This difference can be explained by the limited range of variation of this trait in the set of lines selected for this study, since most of them were selected to increase oil content and they do not constitute a germplasm bank collection. DTF was one of the variables with greatest contribution to the variability explained by the PCs. Similarly, Ayaz et al. (2014) reported that DTF was The adjusted means of the variable Oil for the 70 hybrids evaluated in MET showed no differences associated with the parental group classification obtained by HCA. The highest values were obtained by the crosses between lines belonging to the S3 group. On the other hand, for GY the values obtained for crosses between lines A belonging to group S2 and lines R belonging to group S3 were higher than the grain yields obtained between crosses of lines belonging to the same group. However, it can be seen that crosses of lines A of group S3 with lines R of group S2 showed the lowest yields (Fig. 4). 4 Discussion (2004) suggested that the inconsistent results obtained for the relationship between genetic distance and heterosis could be explained by the narrow genetic base of the germplasm used. Considering the genomic tools currently available, there is a clear need to deepen research on the relationship between genetic distance and heterosis in order to make the parental selection in breeding programs more efficient. one of the variables with more incidence in the variability of the studied germplasm. In addition, the biplot clearly identified in opposite quadrants the restorer and maintainer lines, defined by their different characteristics in terms of oil content, flowering time and achene size. Similar results were expressed by Terzić et al. (2020) taking also into account the branching trait. The PCA allowed us to interpret in a simple form the information collected through the different descriptors used, to identify groups of materials with certain phenotypic charac- teristics to direct future crosses, to identify the traits with less variability and to facilitate the search for specific charac- teristics in the materials available from the breeding program. The distribution of germplasm in the dendrogram did not follow a clear pattern with regard to the predefined groups at the beginning of the work. The largest cluster included most of the restorer lines. Other population studies in sunflower germplasm collections reported the preponderance of this characteristic in cluster delimitation (Mandel et al., 2011; Cadic et al., 2013; Filippi et al., 2015; Terzić et al., 2020). This distinction between maintainer and restorer lines would be expected due to the breeding strategy of keeping these groups as separate heterotic pools to maximize heterosis in hybrid crosses (Fick and Miller, 1997). The close relationship between the restorer lines can be understood from their common ancestor, the RHA 274 line, which has the Rf1 gene, one of the main sources of fertility restorer genes (Cadic et al., 2013). In addition, the similarity between the restorer lines studied is justified by the fact that all of them are branched. The smallest group of accessions is composed of confectionary lines that are characterized by their achene size. These lines represent a small group in this study, their characteristics are different from the rest of the accessions due to the different objectives of confectionary sunflower breeding with respect to oil sunflower breeding. 4 Discussion Germplasm collections are valuable resources for crop breeding. To exploit their potential, it is essential to characterize Page 7 of 11 Page 7 of 11 M. Domínguez et al.: OCL 2021, 28, 33 Fig. 3. Discriminant analysis scatter plot. Fig. 4. Relationship between the adjusted means of oil and grain yield for the 70 hybrids evaluated in MET in relation to the classification cluster of the parental lines. M. Domínguez et al.: OCL 2021, 28, 33 Fig. 3. Discriminant analysis scatter plot. Fig. 4. Relationship between the adjusted means of oil and grain yield for the 70 hybrids evaluated in MET in relation to the classification cluste of the parental lines. Fig. 4. Relationship between the adjusted means of oil and grain yield for the 70 hybrids evaluated in MET in relation to the classification cluster of the parental lines. adjusted means of oil and grain yield for the 70 hybrids evaluated in MET in relation to the classification cluster Fig. 4. Relationship between the adjusted means of oil and grain yield for the 70 hybrids evaluated in MET in relation to the classification cluster of the parental lines. Page 8 of 11 Page 8 of 11 M. Domínguez et al.: OCL 2021, 28, 33 M. Domínguez et al.: OCL 2021, 28, 33 coefficients of coancestry. Similarly, Reif et al. (2013) reported no correlation between the genetic distance obtained through a genomic matrix of 572 AFLP and grain yield, oil yield and oil content for a set of A  R hybrids. In contrast, Darvishzadeh (2012) reported high correlations between distances obtained from morphological and molecular data and the yield of sunflower F1 hybrids. Hladni et al. (2018) in a study carried out on interspecific crosses of sunflower, positive associations were demonstrated between the genetic distance (obtained from a matrix of 37 SSR and 1 STS markers) and grain yield. The results obtained in our work indicate no relationship between the distances obtained and the performance of the hybrids we evaluated. In other crops such as chickpea, cotton and corn, low correlations have also been found between the distance of the parental genotypes and the hybrid performance (Ajmone Marsan et al., 1998; Meredith and Brown, 1998; Sant et al., 1999). In addition, Dias dos Santos et al. 4 Discussion There are numerous papers in the literature on the relationship between genetic distance and heterosis or the response of traits such as yield and oil content. In sunflower, Cheres et al. (2000) found no satisfactory results in predicting grain yield as a function of genetic distance using molecular markers and The discriminant analysis provided functions that allow us to classify the accessions in three groups that will allow a more efficient use of the genetic resources and the preservation of the genetic variability of the germplasm. In addition, it is a low- cost tool to make a better use of the phenotypic information generated in each growing season. In the same sense, Terzić et al. (2020) highlight the importance of using regularly collected phenotypic data in breeding programs. They propose to include as part of the germplasm selection criteria for breeding, traits related to leaf variation, seed coloration and certain flower traits, in order to preserve the variability of the populations used in breeding. Although the clusters defined by the HCA and used in the LDA differed from the groups mentioned at the beginning of the paper, this might be due to the fact that both analyses were performed using the information provided only by the morphological descriptors. In summary, this work points out the need for more in- depth study of genetic variability to be used as a predictor of heterosis. Future genotypic characterizations of the sunflower accessions of the EEA Pergamino germplasm are proposed to develop predictive models that allow a greater efficiency in the selection of parental lines for the production of superior hybrids exploiting heterosis. Also, the results obtained confirm the importance of INTA EEA Pergamino’s sunflower breeding program in the development of improved, locally adapted and diverse materials as valuable resources for sunflower research. 4 Discussion These results revealed the variability of the lines that form the INTA Pergamino sunflower breeding program despite the high selective forces applied to obtain inbred lines for superior hybrids production. The higher oil content obtained by the hybrids whose parental lines belong to the S3 group can be explained by the additive action of the genes that control this characteristic. Similar results were obtained by Reif et al. (2013) evaluating a group of hybrids formed by crosses between and within heterotic groups (A  B, A  R and R  R), that did not present heterosis for oil content for any of the crosses. On the other hand, for grain yield, it was determined that hybrids obtained from the cross between lines of the group 2 and 3 had a greater grain yield. This tendency can be explained by the phenomenon of heterosis that is enhanced by the greater genetic distance between the lines of these groups. The fact that the reciprocal crosses (A lines of S2  R lines of S3) showed a lower yield suggests the need for a further definition of the germplasm groups. The inclusion of molecular data in future studies on this set of lines and more extensive testing of the hybrids would probably allow a more precise differentia- tion of the groups. inbred lines for superior hybrids production. The discriminant analysis provided functions that allow us to classify the accessions in three groups that will allow a more efficient use of the genetic resources and the preservation of the genetic variability of the germplasm. In addition, it is a low- cost tool to make a better use of the phenotypic information generated in each growing season. In the same sense, Terzić et al. (2020) highlight the importance of using regularly collected phenotypic data in breeding programs. They propose to include as part of the germplasm selection criteria for breeding, traits related to leaf variation, seed coloration and certain flower traits, in order to preserve the variability of the populations used in breeding. Although the clusters defined by the HCA and used in the LDA differed from the groups mentioned at the beginning of the paper, this might be due to the fact that both analyses were performed using the information provided only by the morphological descriptors. Developing hybrids is a costly long-term process; therefore, being able to predict heterosis is desirable. References Ajmone Marsan P, Castiglioni P, Fusari F, Kuiper M, Motto M. 1998. Genetic diversity and its relationship to hybrid performance in maize as revealed by RFLP and AFLP markers. Theor Appl Genet 96(2): 219–227. https://doi.org/10.1007/s001220050730. 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Supplementary Material Developing hybrids is a costly long-term process; therefore, being able to predict heterosis is desirable. There are numerous papers in the literature on the relationship between genetic distance and heterosis or the response of traits such as yield and oil content. In sunflower, Cheres et al. (2000) found no satisfactory results in predicting grain yield as a function of genetic distance using molecular markers and Supplementary Table 1. Supplementary Table 2. The Supplementary Material is available at http://www.ocl- journal.org/10.1051/ocl/2021021/olm. Page 9 of 11 Page 9 of 11 M. Domínguez et al.: OCL 2021, 28, 33 Acknowledgements. We are grateful to the Instituto Nacional de Tecnología Agropecuaria for the financial support, to Juan Carlos Righetti and Gabriel Colacilli of the INTA Pergamino Sunflower Breeding Group. We are also grateful to Rumbidzai Matemba-Mutasa for the English revision, María Lujan Farace for the support in the statistical analysis and the anonymous reviewers for the valuable comments that contributed to the improvement of the manuscript. González J, Mancuso N, Alvarez D, Cordes D, Vázquez A. 2015. Contribution of the Argentine germplasm to the improvement of sunflower. Helia 38(62): 121–140. Institute of Field and Vegetable Crops. https://doi.org/10.1515/helia-2014-0026. Grandlund M, Zimmerman DC. 1975. Effects of drying conditions on oil content of sunflower (Helianthus annuus L.) seeds as determined by wideline nuclear magnetic resonance (NMR). Proc North Dakota Acad Sci 27: 128–132. Proc North Dakota Acad Sci 27: 128–132. Hernández F, Presotto A, Poverene M, Mandel JR. 2019. Genetic diversity and population structure of wild sunflower (Heli- anthus annuus L.) in Argentina: reconstructing its invasion history. J Hered 110(6): 746–759. https://doi.org/10.1093/ jhered/esz047. References Dias dos Santos LA, de Toledo Picoli EA, Barros Rocha R, Alfenas AC. 2004. A priori choice of hybrid parents in plants. Genet Mol Res: GMR 3(3). www.funpecrp.com.br. Mandel JR, Dechaine JM, Marek LF, Burke JM. 2011. Genetic diversity and population structure in cultivated sunflower and a comparison to its wild progenitor, Helianthus annuus L. Theor Appl Genet 123(5): 693–704. https://doi.org/10.1007/ s00122-011-1619-3. Dudhe MY, Mulpuri S, Meena HP, Ajjanavara RRG, Kodeboyina VS, Adala VR. 2019. Genetic variability, diversity and identification of trait-specific accessions from the conserved sunflower germplasm for exploitation in the breeding programme. Agric Res. https://doi.org/10.1007/s40003-019-00406-w. Melchinger AE. 1999. Genetic diversity and heterosis. In: Genetics and exploitation of heterosis in crops, pp. 99–118. https://doi.org/ 10.2134/1999.geneticsandexploitation.c10. p g Fick G, Miller J. 1997. Sunflower breeding. In: Schneiter AA, ed. Sunflower technology and production. Madison, Wisconsin, USA. 10.2134/1999.geneticsandexploitation.c10. Melchinger AE, Gumber RK. 2015. Overview of heterosis and heterotic groups in agronomic crops, Issue 25, pp. 29–44. https:// doi.org/10.2135/cssaspecpub25.c3. Filippi CV, Merino GA, Montecchia JF, et al. 2020. Genetic diversity, population structure and linkage disequilibrium assessment among international sunflower breeding collections. Genes 11(3). https://doi.org/10.3390/genes11030283. Meredith WR, Brown JS. 1998. Heterosis and combining ability of cottons originating from different regions of the United States. J Cotton Sci 2(2): 77–84. Filippi CV, Aguirre N, Rivas JG, et al. 2015. Population structure and genetic diversity characterization of a sunflower association mapping population using SSR and SNP markers. BMC Plant Biol 15(1). https://doi.org/10.1186/s12870-014-0360-x. Miller JF. 1999. Oilseeds and heterosis. In: Genetics and exploitation of heterosis in crops, pp. 399–404. https://doi.org/10.2134/1999. geneticsandexploitation.c37. Franco TL, Hidalgo R. 2003. Análisis estadístico de datos de caracterización morfológica de recursos fitogenéticos. IBPGR 89. https://www.bioversityinternational.org/fileadmin/_migrated/ uploads/tx_news/Análisis_estadístico_de_datos_de_caracteriza ción_morfológica_de_recursos_fitogenéticos_894.pdf. Oksanen AJ, Blanchet FG, Friendly M, et al. 2018. Vegan: Community Ecology Package. Ordination methods, diversity analysis and other functions for community and vegetation ecologists, Issue 25(1). https://cran.r-project.org/package=veganER-. Page 10 of 11 M. Domínguez et al.: OCL 2021, 28, 33 Ringnér M. 2008. What is principal component analysis? Nat Biotechnol 26(3): 303–304. https://doi.org/10.1038/nbt0308-303. Palacio F, Apodaca M, Crisci J. 2020. ANÁLISIS MULTIVARIADO PARA DATOS BIOLÓGICOS Teoría y su aplicación utilizando el lenguaje R. Vazquez Mazzini Editores. Sant VJ, Patankar AG, Sarode ND, et al. 1999. Potential of DNA markersindetecting divergenceand inanalysingheterosisinIndian elite chickpea cultivars. Theor Appl Genet 98(8): 1217–1225. https://doi.org/10.1007/s001220051187. g j Paradis E, Schliep K. 2019. ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics 35: 526–528. Cite this article as: Domínguez M, Herrera S, González JH. 2021. Assessment of phenotypic variability among EEA INTA Pergamino sunflower lines: Its relationship with the grain yield and oil content. OCL 28: 33. References Peeters JP, Martinelli JA. 1989. Hierarchical cluster analysis as a tool to manage variation in germplasm collections. Theor Appl Genet 78(1): 42–48. https://doi.org/10.1007/BF00299751. Schneiter AA, Miller JF. 1981. Description of sunflower growth stages 1. Crop Sci 21(6): 901–903. https://doi.org/10.2135/ cropsci1981.0011183x002100060024x. Pilorgé E. 2020. Sunflower in the global vegetable oil system: situation, specificities and perspectives. OCL 27(1): 34. https:// doi.org/10.1051/ocl/2020028. Terzić S, Zorić M, Seiler GJ. 2020. Qualitative traits in sunflower breeding: UGA-SAM1 phenotyping case study. Crop Sci 60(1): 303–319. https://doi.org/10.1002/csc2.20059. g R Core Team. 2020. R: A Language and Environment for Statistical Computing. https://www.r-project.org/. Vear F, Miller JF. 1993. Sunflower. In: Traditional crop breeding practices: an historical review to serve as a baseline for assessing the role of modern biotechnology, pp. 95–111. https://doi.org/ 10.2134/agronmonogr36.c17. Rama Subrahmanyam SVRM, Sudheer Kumar S, Ranganatha ARGR. 2003. Genetic divergence for seed parameters in sunflower (Helianthus annuus L.). Helia 26(38): 73–80. https:// doi.org/10.2298/hel0338073s. Venables WN, Ripley BD. 2002. Modern applied statistics with S, 4th ed. Springer. https://www.stats.ox.ac.uk/pub/MASS4/. Reif JC, Zhao Y, Würschum T, Gowda M, Hahn V. 2013. Genomic prediction of sunflower hybrid performance. Plant Breed 132(1): 107–114. https://doi.org/10.1111/pbr.12007. Wickham H. 2016. ggplot2: elegant graphics for data analysis. New York: Springer-Verlag. https://ggplot2.tidyverse.org. Page 11 of 11
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A robust DayCent model calibration to assess the potential impact of integrated soil fertility management on maize yields, soil carbon stocks and greenhouse gas emissions in Kenya. Here, we present a robust calibration of DayCent to simulate maize productivity under ISFM, using data from four long-term field experiments. The experimental treatments consisted of the addition of low- to high-quality organic resources to the soil, with and without mineral N fertilizer. We assess the potential of DayCent to represent the key aspects of sustainable intensification, including 1) yield, 2) changes in soil carbon, and 3) global warming potential. The model was calibrated and cross-evaluated with the probabilistic Bayesian calibration technique. 10 The standard parameters of DayCent led to poor simulations of maize yield (Nash–Sutcliffe modeling efficiency; EF 0.33) and changes in SOC (EF -1.3) for different ISFM treatments. After calibration of the model, both the simulation of maize yield (EF 0.51) and the change in SOC (EF 0.54) improved significantly compared to the model with the standard parameter values. A leave-one-site-out cross-evaluation indicated the robustness of the approach for spatial upscaling (i.e., the significant improvement, described before, was achieved by calibrating with data from 3 sites and evaluating with the remaining site). 15 The SOC decomposition parameters were altered most severely by the calibration. They were an order of magnitude higher compared to the default parameter set. This confirms that the decomposition of SOC in tropical maize cropping systems is much faster than in temperate systems and that the DayCent temperature function is not suitable to capture this with a single parameter set. Finally, the global warming potential simulated by DayCent was highest in control -N treatments (0.5-2.5 kg CO2 equivalent per kg grain yield, depending on the site) and could be reduced by 14 to 72% by combined application of 20 5 and powerful way to assess the potential of such agricultural management practices at scale, but they need to be calibrated 5 to new environments and their reliability needs to be assured. Here, we present a robust calibration of DayCent to simulate maize productivity under ISFM, using data from four long-term field experiments. The experimental treatments consisted of the addition of low- to high-quality organic resources to the soil, with and without mineral N fertilizer. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. A robust DayCent model calibration to assess the potential impact of integrated soil fertility management on maize yields, soil carbon stocks and greenhouse gas emissions in Kenya. We assess the potential of DayCent to represent the key aspects of sustainable intensification, including 1) yield, 2) changes in soil carbon, and 3) and powerful way to assess the potential of such agricultural management practices at scale, but they need to be calibrated 5 to new environments and their reliability needs to be assured. Here, we present a robust calibration of DayCent to simulate maize productivity under ISFM, using data from four long-term field experiments. The experimental treatments consisted of the addition of low- to high-quality organic resources to the soil, with and without mineral N fertilizer. We assess the potential of DayCent to represent the key aspects of sustainable intensification, including 1) yield, 2) changes in soil carbon, and 3) global warming potential. The model was calibrated and cross-evaluated with the probabilistic Bayesian calibration technique. 10 The standard parameters of DayCent led to poor simulations of maize yield (Nash–Sutcliffe modeling efficiency; EF 0.33) and changes in SOC (EF -1.3) for different ISFM treatments. After calibration of the model, both the simulation of maize yield (EF 0.51) and the change in SOC (EF 0.54) improved significantly compared to the model with the standard parameter values. A leave-one-site-out cross-evaluation indicated the robustness of the approach for spatial upscaling (i.e., the significant global warming potential. The model was calibrated and cross-evaluated with the probabilistic Bayesian calibration technique. 10 The standard parameters of DayCent led to poor simulations of maize yield (Nash–Sutcliffe modeling efficiency; EF 0.33) and changes in SOC (EF -1.3) for different ISFM treatments. After calibration of the model, both the simulation of maize yield (EF 0.51) and the change in SOC (EF 0.54) improved significantly compared to the model with the standard parameter values. A leave-one-site-out cross-evaluation indicated the robustness of the approach for spatial upscaling (i.e., the significant improvement, described before, was achieved by calibrating with data from 3 sites and evaluating with the remaining site). 15 The SOC decomposition parameters were altered most severely by the calibration. They were an order of magnitude higher compared to the default parameter set. This confirms that the decomposition of SOC in tropical maize cropping systems is much faster than in temperate systems and that the DayCent temperature function is not suitable to capture this with a single parameter set. A robust DayCent model calibration to assess the potential impact of integrated soil fertility management on maize yields, soil carbon stocks and greenhouse gas emissions in Kenya. Moritz Laub1, Magdalena Necpalova1,2, Marijn Van de Broek1, Marc Corbeels3,4, Samuel Mathu Ndungu4, Monicah Wanjiku Mucheru-Muna5, Daniel Mugendi6, Rebecca Yegon6, Wycliffe Waswa4, Bernard Vanlauwe4, and Johan Six1 Moritz Laub1, Magdalena Necpalova1,2, Marijn Van de Broek1, Marc Corbeels3,4, Samuel Mathu Ndungu4, Monicah Wanjiku Mucheru-Muna5, Daniel Mugendi6, Rebecca Yegon6, Wycliffe Waswa4, Bernard Vanlauwe4, and Johan Six1 1Department of Environmental Systems Science, ETH Zurich, 8092 Zürich, Switzerland 2University College Dublin, School of Agriculture and Food Science, Ireland 3CIRAD, Avenue d’Agropolis, F-34398 Montpellier, France 4International Institute of Tropical Agriculture (IITA), c/o ICIPE Compound, P. O. Box 30772-00100, Nairobi, Kenya 5Department of Environmental Sciences and Education, Kenyatta University, P.O. Box 43844-00100, Nairobi, Kenya 6Department of Water and Agricultural Resource Management, University of Embu, P.O. Box 6-60100, Embu, Kenya Correspondence: Moritz Laub (moritz.laub@usys.ethz.ch) 1Department of Environmental Systems Science, ETH Zurich, 8092 Zürich, Switzerland 2University College Dublin, School of Agriculture and Food Science, Ireland 3CIRAD, Avenue d’Agropolis, F-34398 Montpellier, France 4International Institute of Tropical Agriculture (IITA), c/o ICIPE Compound, P. O. Box 30772-00100, Nairobi, Kenya 5Department of Environmental Sciences and Education, Kenyatta University, P.O. Box 43844-00100, Nairobi, Kenya 6Department of Water and Agricultural Resource Management, University of Embu, P.O. Box 6-60100, Embu, Kenya Correspondence: Moritz Laub (moritz.laub@usys.ethz.ch) Abstract. Sustainable intensification schemes that increase crop production and soil fertility, such as integrated soil fertility management (ISFM), are a proposed strategy to close yield gaps and achieve food security in sub-Saharan Africa while main- taining soil fertility. However, field trials are insufficient to estimate the potential impact of such technologies at the regional or national scale. Upscaling via biogeochemical models, such as DayCent, from the field-scale to a larger region can be a suitable Abstract. Sustainable intensification schemes that increase crop production and soil fertility, such as integrated soil fertility management (ISFM), are a proposed strategy to close yield gaps and achieve food security in sub-Saharan Africa while main- taining soil fertility. However, field trials are insufficient to estimate the potential impact of such technologies at the regional or national scale. Upscaling via biogeochemical models, such as DayCent, from the field-scale to a larger region can be a suitable and powerful way to assess the potential of such agricultural management practices at scale, but they need to be calibrated 5 to new environments and their reliability needs to be assured. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. mineral N and manure at a medium rate. In three of the four sites, the global warming potential was largely (> 75%) dominated by SOC losses. In summary, our results indicate that DayCent is suitable for estimating the impact of ISFM from the site to the regional level, that trade-offs between yields and global warming potential are stronger in low-fertility sites, and that the reduction of SOC losses is a priority for the sustainable intensification of maize production in Kenya. A robust DayCent model calibration to assess the potential impact of integrated soil fertility management on maize yields, soil carbon stocks and greenhouse gas emissions in Kenya. Finally, the global warming potential simulated by DayCent was highest in control -N treatments (0.5-2.5 kg improvement, described before, was achieved by calibrating with data from 3 sites and evaluating with the remaining site). 15 The SOC decomposition parameters were altered most severely by the calibration. They were an order of magnitude higher compared to the default parameter set. This confirms that the decomposition of SOC in tropical maize cropping systems is much faster than in temperate systems and that the DayCent temperature function is not suitable to capture this with a single parameter set. Finally, the global warming potential simulated by DayCent was highest in control -N treatments (0.5-2.5 kg improvement, described before, was achieved by calibrating with data from 3 sites and evaluating with the remaining site). 15 The SOC decomposition parameters were altered most severely by the calibration. They were an order of magnitude higher compared to the default parameter set. This confirms that the decomposition of SOC in tropical maize cropping systems is much faster than in temperate systems and that the DayCent temperature function is not suitable to capture this with a single parameter set. Finally, the global warming potential simulated by DayCent was highest in control -N treatments (0.5-2.5 kg CO2 equivalent per kg grain yield, depending on the site) and could be reduced by 14 to 72% by combined application of 20 1 1 1 Introduction 25 Similar to many countries of Sub-Saharan Africa (SSA), the low maize yields in Kenya, on average 1.7 t ha-1 compared to the global average of 5.6 t ha-1 over the last decade (2011-2021) (FAO, 2023), are in part responsible for low self-sufficiency of food production. Consequently, around 20% of the Kenyan population lives with severe food insecurity (World-Bank, 2021b). If yields are not improved, increased demand due to population growth will further deteriorate self-sufficiency and in general Similar to many countries of Sub-Saharan Africa (SSA), the low maize yields in Kenya, on average 1.7 t ha-1 compared to the global average of 5.6 t ha-1 over the last decade (2011-2021) (FAO, 2023), are in part responsible for low self-sufficiency of food production. Consequently, around 20% of the Kenyan population lives with severe food insecurity (World-Bank, 2021b). If yields are not improved, increased demand due to population growth will further deteriorate self-sufficiency and in general food security in the coming decades (Zhai et al., 2021): yield declines resulting from more frequent extreme weather events 30 could further exacerbate this situation (Lobell et al., 2011). One of the key limitations to sustainable maize production in SSA is the lack of mineral fertilizer and organic inputs. Integrated soil fertility management (ISFM) is a sustainable intensification practice that can alleviate these limitations by combining the use of mineral fertilizers with organic inputs (Vanlauwe et al., 2010). Different studies reported that ISFM has the potential to more than double maize yields in Kenya, especially in currently food security in the coming decades (Zhai et al., 2021): yield declines resulting from more frequent extreme weather events 30 could further exacerbate this situation (Lobell et al., 2011). One of the key limitations to sustainable maize production in SSA is the lack of mineral fertilizer and organic inputs. Integrated soil fertility management (ISFM) is a sustainable intensification practice that can alleviate these limitations by combining the use of mineral fertilizers with organic inputs (Vanlauwe et al., 2010). Different studies reported that ISFM has the potential to more than double maize yields in Kenya, especially in currently food security in the coming decades (Zhai et al., 2021): yield declines resulting from more frequent extreme weather events 30 could further exacerbate this situation (Lobell et al., 2011). One of the key limitations to sustainable maize production in SSA is the lack of mineral fertilizer and organic inputs. 1 Introduction 25 Integrated soil fertility management (ISFM) is a sustainable intensification practice that can alleviate these limitations by combining the use of mineral fertilizers with organic inputs (Vanlauwe et al., 2010). Different studies reported that ISFM has the potential to more than double maize yields in Kenya, especially in currently unfertile soils, due to the effects on soil fertility, including soil organic matter (SOM) content (Chivenge et al., 2009, 2011). 35 However, in addition to the amount and quality of fertilizer and organic inputs, the effectiveness of ISFM in increasing yields strongly depends on local site conditions, such as soil and climate (Chivenge et al., 2011). Furthermore, increasing SOM also contributes to minimizing the negative effects of climate change, and there may be considerable potential in carbon-depleted soils in SSA (Corbeels et al., 2019). unfertile soils, due to the effects on soil fertility, including soil organic matter (SOM) content (Chivenge et al., 2009, 2011). 35 However, in addition to the amount and quality of fertilizer and organic inputs, the effectiveness of ISFM in increasing yields strongly depends on local site conditions, such as soil and climate (Chivenge et al., 2011). Furthermore, increasing SOM also contributes to minimizing the negative effects of climate change, and there may be considerable potential in carbon-depleted soils in SSA (Corbeels et al., 2019). Hence, to close yield gaps in a resource-efficient way and to assess the climate change mitigation potential of ISFM, we 40 need to understand the effects of long-term implementation of ISFM at larger scales. Ideally, this would be facilitated by implementing a large number of long-term experiments (LTE) across a representative range of soil and climatic conditions. However, due to the high demands in terms of cost, labor, and time associated with maintaining LTE, the number of LTE that evaluate variation in the effects of ISFM practices due to site-specific conditions is very limited. In addition, using statistical Hence, to close yield gaps in a resource-efficient way and to assess the climate change mitigation potential of ISFM, we 40 need to understand the effects of long-term implementation of ISFM at larger scales. Ideally, this would be facilitated by implementing a large number of long-term experiments (LTE) across a representative range of soil and climatic conditions. 1 Introduction 25 However, due to the high demands in terms of cost, labor, and time associated with maintaining LTE, the number of LTE that evaluate variation in the effects of ISFM practices due to site-specific conditions is very limited. In addition, using statistical predictive techniques to upscale results obtained with a low number of sites could lead to low predictive power and large 45 predictive errors, because it is unlikely that the effects of variation in soils and climate on yield and the development of soil organic matter (SOM) would be fully captured by the statistical models. predictive techniques to upscale results obtained with a low number of sites could lead to low predictive power and large 45 predictive errors, because it is unlikely that the effects of variation in soils and climate on yield and the development of soil organic matter (SOM) would be fully captured by the statistical models. Biogeochemical process-based ecosystem models, such as DayCent (Parton et al., 1998; Del Grosso et al., 2001), are able to simulate the influence of the important driving variables of yield and SOM formation using semi-mechanistic functions developed through decades of agronomic and soil research. Because they (partly) embed our current understanding of the 50 Biogeochemical process-based ecosystem models, such as DayCent (Parton et al., 1998; Del Grosso et al., 2001), are able to simulate the influence of the important driving variables of yield and SOM formation using semi-mechanistic functions developed through decades of agronomic and soil research. Because they (partly) embed our current understanding of the 50 complex ecosystem processes, they are more robust for upscaling of the yield potential (Saito et al., 2021) and the SOM building capacity (Lee et al., 2020), compared to statistical predictive techniques. However, to avoid bias in model output, it p g y g developed through decades of agronomic and soil research. Because they (partly) embed our current understanding of the 50 complex ecosystem processes, they are more robust for upscaling of the yield potential (Saito et al., 2021) and the SOM building capacity (Lee et al., 2020), compared to statistical predictive techniques. However, to avoid bias in model output, it developed through decades of agronomic and soil research. 1 Introduction 25 Because they (partly) embed our current understanding of the 50 complex ecosystem processes, they are more robust for upscaling of the yield potential (Saito et al., 2021) and the SOM building capacity (Lee et al., 2020), compared to statistical predictive techniques. However, to avoid bias in model output, it 2 This is especially relevant because a recent study showed considerable uncertainty in DayCent SOM turnover rates, even when using a range of long-term experiments for calibration (Gurung et al., 2020). into biased crop responses to N and biased crop growth rates in any upscaling exercise. A potential solution to this issue is 60 the joint calibration of soil and crop parameters in DayCent, using long-term data from local trials. Ideally, it would include the uncertainty in the model parameters and outputs (Clifford et al., 2014), so a propagation of errors is possible in upscaling exercises (Stella et al., 2019). This is especially relevant because a recent study showed considerable uncertainty in DayCent SOM turnover rates, even when using a range of long-term experiments for calibration (Gurung et al., 2020). In order to use DayCent to assess the potential of ISFM to contribute to closing the yield gap while minimizing environmental 65 impact in Kenya and other SSA countries, the aim of this study was to use Bayesian calibration to derive robust DayCent parameters of SOM cycling and maize growth in Kenya. We used the experimental data of four long-term ISFM trials conducted in Kenya for almost two decades (Laub et al., 2022, 2023). Of these, two sites were in humid western Kenya and two in subhumid to semi-arid central Kenya. Their treatments were different additions of organic resources (1.2 and 4 t C ha-1 yr-1 including farmyard manure, high-quality Tithonia diversifolia and Calliandra calothyrsus residues as well as low-quality low 70 quality stover of Zea mays and sawdust), combined with (+N) or without (-N) 120 kg mineral N fertilizer ha-1 season-1. They thus contain a range of experimental conditions and inputs. The goal was to evaluate to what extent DayCent can replicate the differences in yields and SOM development in response to different qualities and rates of organic resources combined with different rates of N fertilizer and between sites. Furthermore, because ISFM can simultaneously be a source of N2O to the including farmyard manure, high-quality Tithonia diversifolia and Calliandra calothyrsus residues as well as low-quality low 70 quality stover of Zea mays and sawdust), combined with (+N) or without (-N) 120 kg mineral N fertilizer ha-1 season-1. They thus contain a range of experimental conditions and inputs. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. is best practice that models are calibrated and validated to local conditions (Necpálová et al., 2015) whenever they are applied outside the range of usual conditions, for example, in a new climate zone with different soils. is best practice that models are calibrated and validated to local conditions (Necpálová et al., 2015) whenever they are applied outside the range of usual conditions, for example, in a new climate zone with different soils. is best practice that models are calibrated and validated to local conditions (Necpálová et al., 2015) whenever they are applied outside the range of usual conditions, for example, in a new climate zone with different soils. Although DayCent has been used to estimate the development of SOM stock in Kenya on a national scale (Kamoni et al., 55 2007) and recently the efficiency of conservation agriculture in Ethiopia (Lemma et al., 2021), DayCent’s modules of SOM and maize crop growth have never been rigorously calibrated and validated for tropical agroecosystems in SSA. A recent test of DayCent in Kenyan maize growing systems showed that SOM turnover is underpredicted by DayCent (Nyawira et al., 2021). Because SOM in biogeochemical models is coupled to nitrogen (N) mineralization, there is the potential that this translates Although DayCent has been used to estimate the development of SOM stock in Kenya on a national scale (Kamoni et al., 55 2007) and recently the efficiency of conservation agriculture in Ethiopia (Lemma et al., 2021), DayCent’s modules of SOM and maize crop growth have never been rigorously calibrated and validated for tropical agroecosystems in SSA. A recent test of DayCent in Kenyan maize growing systems showed that SOM turnover is underpredicted by DayCent (Nyawira et al., 2021). Because SOM in biogeochemical models is coupled to nitrogen (N) mineralization, there is the potential that this translates 55 g p g p into biased crop responses to N and biased crop growth rates in any upscaling exercise. A potential solution to this issue is 60 the joint calibration of soil and crop parameters in DayCent, using long-term data from local trials. Ideally, it would include the uncertainty in the model parameters and outputs (Clifford et al., 2014), so a propagation of errors is possible in upscaling exercises (Stella et al., 2019). https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 2.1 The experimental sites The present study used data pooled from four experimental sites. The experiments tested different organic resource treatments 85 at different rates in combination with the application of mineral N fertilizer in the context of ISFM. The experimental setup was identical between the sites. The sites are located in agriculturally important areas in western and central Kenya. The Embu and Machanga sites are both located in Embu County, in the central part of Kenya. The Aludeka site is located in Busia County in western Kenya, while Sidada is located in the adjacent Siaya County, just south of Busia (Table A1). The experiments in Embu The present study used data pooled from four experimental sites. The experiments tested different organic resource treatments 85 at different rates in combination with the application of mineral N fertilizer in the context of ISFM. The experimental setup was identical between the sites. The sites are located in agriculturally important areas in western and central Kenya. The Embu and Machanga sites are both located in Embu County, in the central part of Kenya. The Aludeka site is located in Busia County in western Kenya, while Sidada is located in the adjacent Siaya County, just south of Busia (Table A1). The experiments in Embu The present study used data pooled from four experimental sites. The experiments tested different organic resource treatments 85 at different rates in combination with the application of mineral N fertilizer in the context of ISFM. The experimental setup was identical between the sites. The sites are located in agriculturally important areas in western and central Kenya. The Embu and Machanga sites are both located in Embu County, in the central part of Kenya. The Aludeka site is located in Busia County in western Kenya, while Sidada is located in the adjacent Siaya County, just south of Busia (Table A1). The experiments in Embu and Machanga were initiated in early 2002 and the experiments in Aludeka and Sidada were initiated in early 2005. Therefore, 90 19 years of data were available in central and 16 years in western Kenya (2 sites x 16 years + 2 sites x 19 years = 70 site years = 140 site seasons). The sites cover a range of altitudes, temperatures, and precipitations. 2.1 The experimental sites Maize was grown continuously in all experi- 100 ments, with two crops per year, one in the long rainy season and one in the short rainy season. The design was similar in all four sites and has been described in detail for the Embu site (Chivenge et al., 2009; Gentile et al., 2011). Organic resource treatments consisted of high quality Tithonia diversifolia (TD) green manure and Calliandra calothyrsus (CC) prunings, low quality stover of Zea mays (MS) and sawdust from Grevillea robusta trees (SD), locally available farmyard manure (FYM) resources as main plots and the presence or absence of N fertilizer as subplots. Maize was grown continuously in all experi- 100 ments, with two crops per year, one in the long rainy season and one in the short rainy season. The design was similar in all four sites and has been described in detail for the Embu site (Chivenge et al., 2009; Gentile et al., 2011). Organic resource treatments consisted of high quality Tithonia diversifolia (TD) green manure and Calliandra calothyrsus (CC) prunings, low quality stover of Zea mays (MS) and sawdust from Grevillea robusta trees (SD), locally available farmyard manure (FYM) and a control treatment (CT) without any organic resource additions. Organic resources differed in quality by the contents of 105 N, lignin and polyphenols (Table A2). Each organic resource was applied at two rates, 1.2 and 4 t C ha-1 yr-1, while only one amount of applied mineral fertilizer, 120 kg N ha-1 (CaNH4NO3) in each of the two growing seasons was tested. Of that, 40 kg N ha-1 were applied with planting, and the remaining 80 kg N ha-1 about six weeks after planting. Organic resources were applied only once a year, prior to planting for the long rainy season in January or February. They were incorporated to a depth and a control treatment (CT) without any organic resource additions. Organic resources differed in quality by the contents of 105 N, lignin and polyphenols (Table A2). Each organic resource was applied at two rates, 1.2 and 4 t C ha-1 yr-1, while only one amount of applied mineral fertilizer, 120 kg N ha-1 (CaNH4NO3) in each of the two growing seasons was tested. Of that, 40 kg N ha-1 were applied with planting, and the remaining 80 kg N ha-1 about six weeks after planting. The goal was to evaluate to what extent DayCent can replicate the differences in yields and SOM development in response to different qualities and rates of organic resources combined with different rates of N fertilizer and between sites. Furthermore, because ISFM can simultaneously be a source of N2O to the atmosphere (Leitner et al., 2020) and mitigate CO2 emissions from the soil (Laub et al., 2022), the second objective was to 75 evaluate the total global warming potential of different rates of organic material in ISFM to find the optimal balance between limiting greenhouse gas emissions from the soil and optimizing the yield (that is, sustainable intensification). Thus, the specific objectives of this study were (i) to test the capability of an uncalibrated version of DayCent to simulate yield and SOC development of the different ISFM practices, (ii) to calibrate DayCent to represent ISFM under Kenyan condi- atmosphere (Leitner et al., 2020) and mitigate CO2 emissions from the soil (Laub et al., 2022), the second objective was to 75 evaluate the total global warming potential of different rates of organic material in ISFM to find the optimal balance between limiting greenhouse gas emissions from the soil and optimizing the yield (that is, sustainable intensification). Thus, the specific objectives of this study were (i) to test the capability of an uncalibrated version of DayCent to simulate yield and SOC development of the different ISFM practices, (ii) to calibrate DayCent to represent ISFM under Kenyan condi- tions using experimental data from the 4 LTE, displaying the confidence in model parameters by Bayesian calibration, and (iii) 80 to use the calibrated model to gain an understanding of possible trade-offs between yield and SOM increases under ISFM, and to understand the global warming potential of the different ISFM treatments. tions using experimental data from the 4 LTE, displaying the confidence in model parameters by Bayesian calibration, and (iii) 80 to use the calibrated model to gain an understanding of possible trade-offs between yield and SOM increases under ISFM, and to understand the global warming potential of the different ISFM treatments. 3 3 2.1 The experimental sites Embu, with a mean annual temperature (MAT) of 20 °C and 1200 mm annual precipitation, is the coolest of all sites, while Machanga has a MAT of 24°C and receives the lowest amounts of annual precipitation (800 mm). Sidada (23°C, 1700 mm) and Aludeka (24°C, 1700 mm) have a high and Machanga were initiated in early 2002 and the experiments in Aludeka and Sidada were initiated in early 2005. Therefore, 90 19 years of data were available in central and 16 years in western Kenya (2 sites x 16 years + 2 sites x 19 years = 70 site years = 140 site seasons). The sites cover a range of altitudes, temperatures, and precipitations. Embu, with a mean annual temperature (MAT) of 20 °C and 1200 mm annual precipitation, is the coolest of all sites, while Machanga has a MAT of 24°C and receives the lowest amounts of annual precipitation (800 mm). Sidada (23°C, 1700 mm) and Aludeka (24°C, 1700 mm) have a high MAT and receive significantly more precipitation than the sites in central Kenya. There are two rainy seasons at each site, 95 which corresponds to two maize growing seasons per year. The long rainy season runs from March to August/September, the short rainy season runs from October until January/February. In terms of soil texture, Machanga and Aludeka have low clay content (13% clay at both sites), while Sidada and Embu are rich in clay (56 and 60%, respectively). All experiments were set up as a split plot design with three replicates, with different qualities and quantities of organic All experiments were set up as a split plot design with three replicates, with different qualities and quantities of organic resources as main plots and the presence or absence of N fertilizer as subplots. Maize was grown continuously in all experi- 100 ments, with two crops per year, one in the long rainy season and one in the short rainy season. The design was similar in all four sites and has been described in detail for the Embu site (Chivenge et al., 2009; Gentile et al., 2011). Organic resource treatments consisted of high quality Tithonia diversifolia (TD) green manure and Calliandra calothyrsus (CC) prunings, low quality stover of Zea mays (MS) and sawdust from Grevillea robusta trees (SD), locally available farmyard manure (FYM) resources as main plots and the presence or absence of N fertilizer as subplots. 2.2 The DayCent model SOC and soil N in the upper 20 cm are represented by active, slow, and passive SOM pools, while litter and organic resources are represented by structural and metabolic litter pools (Parton et al., 1987). All soil pools are conceptual and have no measurable counterparts, whereas the litter pools are semiquantitative, that is, their division is based on the measurable ratio of lignin to N in organic resources and plant litter. For temperate conditions, DayCent has been shown to adequately simulate 125 crop yields, SOC and soil N dynamics and N2O emissions (Del Grosso et al., 2005; Necpálová et al., 2015; Necpalova et al., 2018; Gurung et al., 2020, 2021). Yet, for tropical conditions the performance has not been studied in similar detail. A recent paper showed that the general model fit of the uncalibrated model was suboptimal (Nyawira et al., 2021). of lignin to N in organic resources and plant litter. For temperate conditions, DayCent has been shown to adequately simulate 125 crop yields, SOC and soil N dynamics and N2O emissions (Del Grosso et al., 2005; Necpálová et al., 2015; Necpalova et al., 2018; Gurung et al., 2020, 2021). Yet, for tropical conditions the performance has not been studied in similar detail. A recent paper showed that the general model fit of the uncalibrated model was suboptimal (Nyawira et al., 2021). of lignin to N in organic resources and plant litter. For temperate conditions, DayCent has been shown to adequately simulate 125 crop yields, SOC and soil N dynamics and N2O emissions (Del Grosso et al., 2005; Necpálová et al., 2015; Necpalova et al., 2018; Gurung et al., 2020, 2021). Yet, for tropical conditions the performance has not been studied in similar detail. A recent paper showed that the general model fit of the uncalibrated model was suboptimal (Nyawira et al., 2021). https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 2.3 Data used for the DayCent model evaluation/calibration In a process that was repeated four times, a large data set was used based on three of the four sites for the model calibration and 130 the validation was performed based on the fourth site. The plot-scale yield of maize grain and the aboveground biomass, both on a dry matter basis (t ha-1), were available for each cropping season between 2002 and 2020 (further details in Laub et al., 2023). In addition to that, plot-scale SOC and total N contents in the top 15 cm were available for several points over time. In Embu and Machanga, soil samples were taken every two to three years since initiation in 2002 until 2021, but in Sidada and In a process that was repeated four times, a large data set was used based on three of the four sites for the model calibration and 130 the validation was performed based on the fourth site. The plot-scale yield of maize grain and the aboveground biomass, both on a dry matter basis (t ha-1), were available for each cropping season between 2002 and 2020 (further details in Laub et al., 2023). In addition to that, plot-scale SOC and total N contents in the top 15 cm were available for several points over time. In Embu and Machanga, soil samples were taken every two to three years since initiation in 2002 until 2021, but in Sidada and Aludeka, only in 2005, 2018, 2019 and 2021 (further details in Laub et al., 2022). Because bulk density was not available for 135 most soil samples and in 2021 there was no significant difference in topsoil bulk density between treatments at any site, the mean bulk density per site was used to calculate SOC stocks of the top 15 cm of soil depth. All simulations were conducted at the site scale, so the plot-scale (i.e. replicate) measurements were aggregated to the site scale, calculating means and variances. DayCent calculates SOC to a depth of 20 cm, so we rescaled the SOC stocks for the top 15 cm to the top 20 cm, using the formula of Jobbágy and Jackson (2000): 140 Aludeka, only in 2005, 2018, 2019 and 2021 (further details in Laub et al., 2022). 2.1 The experimental sites Organic resources were applied only once a year, prior to planting for the long rainy season in January or February. They were incorporated to a depth of 15 cm with hand hoes. Furthermore, a blanket application of 60 kg P ha-1 as triple superphosphate and of 60 kg K ha-1 as 110 muriate potash at planting was provided to all plots once each season. The plots were kept weed free by hand weeding, between two and three times per season, and selective application of pesticides was used when necessary to protect against armyworm, stemborer, and/or termites. of 15 cm with hand hoes. Furthermore, a blanket application of 60 kg P ha-1 as triple superphosphate and of 60 kg K ha-1 as 110 muriate potash at planting was provided to all plots once each season. The plots were kept weed free by hand weeding, between two and three times per season, and selective application of pesticides was used when necessary to protect against armyworm, stemborer, and/or termites. 4 2.2 The DayCent model The DayCent model (version 2020 of DD_EVI) is a terrestrial ecosystem model of intermediate complexity (Del Grosso et al., 115 2001). It simulates C and N fluxes within the soil-plant-atmosphere continuum on a daily basis and has been parameterized for many crops and ecosystems (Necpalova et al., 2018). It has submodules to simulate plant growth, organic resource and soil organic matter (SOM) decomposition, mineralization of N, soil water and temperature, N gas fluxes, and CH oxidation4. The net primary productivity of plants is a function of their genetic potential, a simplified phenology, solar radiation, temperature, and stresses, such as reduced water or N availability. Here, we used the non-growing degree day version of the DayCent crop 120 module, which does not simulate phenology but has a seedling stage with reduced growth until a certain biomass (full canopy) is reached. SOC and soil N in the upper 20 cm are represented by active, slow, and passive SOM pools, while litter and organic resources are represented by structural and metabolic litter pools (Parton et al., 1987). All soil pools are conceptual and have no measurable counterparts, whereas the litter pools are semiquantitative, that is, their division is based on the measurable ratio of lignin to N in organic resources and plant litter For temperate conditions DayCent has been shown to adequately simulate 125 and stresses, such as reduced water or N availability. Here, we used the non-growing degree day version of the DayCent crop 120 module, which does not simulate phenology but has a seedling stage with reduced growth until a certain biomass (full canopy) is reached. SOC and soil N in the upper 20 cm are represented by active, slow, and passive SOM pools, while litter and organic resources are represented by structural and metabolic litter pools (Parton et al., 1987). All soil pools are conceptual and have no measurable counterparts, whereas the litter pools are semiquantitative, that is, their division is based on the measurable ratio and stresses, such as reduced water or N availability. Here, we used the non-growing degree day version of the DayCent crop 120 module, which does not simulate phenology but has a seedling stage with reduced growth until a certain biomass (full canopy) is reached. Here, SOC20 and SOC15 are SOC stocks in kg ha-1 in the top 20 and 15 cm soil depth, respectively. The parameter β is the relative decrease of SOC stocks with depth, for which we took the mean values across sites (0.9725), calculated from the 2021 sampling, where samples from 0-15, 15-30, and 30-50 cm were available for all of the sites. Finally, in the control and the 1.2 t C plots of the Calliandra, farmyard manure and maize stover treatments, continuous soil moisture measurements from sensors placed in each replicate at 10 cm soil depth (EC-5 Soil Moisture Sensor, Meter, Germany), were available for Sidada and Aludeka sites (March 2018 to December 2020). These soil moisture measurements were used to 160 available for Sidada and Aludeka sites (March 2018 to December 2020). These soil moisture measurements were used to 160 determine the optimal pedotransfer functions for soil hydraulic conductivity before the actual model calibration phase. available for Sidada and Aludeka sites (March 2018 to December 2020). These soil moisture measurements were used to 160 determine the optimal pedotransfer functions for soil hydraulic conductivity before the actual model calibration phase. 2.3 Data used for the DayCent model evaluation/calibration Because bulk density was not available for 135 most soil samples and in 2021 there was no significant difference in topsoil bulk density between treatments at any site, the mean bulk density per site was used to calculate SOC stocks of the top 15 cm of soil depth. All simulations were conducted at the site scale, so the plot-scale (i.e. replicate) measurements were aggregated to the site scale, calculating means and variances. DayCent calculates SOC to a depth of 20 cm, so we rescaled the SOC stocks for the top 15 cm to the top 20 cm, using the SOC20(kg ha−1) = 1 −β20 1 −β15 ∗SOC15 SOC20(kg ha−1) = 1 −β20 1 −β15 ∗SOC15 (1) (1) Here, SOC20 and SOC15 are SOC stocks in kg ha-1 in the top 20 and 15 cm soil depth, respectively. The parameter β is the relative decrease of SOC stocks with depth, for which we took the mean values across sites (0.9725), calculated from the 2021 sampling, where samples from 0-15, 15-30, and 30-50 cm were available for all of the sites. 5 5 2.3.1 Model driving variables and model assumptions The site-specific crop management data used to run the DayCent model was obtained from field management operation records for each season at each site. This included specific dates for the yearly application of organic resources (in all but the control The site-specific crop management data used to run the DayCent model was obtained from field for each season at each site. This included specific dates for the yearly application of organic The site-specific crop management data used to run the DayCent model was obtained from field management operation records for each season at each site. This included specific dates for the yearly application of organic resources (in all but the control plots), manual plowing before planting, maize planting, split application of mineral N at rates of 40 kg and 80 kg N ha-1 per 165 season, weeding and harvest dates. Pesticide application events and gap filling plus maize thinning are not part of standard DayCent management and were therefore not included in the DayCent schedule file. Our model runs therefore, assumed no occurrence of pests or diseases and an optimal plant density from the start, which in practice was ensured by manual thinning and gap filling. plots), manual plowing before planting, maize planting, split application of mineral N at rates of 40 kg and 80 kg N ha-1 per 165 season, weeding and harvest dates. Pesticide application events and gap filling plus maize thinning are not part of standard DayCent management and were therefore not included in the DayCent schedule file. Our model runs therefore, assumed no occurrence of pests or diseases and an optimal plant density from the start, which in practice was ensured by manual thinning and gap filling. The climate data used to run DayCent consisted of recorded data at each of the sites. However, filling the data gaps 170 was necessary due to unavailability and loss of recorded data. In Embu and Machanga, manual recordings of daily min- imum and maximum temperature and precipitation were available from 2002 until the end of 2007, but from 2008 un- til 2017, only measured precipitation was available. After 2017, high-quality data from newly installed TAHMO stations (https://tahmo.org/climate-data/) were available near the Machanga and Embu sites, with daily values for temperature and precipitation. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Data on N2O emissions were used in the model evaluation phase, but not for model calibration, because the data were scarce 145 and subject to high variability. The N2O measurements were conducted after N fertilization in 2005 (weekly measurements form March to June in Embu and Machanga and daily measurements in Machanga in November), in 2013 and 2018 (weekly measurements form March to beginning of May in Sidada and Aludeka), and in 2021 (weekly measurements form mid-March to mid-May in Sidada). They were conducted with the static chamber method (Hutchinson and Mosier, 1981). Measuring Data on N2O emissions were used in the model evaluation phase, but not for model calibration, because the data were scarce 145 and subject to high variability. The N2O measurements were conducted after N fertilization in 2005 (weekly measurements form March to June in Embu and Machanga and daily measurements in Machanga in November), in 2013 and 2018 (weekly measurements form March to beginning of May in Sidada and Aludeka), and in 2021 (weekly measurements form mid-March to mid-May in Sidada). They were conducted with the static chamber method (Hutchinson and Mosier, 1981). Measuring frames were permanently installed in the plots for a whole rainy season. The chambers (0.27 × 0.375 × 0.11 m) were made of 150 polyvinylchloride and equipped with a vent tube and a fan to homogenize gas inside them before gas sampling. The measured N2O emissions were evaluated at two levels of aggregation. First, as site means per measurement day (from the three replicates, similar to all other data) and second as cumulative emissions over the whole season, for which we first used the trapezoid method at the plot scale (Levy et al., 2017), specifically, the trapz function of R (Tuszynski, 2021). Site-scale means and variances were then computed for these cumulative N2O emissions, similar to all other measurements. 155 frames were permanently installed in the plots for a whole rainy season. The chambers (0.27 × 0.375 × 0.11 m) were made of 150 polyvinylchloride and equipped with a vent tube and a fan to homogenize gas inside them before gas sampling. The measured N2O emissions were evaluated at two levels of aggregation. First, as site means per measurement day (from the three replicates, similar to all other data) and second as cumulative emissions over the whole season, for which we first used the trapezoid method at the plot scale (Levy et al., 2017), specifically, the trapz function of R (Tuszynski, 2021). Site-scale means and i th t d f th l ti N O i i i il t ll th t 155 frames were permanently installed in the plots for a whole rainy season. The chambers (0.27 × 0.375 × 0.11 m) were made of 150 polyvinylchloride and equipped with a vent tube and a fan to homogenize gas inside them before gas sampling. The measured N2O emissions were evaluated at two levels of aggregation. First, as site means per measurement day (from the three replicates, similar to all other data) and second as cumulative emissions over the whole season, for which we first used the trapezoid method at the plot scale (Levy et al., 2017), specifically, the trapz function of R (Tuszynski, 2021). Site-scale means and variances were then computed for these cumulative N2O emissions, similar to all other measurements. 155 Furthermore, data on soil mineral N (Nmin), measured as NH4+ and NO3-, and measured moisture content at soil depths of 0-15 cm, were available from several measurement campaigns in the years 2012, 2013, 2018, 2019 and 2020. Finally, in the control and the 1.2 t C plots of the Calliandra, farmyard manure and maize stover treatments, continuous soil moisture measurements from sensors placed in each replicate at 10 cm soil depth (EC-5 Soil Moisture Sensor, Meter, Germany), were variances were then computed for these cumulative N2O emissions, similar to all other measurements. 155 Furthermore, data on soil mineral N (Nmin), measured as NH4+ and NO3-, and measured moisture content at soil depths of 0-15 cm, were available from several measurement campaigns in the years 2012, 2013, 2018, 2019 and 2020. Finally, in the control and the 1.2 t C plots of the Calliandra, farmyard manure and maize stover treatments, continuous soil moisture measurements from sensors placed in each replicate at 10 cm soil depth (EC-5 Soil Moisture Sensor, Meter, Germany), were Furthermore, data on soil mineral N (Nmin), measured as NH4+ and NO3-, and measured moisture content at soil depths of 0-15 cm, were available from several measurement campaigns in the years 2012, 2013, 2018, 2019 and 2020. 2.3.1 Model driving variables and model assumptions In Aludeka and Sidada, manual recordings of daily minimum and maximum temperature and precipitation 175 were available for all years from 2005 to 2017, during which high-quality weather stations were installed (Meter climate station, Meter Environment, Munich, Germany). These data gaps were filled by using the NASA POWER product (https: //power.larc.nasa.gov/docs/methodology/). A bias correction for the minimum and maximum temperature of NASA POWER precipitation. In Aludeka and Sidada, manual recordings of daily minimum and maximum temperature and precipitation 175 were available for all years from 2005 to 2017, during which high-quality weather stations were installed (Meter climate station, Meter Environment, Munich, Germany). These data gaps were filled by using the NASA POWER product (https: //power.larc.nasa.gov/docs/methodology/). A bias correction for the minimum and maximum temperature of NASA POWER 6 2.3.2 Initial model parameterization and selection of potentially sensitive parameters 195 The DayCent simulations were conducted at the site scale using averaged values across all three replicate plots for soil texture and SOC and soil N stocks, yield and aboveground biomass. This was done to reduce the computation time of the sensitivity y g p y analysis and calibration. Additionally, initial tests with the default DayCent parameterization showed that applying the model 205 to each experimental replicate individually did not lead to better agreement between measured and simulated values than a single simulation of the average soil parameters of the three replicates. Therefore, final model simulations were conducted with averaged values for soil parameters (i.e., texture, bulk density, soil pH) across the three replicates per site. For data used in model calibration and evaluation, the site-specific variance for each type of measurement was used as a measure of uncertainty analysis and calibration. Additionally, initial tests with the default DayCent parameterization showed that applying the model 205 to each experimental replicate individually did not lead to better agreement between measured and simulated values than a single simulation of the average soil parameters of the three replicates. Therefore, final model simulations were conducted with averaged values for soil parameters (i.e., texture, bulk density, soil pH) across the three replicates per site. For data used in model calibration and evaluation, the site-specific variance for each type of measurement was used as a measure of uncertainty analysis and calibration. Additionally, initial tests with the default DayCent parameterization showed that applying the model 205 to each experimental replicate individually did not lead to better agreement between measured and simulated values than a single simulation of the average soil parameters of the three replicates. Therefore, final model simulations were conducted with averaged values for soil parameters (i.e., texture, bulk density, soil pH) across the three replicates per site. For data used in model calibration and evaluation, the site-specific variance for each type of measurement was used as a measure of uncertainty of the measured data (specifically, the median variance per site and measurement type computed from the three replicates from 210 each time point of each treatment). Site-specific variances were used because a statistical analysis of the data in earlier work of the measured data (specifically, the median variance per site and measurement type computed from the three replicates from 210 each time point of each treatment). https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. data was performed, using a linear regression with measured data as dependent variable (y) and NASA POWER data as in- dependent variable (x). Specifically, the slope and intercept of the regression equation y = mx + b, were used to produce a 180 corrected estimate of these data. In our specific case, the slopes were not significantly different from 1, but intercepts (b) were significantly different from 0. The specific intercepts for maximum temperature were -0.3°C, -0.4°C, +3°C and +6°C for Embu, Machanga, Sidada and Aludeka, respectively. The intercepts for the minimum temperature were -0.25°C, -0.5°C, -3°C and +1°C for Embu, Machanga, Sidada, and Aludeka, respectively. For precipitation, no bias correction was done. p g g p y dependent variable (x). Specifically, the slope and intercept of the regression equation y = mx + b, were used to produce a 180 corrected estimate of these data. In our specific case, the slopes were not significantly different from 1, but intercepts (b) were significantly different from 0. The specific intercepts for maximum temperature were -0.3°C, -0.4°C, +3°C and +6°C for Embu, Machanga, Sidada and Aludeka, respectively. The intercepts for the minimum temperature were -0.25°C, -0.5°C, -3°C and +1°C for Embu, Machanga, Sidada, and Aludeka, respectively. For precipitation, no bias correction was done. The data on the hydraulic properties of the soil needed in DayCent (volumetric field capacity, wilting point, and saturated 185 hydraulic conductivity Ks) were calculated based on the soil texture measured at each site. We tested two pedotransfer functions to see which one provided a better fit: (1) the widely applied function of Saxton and Rawls (2006) and (2) the function of Hodnett and Tomasella (2002), which was specifically designed for tropical soils. Within the Hodnett and Tomasella (2002) equation, Ks was calculated using the Saxton and Rawls (2006) equation, with values of the water retention curve, α and n The data on the hydraulic properties of the soil needed in DayCent (volumetric field capacity, wilting point, and saturated 185 hydraulic conductivity Ks) were calculated based on the soil texture measured at each site. We tested two pedotransfer functions to see which one provided a better fit: (1) the widely applied function of Saxton and Rawls (2006) and (2) the function of Hodnett and Tomasella (2002), which was specifically designed for tropical soils. 2.3.2 Initial model parameterization and selection of potentially sensitive parameters 195 To parameterize the organic inputs, the mean lignin content and C/N ratio of the different organic materials across sites (Table A2) were used. This was justified because measurements were not available for all sites and years and because an analysis of variance of data from the years 2002, 2003, 2004, 2005 and 2006 for Embu and Machanga and from the year 2018 for all sites did not indicate any significant differences in lignin contents and C/N ratios between the sites or years. The C content in maize To parameterize the organic inputs, the mean lignin content and C/N ratio of the different organic materials across sites (Table A2) were used. This was justified because measurements were not available for all sites and years and because an analysis of variance of data from the years 2002, 2003, 2004, 2005 and 2006 for Embu and Machanga and from the year 2018 for all sites did not indicate any significant differences in lignin contents and C/N ratios between the sites or years. The C content in maize To parameterize the organic inputs, the mean lignin content and C/N ratio of the different organic materials across sites (Table A2) were used. This was justified because measurements were not available for all sites and years and because an analysis of variance of data from the years 2002, 2003, 2004, 2005 and 2006 for Embu and Machanga and from the year 2018 for all sites did not indicate any significant differences in lignin contents and C/N ratios between the sites or years. The C content in maize grain was assumed to be 42.5% throughout the simulation period. This was the mean value of measured grain C content across 200 sites (standard deviation 1.8%), which were available from the short rainy season 2018 and long rainy season 2019 (data not shown). The DayCent simulations were conducted at the site scale using averaged values across all three replicate plots for soil texture and SOC and soil N stocks, yield and aboveground biomass. This was done to reduce the computation time of the sensitivity grain was assumed to be 42.5% throughout the simulation period. This was the mean value of measured grain C content across 200 sites (standard deviation 1.8%), which were available from the short rainy season 2018 and long rainy season 2019 (data not shown). Within the Hodnett and Tomasella (2002) equation, Ks was calculated using the Saxton and Rawls (2006) equation, with values of the water retention curve, α and n The data on the hydraulic properties of the soil needed in DayCent (volumetric field capacity, wilting point, and saturated 185 hydraulic conductivity Ks) were calculated based on the soil texture measured at each site. We tested two pedotransfer functions to see which one provided a better fit: (1) the widely applied function of Saxton and Rawls (2006) and (2) the function of Hodnett and Tomasella (2002), which was specifically designed for tropical soils. Within the Hodnett and Tomasella (2002) equation, Ks was calculated using the Saxton and Rawls (2006) equation, with values of the water retention curve, α and n (van Genuchten, 1982), taken from Hodnett and Tomasella (2002), because their equation does not provide a way to estimate 190 Ks. In initial test simulations, we compared the observed versus soil moisture contents in the top 15 cm, for which continuous measurements were available from Sidada and Aludeka from 2018 to 2020. In this comparison, the pedotransfer functions of Hodnett and Tomasella (2002) showed better agreement between the measured and simulated soil moisture contents than Saxton and Rawls (2006) and were consequently used in the application of the model at the four LTE sites. Finally, the parameters for minimum and maximum values of nitrification and loss of nitrified N as N2O, were reformulated as maximum and the difference between assign the same C/N ratios and a constant ratio to the turnover of surface and soil SOM pools. Because the turnover rates of 230 the SOM pools are typically faster on the surface than in the soil, we defined a new parameter that represents the value ratio of the surface to the soil parameters (i.e., decX(2)/decX(1)). This allowed us to jointly evaluate the sensitivity and calibrate parameters related to surface and soil SOM pools without adding too much complexity. Finally, the parameters for minimum and maximum values of nitrification and loss of nitrified N as N2O, were reformulated as maximum and the difference between the minimum and maximum parameters (i.e., N2Oadjust_(max-min) and aneref(1)-anaref(2)). This was done to ensure that 235 maximum was always higher than the minimum value in order to avoid numerical problems. 2.3.2 Initial model parameterization and selection of potentially sensitive parameters 195 Site-specific variances were used because a statistical analysis of the data in earlier work 7 7 This resulted in a selection of 66 parameters (Table 1 and Table A3). Some of these parameters represented groups of the same type of parameters that can each be individually cali- 225 brated in DayCent, e.g., the "tillage multiplier" of SOM turnover can have different values for different SOM pools. However, because the tillage multipliers are usually the same for all SOM pools in the standard DayCent parameterization, we decided to have the same tillage multiplier value for active, slow, and litter pools. In addition, some of the parameters can have different values between the SOM pools of the surface and soil (for example, C / N ratios and turnover). For simplicity, we decided to assign the same C/N ratios and a constant ratio to the turnover of surface and soil SOM pools. Because the turnover rates of 230 the SOM pools are typically faster on the surface than in the soil, we defined a new parameter that represents the value ratio of the surface to the soil parameters (i.e., decX(2)/decX(1)). This allowed us to jointly evaluate the sensitivity and calibrate parameters related to surface and soil SOM pools without adding too much complexity. Finally, the parameters for minimum and maximum values of nitrification and loss of nitrified N as N2O, were reformulated as maximum and the difference between the minimum and maximum parameters (i.e., N2Oadjust_(max-min) and aneref(1)-anaref(2)). This was done to ensure that the 235 maximum was always higher than the minimum value in order to avoid numerical problems. most recent values from Gurung et al. (2021), who showed that older parameters overestimate N2O emissions. To identify 220 which model parameters to include in the global sensitivity analysis (see section 2.4) and in the model calibration, we screened the literature for recently conducted sensitivity analyzes of the DayCent model (Necpálová et al., 2015; Gurung et al., 2020) and additionally used the DayCent manual to identify and add further parameters of potential importance for the processes considered in our study (i.e., plant productivity and soil C and N cycle). This resulted in a selection of 66 parameters (Table 1 most recent values from Gurung et al. (2021), who showed that older parameters overestimate N2O emissions. To identify 220 which model parameters to include in the global sensitivity analysis (see section 2.4) and in the model calibration, we screened the literature for recently conducted sensitivity analyzes of the DayCent model (Necpálová et al., 2015; Gurung et al., 2020) and additionally used the DayCent manual to identify and add further parameters of potential importance for the processes considered in our study (i.e., plant productivity and soil C and N cycle). This resulted in a selection of 66 parameters (Table 1 and Table A3). Some of these parameters represented groups of the same type of parameters that can each be individually cali- 225 brated in DayCent, e.g., the "tillage multiplier" of SOM turnover can have different values for different SOM pools. However, because the tillage multipliers are usually the same for all SOM pools in the standard DayCent parameterization, we decided to have the same tillage multiplier value for active, slow, and litter pools. In addition, some of the parameters can have different values between the SOM pools of the surface and soil (for example, C / N ratios and turnover). For simplicity, we decided to and Table A3). Some of these parameters represented groups of the same type of parameters that can each be individually cali- 225 brated in DayCent, e.g., the "tillage multiplier" of SOM turnover can have different values for different SOM pools. However, because the tillage multipliers are usually the same for all SOM pools in the standard DayCent parameterization, we decided to have the same tillage multiplier value for active, slow, and litter pools. In addition, some of the parameters can have different values between the SOM pools of the surface and soil (for example, C / N ratios and turnover). For simplicity, we decided to assign the same C/N ratios and a constant ratio to the turnover of surface and soil SOM pools. Because the turnover rates of 230 the SOM pools are typically faster on the surface than in the soil, we defined a new parameter that represents the value ratio of the surface to the soil parameters (i.e., decX(2)/decX(1)). This allowed us to jointly evaluate the sensitivity and calibrate parameters related to surface and soil SOM pools without adding too much complexity. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. had shown that variance heterogeneity existed only between sites, but not between treatments at the same site (Laub et al., had shown that variance heterogeneity existed only between sites, but not between treatments at the same site (Laub et al., 2022 2023) had shown that variance heterogeneity existed only between sites, but not between treatments at the same site (Laub et al., 2022, 2023). The standard parameter values of the DayCent 2020 version were taken as initial model parameters. The exceptions were the decomposition parameters of the SOM pools. For these, we used updated estimates from a recent Bayesian calibration (Gurung 215 et al., 2020) that included data from most of the well-known long-term experiments in Europe and the US (and textures from 15-50% clay). To our knowledge Gurung et al. (2020) provide the most up-to-date decomposition parameters, and hence, we assigned the median of reported parameter values for each SOM pool as initial parameter values in our model simulations. Furthermore, for the parameters determining the minimum and maximum proportion of nitrified N lost as N2O, we used the The standard parameter values of the DayCent 2020 version were taken as initial model parameters. The exceptions were the decomposition parameters of the SOM pools. For these, we used updated estimates from a recent Bayesian calibration (Gurung 215 et al., 2020) that included data from most of the well-known long-term experiments in Europe and the US (and textures from 15-50% clay). To our knowledge Gurung et al. (2020) provide the most up-to-date decomposition parameters, and hence, we assigned the median of reported parameter values for each SOM pool as initial parameter values in our model simulations. Furthermore, for the parameters determining the minimum and maximum proportion of nitrified N lost as N2O, we used the most recent values from Gurung et al. (2021), who showed that older parameters overestimate N2O emissions. To identify 220 which model parameters to include in the global sensitivity analysis (see section 2.4) and in the model calibration, we screened the literature for recently conducted sensitivity analyzes of the DayCent model (Necpálová et al., 2015; Gurung et al., 2020) and additionally used the DayCent manual to identify and add further parameters of potential importance for the processes considered in our study (i.e., plant productivity and soil C and N cycle). 2.4 Global sensitivity analysis The parameters that had very small, small and moderate ranges were varied by ±10, 25 and 50% from the default parameter value, respectively. For parameters with large and very large ranges, the upper/lower boundaries were the default parameter values multiplied/divided by 3 and 270 10, respectively. Additionally, we assumed that the maize parameters of the second highest production level (C5 in DayCent) would best represent the production levels in the experiment. The parameter sensitivity was independently determined for the mean maize grain yield, aboveground biomass, as well as for the SOC and soil N stocks at the end of the simulation period. with large and very large ranges, the upper/lower boundaries were the default parameter values multiplied/divided by 3 and 270 10, respectively. Additionally, we assumed that the maize parameters of the second highest production level (C5 in DayCent) would best represent the production levels in the experiment. The parameter sensitivity was independently determined for the mean maize grain yield, aboveground biomass, as well as for the SOC and soil N stocks at the end of the simulation period. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. of these systems. Therefore, the duration of cropping systems after native vegetation was adjusted at each site so that the measured initial SOC stocks corresponded to the simulated SOC stocks at the start of the experiment. Additionally, to achieve suitable levels of soil N stocks after the spin-up, the maximum C/N ratio of the SOM pools had to be increased. It was increased from 14 to 20 for the active SOM pool and from 8 to 13 for the passive SOM pool (parameters varat12&11(1,1) and varat3(1,1), respectively). Due to computational time constraints and to ensure a match between simulated and observed initial SOC and soil N levels, the spin-up and site history simulations were not included in the sensitivity analysis and Bayesian calibration. of these systems. Therefore, the duration of cropping systems after native vegetation was adjusted at each site so that the measured initial SOC stocks corresponded to the simulated SOC stocks at the start of the experiment. Additionally, to achieve suitable levels of soil N stocks after the spin-up, the maximum C/N ratio of the SOM pools had to be increased. It was increased from 14 to 20 for the active SOM pool and from 8 to 13 for the passive SOM pool (parameters varat12&11(1,1) and varat3(1,1), respectively). Due to computational time constraints and to ensure a match between simulated and observed initial SOC and 0 soil N levels, the spin-up and site history simulations were not included in the sensitivity analysis and Bayesian calibration. 250 2.3.3 Spin-up and site history simulation to initialize SOC and soil N contents As is standard practice in DayCent, the initialization of SOM pools was conducted through a spin-up run, which was followed by a simulation of the history of the site before experiment establishment based on the knowledge of site managers. The spin-up has the aim to initialize the SOM pools to equilibrium using the typical input of biomass of the native vegetation 240 type. The type of native vegetation for each site was determined from an available potential vegetation map (Kamoni et al., 2007) and confirmed by site managers as tropical evergreen forest in Embu, dry savanna in Machanga, and humid savanna in Sidada and Aludeka. A 2000-year spin-up simulation was sufficient to reach a steady state of SOM pools. Site managers had a good knowledge of the type of historical cropping systems, e.g., arable vs. grasslands, types of crop rotation (e.g., maize monoculture vs. crop rotation with legumes), manure inputs and management, but without detailed information on the duration 245 As is standard practice in DayCent, the initialization of SOM pools was conducted through a spin-up run, which was followed by a simulation of the history of the site before experiment establishment based on the knowledge of site managers. The spin-up has the aim to initialize the SOM pools to equilibrium using the typical input of biomass of the native vegetation 240 type. The type of native vegetation for each site was determined from an available potential vegetation map (Kamoni et al., 2007) and confirmed by site managers as tropical evergreen forest in Embu, dry savanna in Machanga, and humid savanna in Sidada and Aludeka. A 2000-year spin-up simulation was sufficient to reach a steady state of SOM pools. Site managers had a good knowledge of the type of historical cropping systems, e.g., arable vs. grasslands, types of crop rotation (e.g., maize monoculture vs. crop rotation with legumes), manure inputs and management, but without detailed information on the duration 245 8 8 2.4 Global sensitivity analysis To reduce the dimensionality of the calibration of the model, we performed a parameter screening (van Oijen, 2020). For this purpose, a global sensitivity analysis was conducted to quantify the relative importance of different model parameters to the relevant model outputs regarding our study focus on maize yield and ISFM greenhouse gas mitigation potential. The goal was 255 to fix less influential model parameters to their default values, reducing the computational cost for performing the consecutive Bayesian calibration (see section 2.5). Global sensitivity analysis was performed using the Sobol method (Saltelli, 2002a, b), which allows for the estimation of the proportion of variance in the model outputs that is explained by each model parameter, considering the interaction terms of first-order and higher-order (Gurung et al., 2020). The "sensitivity" package (function sobolSalt; Iooss et al., 2021) of R version 4.0 (R Core Team, 2020) was applied. This function implements a simultaneous 260 Monte Carlo estimation of first-order and total-effect Sobol indices. The computational cost is N(p + 2) model runs, N being the dimension of the two matrices to construct the Sobol sequence, p being the number of parameters (66 in our case). Our tests indicated similar results for N = 500/1000, so we chose a dimension of 1000. The preselection of model parameters to include (Tables 1 and A3) is described above. Independent uniform prior distributions were used for the global sensitivity analysis, with the upper and lower parameter boundaries centered around the default parameter value obtained as described 265 below. We based the global sensitivity analysis parameter ranges on previous sensitivity analyses (e.g. Necpálová et al., 2015; Gurung et al., 2020), on plausible ranges reported in the DayCent manual and on how the parameters varied between different maize parameterizations. The parameters were then grouped by how large the ranges were. The parameters that had very small, small and moderate ranges were varied by ±10, 25 and 50% from the default parameter value, respectively. For parameters analysis, with the upper and lower parameter boundaries centered around the default parameter value obtained as described 265 below. We based the global sensitivity analysis parameter ranges on previous sensitivity analyses (e.g. Necpálová et al., 2015; Gurung et al., 2020), on plausible ranges reported in the DayCent manual and on how the parameters varied between different maize parameterizations. The parameters were then grouped by how large the ranges were. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table 1. DayCent model parameters and possible range of values. Displayed are parameters considered for calibration due to total sensitivity index > 2.5% (top) and with a total sensitivity index > 1% (bottom). The remainder of parameters (<1%) can be found in the supplementary (Table A3). Table 1. DayCent model parameters and possible range of values. Displayed are parameters considered for calibration due to total sensitivity index > 2.5% (top) and with a total sensitivity index > 1% (bottom). The remainder of parameters (<1%) can be found in the supplementary (Table A3). Possible ranges Initial Lower Upper Calibrated Parameter Description of values Units value value value value+ Included in calibration (total sensitivity >2.5%) himax Maximum harvest index for maize moderate g g-1 (C) 0.40 0.20 0.60 0.52 ppdf(1) Optimum temperature for growth of maize very small °C 30.00 27.00 33.00 29.10 ppdf(2) Maximum temperature for growth of maize very small °C 45.00 40.50 49.50 48.15 prdx(1) Potential aboveground production of maize large g C m-2 langley-1 2.25 0.75 6.75 2.45 clteff(1,2&4) Tillage multiplier for SOM turnover large unitless 10.00 3.33 30.00 18.10 aneref(3) Min. impact of soil anaerobiosis on SOM turnover fixed unitless 1.00 0.20 1.00 0.73 dec4 Max. turnover rate of passive SOM pool very large g g-1 yr-1 0.0035 0.00035 0.035 0.033 dec5(2) Max. turnover rate of slow SOM pool large g g-1 yr-1 0.10 0.03 0.30 0.19 fwloss(4) Scaling factor potential evapotranspiration moderate unitless 0.75 0.38 1.125 1.02 pmco2(1&2) C lost as CO2 with metabolic litter turnover* fixed g g-1 (C) 0.54 0.30 0.95 0.62 ps1co2(1&2) C lost as CO2 with structural litter turnover* fixed g g-1 (C) 0.50 0.40 0.99 0.89 & rsplig Not included in calibration (total sensitivity <2.5% & > 1% ) frtc(1) C allocated to roots at planting, without stress small fraction of NPP 0.50 0.38 0.62 - frtc(3) Time after planting at which maturity is reached small number of days 90.00 67.50 112.50 - pramn(1,2) Min. aboveground C/N ratio at maturity small C/N ratio 62.50 46.88 78.12 - hiwsf Max. harvest index reduction with water stress moderate g g-1 (C) 0.60 0.30 0.90 - teff(1) Temperature inflection point (effect on SOM turnover) moderate unitless 17.05 8.53 25.58 - varat21&22(2,1) Min. 2.5 Combined Bayesian calibration of plant and soil model parameters Joint Bayesian calibration of the sensitive DayCent parameters was performed using all available data on maize grain yield, 275 aboveground biomass, and SOC stocks. The main reason for only using these data was that the yields, SOC stocks, and their trade-offs were the focus of this study. A second, technical reason, was that the creation and readout of daily simulations outputs, needed to match simulated and measured soil moisture content, mineral N content and /N2O fluxes, slowed down the 9 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. While the prior, p(θ), is chosen based on previous knowledge of the model parameters, the likelihood function, p(D,M|θ), measures how well the model and the data match. In practice it is derived for a given set of parameter sampled from the prior, by running and evaluating the model using the measured data, the simulated counterpart and the variance-covariance matrix 290 of the data We used the median variances per site for each type of measurement (computed from the three replicates) as the While the prior, p(θ), is chosen based on previous knowledge of the model parameters, the likelihood function, p(D,M|θ), measures how well the model and the data match. In practice it is derived for a given set of parameter sampled from the prior, by running and evaluating the model using the measured data, the simulated counterpart and the variance-covariance matrix 290 of the data. We used the median variances per site for each type of measurement (computed from the three replicates) as the diagonal elements of the variance-covariance matrix. Due to the large number of observations and the mostly balanced dataset, the off-diagonal elements were set to 0. While the prior, p(θ), is chosen based on previous knowledge of the model parameters, the likelihood function, p(D,M|θ), measures how well the model and the data match. In practice it is derived for a given set of parameter sampled from the prior, by running and evaluating the model using the measured data, the simulated counterpart and the variance-covariance matrix 290 of the data. We used the median variances per site for each type of measurement (computed from the three replicates) as the diagonal elements of the variance-covariance matrix. Due to the large number of observations and the mostly balanced dataset, the off-diagonal elements were set to 0. The sampling importance resampling method, which was used in this study, is a direct fo The sampling importance resampling method, which was used in this study, is a direct form of Bayesian calibration, which has recently been used by Gurung et al. (2020), to calibrate the parameters of the SOM module of DayCent using a large 295 collection of temperate long-term experiments. C/N ratio for material entering slow SOM pool small C/N 12.00 9.00 15.00 - basef Soil water of bottom layer lost via base flow moderate fraction H2O 0.30 0.15 0.45 - N2Oadjust_max Proportion of nitrified N that is lost as N2O large g g-1 (N) 0.004 0.0013 0.012 - MaxNitAmt Maximum daily nitrification amount large g N m-2 0.40 0.13 1.20 - *(1 - microbial carbon use efficiency); +highest likelihood parameter set across all four sites whole calibration by a factor of 5, which made Bayesian calibration unfeasible (one iteration would have taken more than three whole calibration by a factor of 5, which made Bayesian calibration unfeasible (one iteration would have taken more than three months on our virtual machine with 64 cores). Following (Gurung et al., 2020), model parameters that had a total sensitivity 280 index of at least 2.5% for either yield, aboveground biomass, or SOC were considered influential and thus were altered in Bayesian calibration. This resulted in 11 parameters to be calibrated (Table 1). Bayesian calibration is a probabilistic inverse modeling/data assimilation technique which is used to estimate the joint pos- months on our virtual machine with 64 cores). Following (Gurung et al., 2020), model parameters that had a total sensitivity 280 index of at least 2.5% for either yield, aboveground biomass, or SOC were considered influential and thus were altered in Bayesian calibration. This resulted in 11 parameters to be calibrated (Table 1). Bayesian calibration is a probabilistic inverse modeling/data assimilation technique, which is used to estimate the joint pos- terior distribution of model parameters (θ) given the measured data (D) and the model structure (M), expressed as p(θ|D,M). months on our virtual machine with 64 cores). Following (Gurung et al., 2020), model parameters that had a total sensitivity 280 index of at least 2.5% for either yield, aboveground biomass, or SOC were considered influential and thus were altered in Bayesian calibration. This resulted in 11 parameters to be calibrated (Table 1). Bayesian calibration is a probabilistic inverse modeling/data assimilation technique, which is used to estimate the joint pos- terior distribution of model parameters (θ) given the measured data (D) and the model structure (M), expressed as p(θ|D,M). It uses the proportionality form of Bayes‘ theorem, where p(θ|D,M) is proportional to the prior belief about model parameters, 285 p(θ) times the likelihood function, p(D,M|θ): p(θ|D,M) ∝p(θ) ∗p(D,M|θ) (2) 10 It samples the prior by running the model for a large sample of parameter sets of size I from the prior, computing the likelihood for each sample, and filtering the prior based on importance weights w(θz) has recently been used by Gurung et al. (2020), to calibrate the parameters of the SOM module of DayCent using a large 295 collection of temperate long-term experiments. It samples the prior by running the model for a large sample of parameter sets of size I from the prior, computing the likelihood for each sample, and filtering the prior based on importance weights w(θz) (3) (3) where p(D,M|θz) is the likelihood function of the zth parameter set and w(θz) is the corresponding importance weight. It where p(D,M|θz) is the likelihood function of the zth parameter set and w(θz) is the corresponding importance weight. It is consistent with the proportionality form of Bayes‘ theorem in that it uses the importance weights w(θz) as probabilities for 300 sampling from the prior, without replacement, to derive the posterior. Overall, a total of 200000 simulations were performed, from which, as in 0.1% (200) of the parameter sets were sampled to derive the posterior distribution through resampling (Gurung et al., 2020). It was assured that this number of simulations was sufficient by splitting the simulations into two halves and visually assessing the similarity of derived posteriors for these subsets. In our experience, the sampling importance is consistent with the proportionality form of Bayes‘ theorem in that it uses the importance weights w(θz) as probabilities for 300 sampling from the prior, without replacement, to derive the posterior. Overall, a total of 200000 simulations were performed, from which, as in 0.1% (200) of the parameter sets were sampled to derive the posterior distribution through resampling (Gurung et al., 2020). It was assured that this number of simulations was sufficient by splitting the simulations into two halves and visually assessing the similarity of derived posteriors for these subsets. In our experience, the sampling importance resampling algorithm is ideal for DayCent, which is prone to model crash when using inappropriate parameter combinations. 305 This method does not depend on chains, but rather on model runs that are independent of each other. This means that an erroneous run does not stop the algorithm, as would be the case in chain-dependent methods such as Markov chain Monte Carlo. In addition, this strategy allows for an efficient cross-validation of the posterior parameter set. To evaluate the model by comparing measured vs. modeled data, we conducted a leave-one-site-out cross-validation. This resampling algorithm is ideal for DayCent, which is prone to model crash when using inappropriate parameter combinations. 305 This method does not depend on chains, but rather on model runs that are independent of each other. This means that an erroneous run does not stop the algorithm, as would be the case in chain-dependent methods such as Markov chain Monte Carlo. In addition, this strategy allows for an efficient cross-validation of the posterior parameter set. To evaluate the model by comparing measured vs. modeled data, we conducted a leave-one-site-out cross-validation. This To evaluate the model by comparing measured vs. modeled data, we conducted a leave-one-site-out cross-validation. This means that each of the sites is left out one by one for an independent evaluation, while the other three sites were used to compute 310 the resampling weights and derive the posterior parameter distributions. Hence, the evaluation is representative of up-scaling exercises of the DayCent model to other sites, because it involves evaluation of the model performance across different sites. Here, the SIR algorithm was also advantageous. We stored the model results for each parameter set by site, which meant that the cross-evaluation could be done by a simple filter by site, without rerunning the model for each iteration. In contrast to model means that each of the sites is left out one by one for an independent evaluation, while the other three sites were used to compute 310 the resampling weights and derive the posterior parameter distributions. Hence, the evaluation is representative of up-scaling exercises of the DayCent model to other sites, because it involves evaluation of the model performance across different sites. Here, the SIR algorithm was also advantageous. We stored the model results for each parameter set by site, which meant that the cross-evaluation could be done by a simple filter by site, without rerunning the model for each iteration. In contrast to model means that each of the sites is left out one by one for an independent evaluation, while the other three sites were used to compute 310 the resampling weights and derive the posterior parameter distributions. Hence, the evaluation is representative of up-scaling exercises of the DayCent model to other sites, because it involves evaluation of the model performance across different sites. Here, the SIR algorithm was also advantageous. We stored the model results for each parameter set by site, which meant that the cross-evaluation could be done by a simple filter by site, without rerunning the model for each iteration. In contrast to model cross-validation, the parameter posterior distributions are not displayed by site. They are displayed for all four combined sites; 315 hence the full dataset without leaving any site out was used to derive them. This was done to present the most representative posterior distributions and to make efficient use of all available data. cross-validation, the parameter posterior distributions are not displayed by site. They are displayed for all four combined sites; 315 hence the full dataset without leaving any site out was used to derive them. This was done to present the most representative posterior distributions and to make efficient use of all available data. cross-validation, the parameter posterior distributions are not displayed by site. They are displayed for all four combined sites; 315 hence the full dataset without leaving any site out was used to derive them. This was done to present the most representative posterior distributions and to make efficient use of all available data. 11 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 2.6 Model evaluation Here, ¯P y is the mean predicted value of the y-th measurement type, b the slope of the regression of P on O and r the 0 correlation coefficient between O and P. The indicators LC, SB and NU show the nature of model errors, that is, a high LC shows that it is mostly random, a high SB a systematic bias, while a high NU shows issues of model sensitivity. Here, ¯P y is the mean predicted value of the y-th measurement type, b the slope of the regression of P on O and r the 0 correlation coefficient between O and P. The indicators LC, SB and NU show the nature of model errors, that is, a high LC shows that it is mostly random, a high SB a systematic bias, while a high NU shows issues of model sensitivity. 330 2.6 Model evaluation We used the following standard model evaluation statistics (Loague and Green, 1991): MSEy = 1 n n X z=1 (Oyz −Pyz)2 320 (4) RMSEy = p MSEy (5) EFy = 1 − Pn z=1(Oyz −Pyz)2 Pn z=1(Oyz −¯Oy)2 (6) EFy = 1 − Pn z=1(Oyz −Pyz)2 Pn z=1(Oyz −¯Oy)2 (6) Here, MSEy is the mean-squared-error and RMSE is its root. EFy is the Nash-Sutcliffe modeling efficiency. Oyz is the measured value of the z-th measurement of the y-th type of measurement, ¯Oy the mean of the y-th type of measurement and Pyz the simulated value corresponding to Oyz. We further divided MSEy into squared bias (SB), nonunity slope (NU) and 25 lack of correlation (LC), as suggested by Gauch et al. (2003). We expressed them as a percentage of the MSEy: Here, MSEy is the mean-squared-error and RMSE is its root. EFy is the Nash-Sutcliffe modeling efficiency. Oyz is the measured value of the z-th measurement of the y-th type of measurement, ¯Oy the mean of the y-th type of measurement and Pyz the simulated value corresponding to Oyz. We further divided MSEy into squared bias (SB), nonunity slope (NU) and 325 lack of correlation (LC), as suggested by Gauch et al. (2003). We expressed them as a percentage of the MSEy: SBy(%) = ( ¯Oy −¯Py)2 MSEy ∗100 (7) NUy(%) = (1 −by)2 ∗( Pn z=1(O2 yz) n ) MSEy ∗100 (8) LCy(%) = (1 −ry)2 ∗( Pn z=1(P 2 yz) n ) MSEy ∗100 (9) SBy(%) = ( ¯Oy −¯Py)2 MSEy ∗100 NUy(%) = (1 −by)2 ∗( Pn z=1(O2 yz) n ) MSEy ∗100 LCy(%) = (1 −ry)2 ∗( Pn z=1(P 2 yz) n ) MSEy ∗100 SBy(%) = ( ¯Oy −¯Py)2 MSEy ∗100 (7) NUy(%) = (1 −by)2 ∗( Pn z=1(O2 yz) n ) MSEy ∗100 (8) (9) Here, ¯P y is the mean predicted value of the y-th measurement type, b the slope of the regression of P on O and r the correlation coefficient between O and P. The indicators LC, SB and NU show the nature of model errors, that is, a high LC shows that it is mostly random, a high SB a systematic bias, while a high NU shows issues of model sensitivity. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Aboveground biomass Grain yield SOC stock Soil N stock 0.0 0.4 0.0 0.4 0.0 0.4 0.0 0.4 hiwsf basef varat21(2,1)&22(2,1) MaxNitAmt himax N2Oadjust_max frtc(3) pramn(1,2) teff(1) frtc(1) ps1co2(1&2)&rsplig aneref(3) fwloss(4) ppdf(2) ppdf(1) dec4 clteff(1,2&4) dec5(2) pmco2(1&2) prdx(1) Sobol sensitivity index Parameters 1st Order Total Figure 1. Results of the uncertainty-based global sensitivity analysis of the most relevant DayCent model parameters. Parameter sensitivity was independently determined for the mean maize aboveground biomass, grain yield, and SOC and soil N stocks at the end of the simulation period. Only parameters with a Sobol sensitivity index >1% are displayed. Aboveground biomass Grain yield SOC stock Soil N stock 0.0 0.4 0.0 0.4 0.0 0.4 0.0 0.4 hiwsf basef varat21(2,1)&22(2,1) MaxNitAmt himax N2Oadjust_max frtc(3) pramn(1,2) teff(1) frtc(1) ps1co2(1&2)&rsplig aneref(3) fwloss(4) ppdf(2) ppdf(1) dec4 clteff(1,2&4) dec5(2) pmco2(1&2) prdx(1) Sobol sensitivity index Parameters 1st Order Total Figure 1. Results of the uncertainty-based global sensitivity analysis of the most relevant DayCent model parameters. Parameter sensitivity Figure 1. Results of the uncertainty-based global sensitivity analysis of the most relevant DayCent model parameters. Parameter sensitivity was independently determined for the mean maize aboveground biomass, grain yield, and SOC and soil N stocks at the end of the simulation period. Only parameters with a Sobol sensitivity index >1% are displayed. Here, ∆SOC is the change in SOC content (kg C ha-1 yr-1), N2O the cumulative N2O flux (kg N2O ha-1 yr-1). The CH4 oxidation capacity was not considered, because it usually makes a very limited contribution to GWP in rainfed maize cropping 340 systems and we did not have data to evaluate the reliability of this simulated flux. In addition to GWP we calculated yield- scaled GWP (in CO2eq kg-1 maize grain yield) by dividing the cumulative GWP over the entire simulation period by cumulative simulated yields (dry matter base). Here, ∆SOC is the change in SOC content (kg C ha-1 yr-1), N2O the cumulative N2O flux (kg N2O ha-1 yr-1). The CH4 oxidation capacity was not considered, because it usually makes a very limited contribution to GWP in rainfed maize cropping 340 systems and we did not have data to evaluate the reliability of this simulated flux. In addition to GWP we calculated yield- scaled GWP (in CO2eq kg-1 maize grain yield) by dividing the cumulative GWP over the entire simulation period by cumulative simulated yields (dry matter base). 2.7 Net global warming potential To compare different ISFM treatments in terms of their greenhouse gas emissions, their net global warming potential (GWP) on To compare different ISFM treatments in terms of their greenhouse gas emissions, their net global warming potential (GWP) on a yearly basis (CO2eq ha-1 yr-1) was derived from the outputs over the whole simulation period. It was calculated from changes 335 in the SOC content and cumulative emissions of N2O using 100-year time horizon of global warming potentials (Necpalova et al., 2018): a yearly basis (CO2eq ha-1 yr-1) was derived from the outputs over the whole simulation period. It was calculated from changes 335 in the SOC content and cumulative emissions of N2O using 100-year time horizon of global warming potentials (Necpalova et al., 2018): GWP = 44 12 ∗∆SOC + 265 ∗N2O (10) 12 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. a suitable selection of parameters for model calibration (Gurung et al., 2020). The parameters that turned out to be the most sensitive, with a Sobol total sensitivity index >10% for at least one type of measurement were radiation use efficiency (prdx(1); 350 for all measurement types); the optimal and maximum temperature for maize growth (ppdf(1) and ppdf(2), respectively; only for grain yield of maize and aboveground biomass), and maximum harvest index (himax; only for crop yield). Further, the turnover rate of the slow and passive SOM pools (dec5(2) and dec4, respectively; only for SOC and soil N), the decomposition multiplier after tillage events (clteff(1,2&4); only for SOC and soil N) and the fraction lost as CO2 upon metabolic litter pool turnover (pmco2(1&2), i.e., 1 - microbial carbon use efficiency (CUE); only for SOC and soil N) belonged to the most 355 sensitive model parameters. The parameters of further importance, with a Sobol total sensitivity index <10% and >2.5%, were the minimum value for the impact of anaerobic soil conditions (aneref(3); only for SOC and soil N), the scaling factor for potential evapotranspiration (fwloss(4); only for maize grain yield), and the fraction lost as CO2 upon structural litter and lignin turnover (ps1co(1&2)&resplig, i.e., 1-CUE; only for SOC and soil N). The fact that the Sobol 1st order and total sensitivity pool turnover (pmco2(1&2), i.e., 1 - microbial carbon use efficiency (CUE); only for SOC and soil N) belonged to the most 355 sensitive model parameters. The parameters of further importance, with a Sobol total sensitivity index <10% and >2.5%, were the minimum value for the impact of anaerobic soil conditions (aneref(3); only for SOC and soil N), the scaling factor for potential evapotranspiration (fwloss(4); only for maize grain yield), and the fraction lost as CO2 upon structural litter and lignin turnover (ps1co(1&2)&resplig, i.e., 1-CUE; only for SOC and soil N). The fact that the Sobol 1st order and total sensitivity indexes were similar for most parameters suggested only a limited number of interactions between the parameters identified 360 by the global sensitivity analysis. 3.1 Most sensitive DayCent parameters 345 The results of the global sensitivity analysis showed that of the 66 model parameters included, only 20 parameters had a Sobol total sensitivity index >1% for either maize grain yield, aboveground biomass, SOC or soil N stocks (Fig. 1). Of these, only 11 parameters had a Sobol total sensitivity index >2.5%, a threshold that captures the most influential parameters and represents 13 3.2 Posterior parameter distributions from the Bayesian model calibration Following the global sensitivity analysis, 11 selected parameters were calibrated using the same ranges of possible values as defined for the sensitivity analysis. Four parameters were fully constrained by the Bayesian calibration, one was partly Following the global sensitivity analysis, 11 selected parameters were calibrated using th as defined for the sensitivity analysis. Four parameters were fully constrained by the Ba Following the global sensitivity analysis, 11 selected parameters were calibrated using the same ranges of possible values as defined for the sensitivity analysis. Four parameters were fully constrained by the Bayesian calibration, one was partly constrained and tended towards the upper boundary. Four showed a tendency, but values from the whole prior range were 365 present in the posterior distribution. Finally, two showed almost no difference to the uniform prior distribution (Fig. 2). Very clearly constrained were the potential maximum maize production and the maximum maize harvest index (2.5 g C m-2 langley-1 for prdx(1) and 0.48 g g-1 for himax), with values slightly higher than the default values and somewhere in between the maize with the highest and second highest production levels (default DayCent maize parameters, named C6 and C5). Furthermore, the scaling factor for potential evapotranspiration was clearly constrained and slightly higher than the default value (fwloss(4), 370 0.9 vs 0.75). The turnover rate of the slow SOM pool was also clearly constrained, but was centered around a value twice the default (0.2 vs 0.1 g g-1 yr-1 for dec5(2)). The turnover rate of the passive SOM pool was clearly constrained at the lower end of possible values, it was centered around a much higher value than the default value (0.03 vs 0.0035 g g-1 yr-1 for dec4) and tended towards the upper boundary. A test with even broader ranges (up to 0.1 g g-1 yr-1) showed that the value around the scaling factor for potential evapotranspiration was clearly constrained and slightly higher than the default value (fwloss(4), 370 0.9 vs 0.75). The turnover rate of the slow SOM pool was also clearly constrained, but was centered around a value twice the default (0.2 vs 0.1 g g-1 yr-1 for dec5(2)). The turnover rate of the passive SOM pool was clearly constrained at the lower end of possible values, it was centered around a much higher value than the default value (0.03 vs 0.0035 g g-1 yr-1 for dec4) and tended towards the upper boundary. 3.2 Posterior parameter distributions from the Bayesian model calibration A test with even broader ranges (up to 0.1 g g-1 yr-1) showed that the value around 0.03 g g-1 yr-1 was in fact the center of the posterior distribution of this parameter (Fig. A1), but allowing the parameters 375 to vary that much reduced the model performance to unfeasible levels (model output not shown). The optimal production temperature for maize (ppdf(1)) tended to have values lower than the default value, while the opposite was true for the maximum production temperature (ppdf(2)). Also the parameters representing CO2 loss upon turnover of metabolic and structural litter pools (pmco2(1&2) and ps1co2(1&2)&rsplig) tended towards higher values than the default values of these parameters (i.e., 0.03 g g-1 yr-1 was in fact the center of the posterior distribution of this parameter (Fig. A1), but allowing the parameters 375 to vary that much reduced the model performance to unfeasible levels (model output not shown). The optimal production temperature for maize (ppdf(1)) tended to have values lower than the default value, while the opposite was true for the maximum production temperature (ppdf(2)). Also the parameters representing CO2 loss upon turnover of metabolic and structural litter pools (pmco2(1&2) and ps1co2(1&2)&rsplig) tended towards higher values than the default values of these parameters (i.e., lower carbon use efficiencies, because the parameters represent 1-CUE), but they were not clearly constrained. Finally, the 380 parameters representing the increase of the SOM pools turnover after tillage (clteff(1,2,&4)) and the maximum rate limitation of soil under anaerobic conditions (aneref(3)), were poorly constrained. lower carbon use efficiencies, because the parameters represent 1-CUE), but they were not clearly constrained. Finally, the 380 parameters representing the increase of the SOM pools turnover after tillage (clteff(1,2,&4)) and the maximum rate limitation of soil under anaerobic conditions (aneref(3)), were poorly constrained. 14 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 2. Prior compared to the posterior model parameter distribution resulting from the uncertainty-based Bayesian model calibration of DayCent. Dashed vertical lines represent the values of the default parameter set. The posterior distributions are based on all four study sites combined. For the description of the parameters see Table 1. Figure 2. Prior compared to the posterior model parameter distribution resulting from the uncertainty-based Bayesian model calibration of DayCent. Dashed vertical lines represent the values of the default parameter set. 3.2 Posterior parameter distributions from the Bayesian model calibration The posterior distributions are based on all four study sites combined. For the description of the parameters see Table 1. Only a few strong correlations existed between the parameters of the posterior parameter set (Fig. A2). Namely, there was a strong positive correlation (r = 0.63) between the potential maximum production of maize (prdx(1)) and the optimal temperature for maize growth (ppdf(1)), the latter also being positively correlated with the maximum temperature for maize 5 growth (ppdf(2); r = 0.45). Furthermore, there was a negative correlation (r = -0.43) between the effect of tillage on SOM turnover and the amount of CO2 loss upon turnover of the metabolic litter pool (pmco2(1&2)). The other correlations of the parameters were weak (i.e., below ± 0.4) and therefore were of low importance. Only a few strong correlations existed between the parameters of the posterior parameter set (Fig. A2). Namely, there was a strong positive correlation (r = 0.63) between the potential maximum production of maize (prdx(1)) and the optimal temperature for maize growth (ppdf(1)), the latter also being positively correlated with the maximum temperature for maize 385 growth (ppdf(2); r = 0.45). Furthermore, there was a negative correlation (r = -0.43) between the effect of tillage on SOM turnover and the amount of CO2 loss upon turnover of the metabolic litter pool (pmco2(1&2)). The other correlations of the parameters were weak (i.e., below ± 0.4) and therefore were of low importance. temperature for maize growth (ppdf(1)), the latter also being positively correlated with the maximum temperature for maize 385 growth (ppdf(2); r = 0.45). Furthermore, there was a negative correlation (r = -0.43) between the effect of tillage on SOM turnover and the amount of CO2 loss upon turnover of the metabolic litter pool (pmco2(1&2)). The other correlations of the parameters were weak (i.e., below ± 0.4) and therefore were of low importance. 3.3 Simulation of maize grain yields and aboveground biomass at harvest Although the overall variation of maize grain yields between sites and treatments could be captured with the set of default 390 model parameters, the comparison of model results obtained with the default parameters compared to the set of parameters chosen for each site by leave-one-site-out cross-validation showed that the Bayesian calibration significantly improved the fit of the model for both maize grain yield and aboveground biomass (Fig. 3). The calibration improved the simulation of grain yield for all sites and for aboveground biomass for all sites except Machanga (Fig. A9). It should be noted that despite excluding the Although the overall variation of maize grain yields between sites and treatments could be captured with the set of default 390 model parameters, the comparison of model results obtained with the default parameters compared to the set of parameters chosen for each site by leave-one-site-out cross-validation showed that the Bayesian calibration significantly improved the fit of the model for both maize grain yield and aboveground biomass (Fig. 3). The calibration improved the simulation of grain yield for all sites and for aboveground biomass for all sites except Machanga (Fig. A9). It should be noted that despite excluding the evaluation site in the calibration step, the calibrated model was unbiased and errors mostly random for both yield (i.e., lack of 395 correlation (LC) increased from 90% to 97%) and aboveground biomass (LC increased from 71% to 91%). Furthermore, while DayCent was inclined to overestimate the lowest and underestimate the highest values of yields and aboveground biomass, the mean yields and aboveground biomass per treatment throughout the simulation period were sim- ulated well for most treatments (Fig. A4). The exception to this was that DayCent poorly distinguished the mean yields and evaluation site in the calibration step, the calibrated model was unbiased and errors mostly random for both yield (i.e., lack of 395 correlation (LC) increased from 90% to 97%) and aboveground biomass (LC increased from 71% to 91%). Furthermore, while DayCent was inclined to overestimate the lowest and underestimate the highest values of yields and aboveground biomass, the mean yields and aboveground biomass per treatment throughout the simulation period were sim- ulated well for most treatments (Fig. A4). https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. y = c(1.297) + c(0.5703) ⋅x, r2 = 0.403 EF: 0.333; RMSE: 1.842 SB(%): 0; NU(%): 10; LC(%): 90 0 5 10 0 5 10 Measured yield (t ha−1 ) Simulated yield (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N y = c(1.033) + c(0.5937) ⋅x, r2 = 0.525 EF: 0.512; RMSE: 1.575 SB(%): 1; NU(%): 2; LC(%): 97 29.7 % are actually in 95% CI 0 5 10 0 5 10 Measured yield (t ha−1 ) Simulated yield (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N y = c(2.869) + c(0.3678) ⋅x, r2 = 0.243 EF: −0.064; RMSE: 4.771 SB(%): 23; NU(%): 6; LC(%): 71 0 5 10 15 20 25 0 5 10 15 20 25 Measured AGB (t ha−1 ) Simulated AGB (t ha−1 ) y = c(3.639) + c(0.4357) ⋅x, r2 = 0.354 EF: 0.292; RMSE: 3.892 SB(%): 6; NU(%): 3; LC(%): 91 29.4 % are actually in 95% CI 0 5 10 15 20 25 0 5 10 15 20 25 Measured AGB (t ha−1 ) Simulated AGB (t ha−1 ) Figure 3. Simulated compared to measured maize grain yields at the four study sites for the default DayCent parameter set (top-left) vers the calibrated parameter set by leave-one-site-out cross-validation (top-right). The same is displayed for maize aboveground biomass (AGB showing the default DayCent parameter set (bottom-left) versus the calibrated parameter set (bottom-right). The 2985 data points correspon to the observations from the experimental treatments over 32 to 38 seasons, depending on the site. Symbols represent the different organ resource and chemical nitrogen fertilizer treatments. Grey bands show the 95% confidence intervals of measured (horizontal) values an the 95% credibility intervals of posterior distribution (vertical). Abbreviations: EF, Nash-Sutcliffe modeling efficiency; RMSE, root mea squared error; SB, squared bias; NU, non-unity slope; LC, lack of correlation. 3.3 Simulation of maize grain yields and aboveground biomass at harvest The exception to this was that DayCent poorly distinguished the mean yields and aboveground biomass of treatments with high compared to very high rates of N inputs (i.e., differences between the different 400 organic resources and the control within the +N treatment). An additional test of the model sensitivity of mean yields to dif- ferent levels of mineral N fertilizer in the control provided further insights into this (Fig. A6). In this test, the yields stopped aboveground biomass of treatments with high compared to very high rates of N inputs (i.e., differences between the different 400 organic resources and the control within the +N treatment). An additional test of the model sensitivity of mean yields to dif- ferent levels of mineral N fertilizer in the control provided further insights into this (Fig. A6). In this test, the yields stopped 15 Interestingly, the 95% credibility intervals for yield and aboveground biomass produced by the leave-one-site-out cross-validation contained only about 30% of measured data and also tended to be considerably smaller than variance-based 95% confidence intervals for the measured data. 3.4 Simulated SOC stocks in response to integrated soil fertility management 415 Most of the treatments were well simulated, but it is noteworthy that there is considerable variability in the measured SOC stocks between different time points. SOC losses, while in Aludeka most treatments except the control -N treatment showed weaker predicted than observed SOC 425 losses (Fig. A3). These tendencies are also evident when comparing the temporal dynamics of measured versus simulated SOC stocks (Fig. 6). Most of the treatments were well simulated, but it is noteworthy that there is considerable variability in the measured SOC stocks between different time points. y = c(1.297) + c(0.5703) ⋅x, r2 = 0.403 EF: 0.333; RMSE: 1.842 SB(%): 0; NU(%): 10; LC(%): 90 0 5 10 0 5 10 Measured yield (t ha−1 ) Simulated yield (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N y = c(1.033) + c(0.5937) ⋅x, r2 = 0.525 EF: 0.512; RMSE: 1.575 SB(%): 1; NU(%): 2; LC(%): 97 29.7 % are actually in 95% CI 0 5 10 0 5 10 Measured yield (t ha−1 ) Simulated yield (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N 1 Simulated yield (t ha−1 ) Simulated yield (t ha−1 ) Measured yield (t ha ) y = c(2.869) + c(0.3678) ⋅x, r2 = 0.243 EF: −0.064; RMSE: 4.771 SB(%): 23; NU(%): 6; LC(%): 71 0 5 10 15 20 25 0 5 10 15 20 25 Measured AGB (t ha−1 ) Simulated AGB (t ha−1 ) y = c(3.639) + c(0.4357) ⋅x, r2 = 0.354 EF: 0.292; RMSE: 3.892 SB(%): 6; NU(%): 3; LC(%): 91 29.4 % are actually in 95% CI 0 5 10 15 20 25 0 5 10 15 20 25 Measured AGB (t ha−1 ) Simulated AGB (t ha−1 ) Simulated AGB (t ha−1 ) Simulated AGB (t ha−1 ) Figure 3. Simulated compared to measured maize grain yields at the four study sites for the default DayCent parameter set (top-left) versus the calibrated parameter set by leave-one-site-out cross-validation (top-right). The same is displayed for maize aboveground biomass (AGB), showing the default DayCent parameter set (bottom-left) versus the calibrated parameter set (bottom-right). The 2985 data points correspond to the observations from the experimental treatments over 32 to 38 seasons, depending on the site. Symbols represent the different organic resource and chemical nitrogen fertilizer treatments. Grey bands show the 95% confidence intervals of measured (horizontal) values and the 95% credibility intervals of posterior distribution (vertical). Abbreviations: EF, Nash-Sutcliffe modeling efficiency; RMSE, root mean squared error; SB, squared bias; NU, non-unity slope; LC, lack of correlation. 16 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. increasing at mineral N rates that were lower than the maximum N rates provided in the organic resource +N treatments by mineral N and organic resources combined (up to >500 kg N per year or > 250kg N per season). In Machanga and Embu, simulated mean yields stopped increasing at around 100 kg N ha-1 per season, which is less the 120 kg N ha-1 per season that 405 the control +N received. In Aludeka and Sidada, simulated mean yields stopped increasing at 200 to 250 kg N ha-1 per season, but most of the response to N was below 120 kg N ha-1 per season (Fig. A6). Although the mean yields of the high-quality inputs in the -N treatments were well simulated, some of the low-quality input increasing at mineral N rates that were lower than the maximum N rates provided in the organic resource +N treatments by mineral N and organic resources combined (up to >500 kg N per year or > 250kg N per season). In Machanga and Embu, simulated mean yields stopped increasing at around 100 kg N ha-1 per season, which is less the 120 kg N ha-1 per season that 405 the control +N received. In Aludeka and Sidada, simulated mean yields stopped increasing at 200 to 250 kg N ha-1 per season, but most of the response to N was below 120 kg N ha-1 per season (Fig. A6). treatments in Aludeka and Sidada, namely maize stover at 1.2 t C ha-1 yr-1 , and sawdust at 1.2 and 4 t C ha-1 yr-1, had lower simulated yields than the observed mean yields in their -N treatments (Fig. 4). The same was true for the control -N in Aludeka, 410 but the yields for sawdust -N at 4 t C ha-1 yr-1 in Machanga were overpredicted compared to measurements. Interestingly, the 95% credibility intervals for yield and aboveground biomass produced by the leave-one-site-out cross-validation contained only about 30% of measured data and also tended to be considerably smaller than variance-based 95% confidence intervals for the measured data. simulated yields than the observed mean yields in their -N treatments (Fig. 4). The same was true for the control -N in Aludeka, 410 but the yields for sawdust -N at 4 t C ha-1 yr-1 in Machanga were overpredicted compared to measurements. 3.4 Simulated SOC stocks in response to integrated soil fertility management 415 In contrast to the simulation of maize yields, the change in SOC stocks following the application of organic resources at different rates (1.2 and 4 t ha-1 yr-1) was poorly simulated by DayCent with the set of default parameters. The simulated SOC losses were too low compared to the observations (Fig. 5). The posterior parameter set obtained through Bayesian calibration with strongly increased SOM turnover rates showed a tendency for a higher loss of CO2 from the turnover of metabolic and structural litter pools. With the posterior parameter set, the model, in the leave-one-site-out cross-validation, simulated the 420 change in SOC much more accurately than with the default parameter set (EF of 0.54 vs -1.3; LC of 88% vs 42%; Fig. 5). The 95% credibility intervals of the simulated values contained 47% of the measured data. It should be noted that under- or overestimation of the change in SOC stocks from the start to the last year of the experiment was rather related to site than to treatment. In Embu, for example, all treatments except the control +N treatment tended to have lower observed than simulated structural litter pools. With the posterior parameter set, the model, in the leave-one-site-out cross-validation, simulated the 420 change in SOC much more accurately than with the default parameter set (EF of 0.54 vs -1.3; LC of 88% vs 42%; Fig. 5). The 95% credibility intervals of the simulated values contained 47% of the measured data. It should be noted that under- or overestimation of the change in SOC stocks from the start to the last year of the experiment was rather related to site than to treatment. In Embu, for example, all treatments except the control +N treatment tended to have lower observed than simulated SOC losses, while in Aludeka most treatments except the control -N treatment showed weaker predicted than observed SOC 425 losses (Fig. A3). These tendencies are also evident when comparing the temporal dynamics of measured versus simulated SOC stocks (Fig. 6). Most of the treatments were well simulated, but it is noteworthy that there is considerable variability in the measured SOC stocks between different time points. SOC losses, while in Aludeka most treatments except the control -N treatment showed weaker predicted than observed SOC 425 losses (Fig. A3). These tendencies are also evident when comparing the temporal dynamics of measured versus simulated SOC stocks (Fig. 6). https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. y = c(−1.64) + c(0.08953) ⋅x, r2 = 0.0218 EF: −1.312; RMSE: 11.711 SB(%): 49; NU(%): 9; LC(%): 42 −10 0 10 20 30 −10 0 10 20 30 Measured loss of SOC stocks (t ha−1 in 0−20 cm) Simulated loss of SOC stocks (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N y = c(3.507) + c(0.711) ⋅x, r2 = 0.593 EF: 0.539; RMSE: 5.031 SB(%): 7; NU(%): 5; LC(%): 88 45.3 % are actually in 95% CI −10 0 10 20 30 −10 0 10 20 30 Measured loss of SOC stocks (t ha−1 in 0−20 cm) Simulated loss of SOC stocks (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Figure 5. Simulated compared to measured changes in SOC stocks since the start of the experiment at the four study sites for the default DayCent parameter set (left) versus the calibrated parameter set by leave-one-site-out cross-validation (right). The 724 data points correspond to the observations from the experimental treatments over 32 to 38 seasons, depending on the site. Symbols represent the different organic resource and chemical nitrogen fertilizer treatments. Grey bands show the 95% confidence intervals of measured (horizontal) values and the 95% credibility intervals of posterior distribution (vertical). Abbreviations: EF, Nash-Sutcliffe modeling efficiency; RMSE, root mean squared error; SB, squared bias; NU, non-unity slope; LC, lack of correlation. y = c(−1.64) + c(0.08953) ⋅x, r2 = 0.0218 EF: −1.312; RMSE: 11.711 SB(%): 49; NU(%): 9; LC(%): 42 −10 0 10 20 30 −10 0 10 20 30 Measured loss of SOC stocks (t ha−1 in 0−20 cm) Simulated loss of SOC stocks (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N y = c(3.507) + c(0.711) ⋅x, r2 = 0.593 EF: 0.539; RMSE: 5.031 SB(%): 7; NU(%): 5; LC(%): 88 45.3 % are actually in 95% CI −10 0 10 20 30 −10 0 10 20 30 Measured loss of SOC stocks (t ha−1 in 0−20 cm) Simulated loss of SOC stocks (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N of SOC stocks (t ha−1 in 0−20 cm) Simulated loss of SOC stocks (t ha−1 in 0−20 cm) Simulated loss of SOC stocks (t ha−1 Figure 5. Simulated compared to measured changes in SOC stocks since the start of the experiment at the four study sites for the default DayCent parameter set (left) versus the calibrated parameter set by leave-one-site-out cross-validation (right). The 724 data points correspond to the observations from the experimental treatments over 32 to 38 seasons, depending on the site. Symbols represent the different organic resource and chemical nitrogen fertilizer treatments. Grey bands show the 95% confidence intervals of measured (horizontal) values and the 95% credibility intervals of posterior distribution (vertical). Abbreviations: EF, Nash-Sutcliffe modeling efficiency; RMSE, root mean squared error; SB, squared bias; NU, non-unity slope; LC, lack of correlation. Figure 5. Simulated compared to measured changes in SOC stocks since the start of the experiment at the four study sites for the default DayCent parameter set (left) versus the calibrated parameter set by leave-one-site-out cross-validation (right). The 724 data points correspond to the observations from the experimental treatments over 32 to 38 seasons, depending on the site. Symbols represent the different organic resource and chemical nitrogen fertilizer treatments. Grey bands show the 95% confidence intervals of measured (horizontal) values and the 95% credibility intervals of posterior distribution (vertical). Abbreviations: EF, Nash-Sutcliffe modeling efficiency; RMSE, root mean squared error; SB, squared bias; NU, non-unity slope; LC, lack of correlation. Fig. 7). Additionally, the correlation between cumulative simulated and measured N2O emissions was much higher than for daily emission fluxes (R2 between 0.26 for Sidada and 0.81 for Aludeka, compared to R2 close to 0). The simulated changes in SOC and simulated cumulative N2O emissions, showed positive global warming potentials (GWP) for all treatments (Fig. 8). No treatment acted as a greenhouse gas sink, but the amount of emissions, as well as the contribution The simulated changes in SOC and simulated cumulative N2O emissions, showed positive global warming potentials (GWP) for all treatments (Fig. 8). No treatment acted as a greenhouse gas sink, but the amount of emissions, as well as the contribution of N2O and CO2 differed strongly between sites and treatments. In the control -N treatment, it ranged from 1.4 t CO2 equivalent 440 ha-1 yr-1 in Aludeka to 5.1 t CO2 equivalent ha-1 yr-1 in Sidada and Embu. The relative contribution of N2O from site to site also differed strongly by site. 3.5 Simulated N2O emissions and global warming potential The simulations and measurements of N2O emissions on a daily basis were not well aligned. This is clear from the negative 430 modeling efficiencies and the absence of a clear correlation between observed and measured values (Fig. 7). Peaks of N2O emissions were often simulated on different dates than the measurements, but treatments with higher N loads tended to have higher simulated and higher measured N2O fluxes. This was most noticeable in +N compared to -N treatments (Fig. A10). For cumulative N2O emissions per season on the other hand, there was a much better alignment between the simulated and measured values. All sites showed positive modeling efficiency (highest EF 0.34 for Aludeka, lowest EF 0.21 for Machanga; 435 measured values. All sites showed positive modeling efficiency (highest EF 0.34 for Aludeka, lowest EF 0.21 for Machanga; 435 17 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. −N 0 t C −N 1.2 t C −N 4 t C +N 0 t C +N 1.2 t C +N 4 t C Aludeka Embu Machanga Sidada Control Calliandra Farmyard manure Maize stover Sawdust Tithonia Calliandra Farmyard manure Maize stover Sawdust Tithonia Control Calliandra Farmyard manure Maize stover Sawdust Tithonia Calliandra Farmyard manure Maize stover Sawdust Tithonia 0 2 4 6 0 2 4 6 0 1 2 3 4 0.0 2.5 5.0 7.5 Mean yield (t ha−1 ) measured simulated −N 0 t C −N 1.2 t C −N 4 t C +N 0 t C +N 1.2 t C +N 4 t C Aludeka Embu Machanga Sidada Control Calliandra Farmyard manure Maize stover Sawdust Tithonia Calliandra Farmyard manure Maize stover Sawdust Tithonia Control Calliandra Farmyard manure Maize stover Sawdust Tithonia Calliandra Farmyard manure Maize stover Sawdust Tithonia 0 5 10 15 0 5 10 15 0 3 6 9 0 5 10 15 Mean AGB (t ha−1 ) measured simulated gure 4. Barplots of mean simulated and mean measured yield and aboveground biomass (AGB) from cross-validation. Error bars repres ndard deviation. Figure 4. Barplots of mean simulated and mean measured yield and aboveground biomass (AGB) from cross-validation. Error bars represent standard deviation. 18 In Aludeka, for example, between 32 (CT+N) and 82% (FYM4+N) of the simulated GWP in the +N treatments came from N2O, while at all other sites, none of the treatments had more than 25% of GWP associated with N2O emissions. Compared to the control -N treatment, which is closest to the low-input agriculture practiced by most of N2O and CO2 differed strongly between sites and treatments. In the control -N treatment, it ranged from 1.4 t CO2 equivalent 440 ha-1 yr-1 in Aludeka to 5.1 t CO2 equivalent ha-1 yr-1 in Sidada and Embu. The relative contribution of N2O from site to site also differed strongly by site. In Aludeka, for example, between 32 (CT+N) and 82% (FYM4+N) of the simulated GWP in the +N treatments came from N2O, while at all other sites, none of the treatments had more than 25% of GWP associated with N2O emissions. Compared to the control -N treatment, which is closest to the low-input agriculture practiced by most 440 smallholder farmers, all organic resource input treatments in the -N treatments were projected to have lower emissions (Fig. 445 8). The reductions ranged from -0.2 t CO2 equivalent ha-1 yr-1 to 1 t CO2 equivalent ha-1 yr-1. Apart from that, the only site where the addition of mineral N (+N treatment) could lead to a higher simulated GWP per ha than the control -N treatment was Embu, and this was only the case for the treatments of Calliandra, Tithonia and farmyard manure in both 1.2 and 4 t of C 19 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0 t C Control 4 t C Calliandra 4 t C Farmyard manure 4 t C Maize stover 4 t C Sawdust 4 t C Tithonia −N Aludeka +N Aludeka −N Embu +N Embu −N Machanga +N Machanga −N Sidada +N Sidada 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 0 10 20 30 0 10 20 30 0 20 40 60 80 0 20 40 60 80 0 10 20 30 0 10 20 30 40 0 20 40 60 0 20 40 60 SOC stocks (t ha−1 in 0 − 20 cm) Figure 6. y = c(3.625) + c(0.1086) ⋅x, r2 = 0.0802 EF: −0.024; RMSE: 13.567 SB(%): 9; NU(%): 1; LC(%): 90 y = c(6.716) + c(0.01222) ⋅x, r2 = 0.00394 EF: −0.038; RMSE: 33.525 SB(%): 2; NU(%): 2; LC(%): 96 y = c(5.466) + c(0.01846) ⋅x, r2 = 0.00319 EF: −0.071; RMSE: 16.787 SB(%): 0; NU(%): 7; LC(%): 93 y = c(4.283) + c(0.05399) ⋅x, r2 = 0.027 EF: 0; RMSE: 12.169 SB(%): 0; NU(%): 3; LC(%): 97 Aludeka Embu Machanga Sidada 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 Measured N2O emissions (g N2O−N ha−1 d−1 ) Simulated N2O emissions (g N2O−N ha−1 d−1 ) Calliandra Control Farmyard manure Maize stover Tithonia −N +N y = c(124.2) + c(0.3607) ⋅x, r2 = 0.815 EF: 0.342; RMSE: 409.445 SB(%): 33; NU(%): 39; LC(%): 28 y = c(126.9) + c(0.2903) ⋅x, r2 = 0.739 EF: 0.325; RMSE: 753.489 SB(%): 21; NU(%): 40; LC(%): 39 y = c(176.2) + c(0.1499) ⋅x, r2 = 0.244 EF: 0.208; RMSE: 237.625 SB(%): 0; NU(%): 5; LC(%): 95 y = c(225.1) + c(0.1563) ⋅x, r2 = 0.255 EF: 0.217; RMSE: 403.042 SB(%): 0; NU(%): 5; LC(%): 95 Aludeka Embu Machanga Sidada 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 Cumulative measured emissions (g N2O−N ha−1) Simulated cumulative emissions (g N2O−N ha−1) Figure 7. Simulated compared to measured N2O emissions at the four study sites for the different organic resource and chemical nitrogen fertilizer treatments, based on the calibrated parameter set using leave-one-site-out cross-validation. Displayed are the measured versus modelled per treatment for the days where measurements were conducted (top) and for the mean of cumulative flux measurements per season using the trapeziod method (bottom). The 808 data points (top) correspond to the daily measurements from the experimental treatments over one to two seasons, depending on the site. Symbols represent the different organic resource and chemical nitrogen fertilizer treatments. Error bars represent 95% confidence intervals based on BC (simulations) and variance (measurements). Abbreviations: EF, Nash-Sutcliffe modeling efficiency; RMSE, root mean squared error; SB, squared bias; NU, non-unity slope; LC, lack of correlation. Measured (dots) versus simulated SOC stocks over time at the four study sites for the different organic resource and chemical nitrogen fertilizer treatments. Error bars represent 95% confidence intervals for measured data, the black solid line the simulation by the best parameter set for each site. Grey bands represent the 95% credibility intervals of the model posterior simulations, calibrated by leave-one- site-out cross-validation. Sidada Sidada Figure 6. Measured (dots) versus simulated SOC stocks over time at the four study sites for the different organic resource and chemical nitrogen fertilizer treatments. Error bars represent 95% confidence intervals for measured data, the black solid line the simulation by the best parameter set for each site. Grey bands represent the 95% credibility intervals of the model posterior simulations, calibrated by leave-one- site-out cross-validation. 20 y = c(3.625) + c(0.1086) ⋅x, r2 = 0.0802 EF: −0.024; RMSE: 13.567 SB(%): 9; NU(%): 1; LC(%): 90 y = c(6.716) + c(0.01222) ⋅x, r2 = 0.00394 EF: −0.038; RMSE: 33.525 SB(%): 2; NU(%): 2; LC(%): 96 y = c(5.466) + c(0.01846) ⋅x, r2 = 0.00319 EF: −0.071; RMSE: 16.787 SB(%): 0; NU(%): 7; LC(%): 93 y = c(4.283) + c(0.05399) ⋅x, r2 = 0.027 EF: 0; RMSE: 12.169 SB(%): 0; NU(%): 3; LC(%): 97 Aludeka Embu Machanga Sidada 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 Measured N2O emissions (g N2O−N ha−1 d−1 ) Simulated N2O emissions (g N2O−N ha−1 d−1 ) Calliandra Control Farmyard manure Maize stover Tithonia −N +N y = c(124.2) + c(0.3607) ⋅x, r2 = 0.815 EF: 0.342; RMSE: 409.445 SB(%): 33; NU(%): 39; LC(%): 28 y = c(126.9) + c(0.2903) ⋅x, r2 = 0.739 EF: 0.325; RMSE: 753.489 SB(%): 21; NU(%): 40; LC(%): 39 y = c(176.2) + c(0.1499) ⋅x, r2 = 0.244 EF: 0.208; RMSE: 237.625 SB(%): 0; NU(%): 5; LC(%): 95 y = c(225.1) + c(0.1563) ⋅x, r2 = 0.255 EF: 0.217; RMSE: 403.042 SB(%): 0; NU(%): 5; LC(%): 95 Aludeka Embu Machanga Sidada 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 Cumulative measured emissions (g N2O−N ha−1) Simulated cumulative emissions (g N2O−N ha−1) Cumulative measured emissions (g N2O−N ha−1) Figure 7. Simulated compared to measured N2O emissions at the four study sites for the different organic resource and chemical nitrogen fertilizer treatments, based on the calibrated parameter set using leave-one-site-out cross-validation. Displayed are the measured versus modelled per treatment for the days where measurements were conducted (top) and for the mean of cumulative flux measurements per season using the trapeziod method (bottom). The 808 data points (top) correspond to the daily measurements from the experimental treatments over one to two seasons, depending on the site. Symbols represent the different organic resource and chemical nitrogen fertilizer treatments. Error bars represent 95% confidence intervals based on BC (simulations) and variance (measurements). Abbreviations: EF, Nash-Sutcliffe modeling efficiency; RMSE, root mean squared error; SB, squared bias; NU, non-unity slope; LC, lack of correlation. 21 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. input ha-1 yr-1. Furthermore, Embu and Machanga tended to have high simulated emissions per kg of maize yields (0.9 to 1.6 and 0.8 to 2.5 kg CO2 equivalent per kg of yield, respectively, due to lower yield). On the contrary, the simulated emissions 450 ranged between 0.1 and 0.7 kg CO2 equivalent per kg of yield in Aludeka and between 0.4 and 0.9 kg CO2 equivalent per kg of yield in Sidada. Notably, none of the +N treatments in Sidada and Aludeka had higher emissions than 0.5 kg CO2 equivalent per kg of yield. input ha-1 yr-1. Furthermore, Embu and Machanga tended to have high simulated emissions per kg of maize yields (0.9 to 1.6 and 0.8 to 2.5 kg CO2 equivalent per kg of yield, respectively, due to lower yield). On the contrary, the simulated emissions 450 ranged between 0.1 and 0.7 kg CO2 equivalent per kg of yield in Aludeka and between 0.4 and 0.9 kg CO2 equivalent per kg of yield in Sidada. Notably, none of the +N treatments in Sidada and Aludeka had higher emissions than 0.5 kg CO2 equivalent per kg of yield. CC BY 4.0 License. 0 −N 0 +N 1.2 −N 1.2 +N 4 −N 4 +N Aludeka Embu Machanga Sidada 0 2 4 0 2 4 0 2 4 0 2 4 Global warming potential (t COeq −2 ha−1 yr−1) N2O SOC 0 +N 1.2 −N 1.2 +N 4 −N 4 +N Aludeka Embu Machanga Sidada −1.00 −0.75 −0.50 −0.25 0.00 −1.00 −0.75 −0.50 −0.25 0.00 −1.00 −0.75 −0.50 −0.25 0.00 −1.00 −0.75 −0.50 −0.25 0.00 Difference to CT−N (t COeq −2 ha−1 yr−1) 0 −N 0 +N 1.2 −N 1.2 +N 4 −N 4 +N Aludeka Embu Machanga Sidada Control Control Calliandra Farmyard manure Maize stover Saw dust Tithonia Calliandra Farmyard manure Maize stover Saw dust Tithonia Calliandra Farmyard manure Maize stover Saw dust Tithonia Calliandra Farmyard manure Maize stover Saw dust Tithonia 0 1 2 0 1 2 0 1 2 0 1 2 Yield scaled global warming potential (kg COeq −2 kg−1 grain yield) N2O SOC Figure 8. Cumulative simulated global warming potential of N2O emissions and CO2 emissions due to loss of SOC at the four study sites for different organic resource and chemical nitrogen fertilizer treatments, combined throughout the simulated period (16 years for Aludeka/Sidada; 19 years for Embu/Machanga). The global warming potential is expressed in CO2 equivalent over a 100-year horizon. Figure 8. Cumulative simulated global warming potential of N2O emissions and CO2 emissions due to loss of SOC at the four study sites for different organic resource and chemical nitrogen fertilizer treatments, combined throughout the simulated period (16 years for Aludeka/Sidada; 19 years for Embu/Machanga). The global warming potential is expressed in CO2 equivalent over a 100-year horizon. 22 4.1 Robustness of the Bayesian calibration shown by cross evaluation 455 This suggests 465 that a reasonable threshold for including a parameter in model calibration may even be greater than a total Sobol sensitivity index of 2.5%. This result is similar to the study of Gurung et al. (2020), where the parameters with a total sensitivity index <10% were also poorly constrained. In our study, it was possible to constrain the most sensitive parameters (prdx(1), himax, dec(5)); exceptions were the two parameters pmco2(1&2) and clteff(1,2&4), but their correlation (r = -0.43) is a reasonable explanation of why they could not be well constrained despite high sensitivity. Possibly, the algorithm cannot distinguish 470 whether organic resources are poorly stabilized due to tillage disturbance, a fast turnover rate of the slow SOM pool, or a low carbon use efficiency of metabolic and/or structural litter pools. Such a correlation between SOM stabilization and turnover parameters is common in soil models (e.g. Ahrens et al., 2014; Laub et al., 2021), especially if the SOM pools do not represent measurable fractions (Laub et al., 2020). explanation of why they could not be well constrained despite high sensitivity. Possibly, the algorithm cannot distinguish 470 whether organic resources are poorly stabilized due to tillage disturbance, a fast turnover rate of the slow SOM pool, or a low carbon use efficiency of metabolic and/or structural litter pools. Such a correlation between SOM stabilization and turnover parameters is common in soil models (e.g. Ahrens et al., 2014; Laub et al., 2021), especially if the SOM pools do not represent measurable fractions (Laub et al., 2020). Robust predictions of crop yields and changes in SOC stocks, as achieved by our cross-validation, are an important basis for 475 potential future upscaling exercises of crop yield and SOC model predictions to national scales. While many model calibration exercises often display an overly optimistic picture of the capacity of models to represent SOC dynamics, by displaying how well the models represent absolute levels of SOC (e.g., in Nyawira et al., 2021; Ma et al., 2022; Levavasseur et al., 2021), the changes in SOC since the start of an experiment, shown in this study, are much more robust evaluation. Since models are Robust predictions of crop yields and changes in SOC stocks, as achieved by our cross-validation, are an important basis for 475 potential future upscaling exercises of crop yield and SOC model predictions to national scales. 4.1 Robustness of the Bayesian calibration shown by cross evaluation 455 As shown by our leave-one-site-out cross-validation (Figs. 3 and A7), the Bayesian calibration considerably improved the capability of DayCent to predict maize grain yield, aboveground biomass and changes in SOC stocks at the four long-term ISFM experimental sites. The model evaluation statistics from this calibration exercise were comparable to those of other recent publications that combined the predictions of yield and SOC (Necpalova et al., 2018; Levavasseur et al., 2021; Nyawira et al., 2021). However, these studies generally showed a better simulation of crop yield than SOC, which is in contrast to our 460 results, where crop yield and SOC were simulated equally well. Of the three types of measurements, simulated changes in SOC stocks related to different treatments were most improved after model calibration, reducing the strong bias towards simulated gains in SOC, where instead losses were measured. In general, the screening of the model parameters with the global sensitivity analysis proved valuable in reducing the complexity of the calibration problem. The most sensitive parameters (Table 1) were et al., 2021). However, these studies generally showed a better simulation of crop yield than SOC, which is in contrast to our 460 results, where crop yield and SOC were simulated equally well. Of the three types of measurements, simulated changes in SOC stocks related to different treatments were most improved after model calibration, reducing the strong bias towards simulated gains in SOC, where instead losses were measured. In general, the screening of the model parameters with the global sensitivity analysis proved valuable in reducing the complexity of the calibration problem. The most sensitive parameters (Table 1) were well constrained, while the least sensitive parameters (aneref(3), ppdf(2) and ps1co2) were difficult to constrain. This suggests 465 that a reasonable threshold for including a parameter in model calibration may even be greater than a total Sobol sensitivity index of 2.5%. This result is similar to the study of Gurung et al. (2020), where the parameters with a total sensitivity index <10% were also poorly constrained. In our study, it was possible to constrain the most sensitive parameters (prdx(1), himax, dec(5)); exceptions were the two parameters pmco2(1&2) and clteff(1,2&4), but their correlation (r = -0.43) is a reasonable well constrained, while the least sensitive parameters (aneref(3), ppdf(2) and ps1co2) were difficult to constrain. 4.1 Robustness of the Bayesian calibration shown by cross evaluation 455 While many model calibration exercises often display an overly optimistic picture of the capacity of models to represent SOC dynamics, by displaying how well the models represent absolute levels of SOC (e.g., in Nyawira et al., 2021; Ma et al., 2022; Levavasseur et al., 2021), the changes in SOC since the start of an experiment, shown in this study, are much more robust evaluation. Since models are Robust predictions of crop yields and changes in SOC stocks, as achieved by our cross-validation, are an important basis for 475 potential future upscaling exercises of crop yield and SOC model predictions to national scales. While many model calibration exercises often display an overly optimistic picture of the capacity of models to represent SOC dynamics, by displaying how well the models represent absolute levels of SOC (e.g., in Nyawira et al., 2021; Ma et al., 2022; Levavasseur et al., 2021), the changes in SOC since the start of an experiment, shown in this study, are much more robust evaluation. Since models are typically parameterized to fit the observed SOC stock at the start of the experiment, absolute SOC will always closely resemble 480 measurements, with the possible exception of experiments that run for many decades. The fallacy of this approach was shown typically parameterized to fit the observed SOC stock at the start of the experiment, absolute SOC will always closely resemble 480 measurements, with the possible exception of experiments that run for many decades. The fallacy of this approach was shown 23 4.2 Bayesian calibration suggests that SOC is lost at higher rates in tropical than in temperate soils To estimate the potential yield and long-term sustainability of cropping systems without major bias using biogeoch models, regionalized model calibrations are needed (Rattalino Edreira et al., 2021; Yang et al., 2021). Therefore, while p studies have simulated crop productivity under ISFM and similar practices with default parameter sets (e.g. Nezomba et al., 495 2018; Nyawira et al., 2021), the results of our study indicate that especially to estimate the effect on SOC, local calibration is very much needed (Fig. 5). In fact, the turnover of the slow and passive SOM pools was approximately two and eight times higher than in a recent Bayesian calibration of DayCent for temperate systems (Gurung et al., 2020), indicating a faster SOM turnover under tropical conditions, as expected. However, since the SOM pools in DayCent do not correspond to measurable fractions and we did not have additional data (e.g., 14 C), constraining the related parameters is expected to be subject to high 500 uncertainty (e.g. Ahrens et al., 2014). Nevertheless, our results of high SOM turnover in Kenya are in line with the results of two recent studies, which also found that the default parameterizations of the DayCent (Nyawira et al., 2021) and the LPJGUESS model (Ma et al., 2022) underestimated the loss of SOC in two other long-term experiments in Kenya. In our study, the low Sobol total sensitivity indices of the maize productivity parameters for the SOC stocks suggest a limited importance of root fractions and we did not have additional data (e.g., 14 C), constraining the related parameters is expected to be subject to high 500 uncertainty (e.g. Ahrens et al., 2014). Nevertheless, our results of high SOM turnover in Kenya are in line with the results of two recent studies, which also found that the default parameterizations of the DayCent (Nyawira et al., 2021) and the LPJGUESS model (Ma et al., 2022) underestimated the loss of SOC in two other long-term experiments in Kenya. In our study, the low Sobol total sensitivity indices of the maize productivity parameters for the SOC stocks suggest a limited importance of root input in the soil system for the storage of SOC. 2) should be used. A computationally less expensive alternative is to use 490 only the single parameter set with the highest likelihood (Table 1). https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. by Necpalova et al. (2018); Gurung et al. (2020), where the absolute SOC overoptimistically masks the bias of change in SOC stocks. This is also the case for the present study, as is clear from the results of the model simulations for the absolute SOC stocks compared to the changes in the SOC stocks (Fig. A7). Since our calibration shows a good fit and is free from serious bias even for changes in SOC stocks (that is, 88% of the errors are LC and not systematic; EF is 0.55), the calibrated DayCent model 485 can be used in a robust manner to estimate the change of SOC stocks under different ISFM management in Kenya. Furthermore, because the model evaluation and calibration were performed at different sites, it seems reasonable to use DayCent for other sites with similar climate and soil conditions, even beyond Kenya. In that respect, the leave-one-site-out cross-validation is using the data most efficiently: for evaluation, we leave one site out, but for further model upscaling exercises, ideally the full posterior model parameter set including all sites (Fig. 2) should be used. A computationally less expensive alternative is to use 490 only the single parameter set with the highest likelihood (Table 1). by Necpalova et al. (2018); Gurung et al. (2020), where the absolute SOC overoptimistically masks the bias of change in SOC stocks. This is also the case for the present study, as is clear from the results of the model simulations for the absolute SOC stocks compared to the changes in the SOC stocks (Fig. A7). Since our calibration shows a good fit and is free from serious bias even for changes in SOC stocks (that is, 88% of the errors are LC and not systematic; EF is 0.55), the calibrated DayCent model 485 can be used in a robust manner to estimate the change of SOC stocks under different ISFM management in Kenya. Furthermore, because the model evaluation and calibration were performed at different sites, it seems reasonable to use DayCent for other sites with similar climate and soil conditions, even beyond Kenya. In that respect, the leave-one-site-out cross-validation is using the data most efficiently: for evaluation, we leave one site out, but for further model upscaling exercises, ideally the full posterior model parameter set including all sites (Fig. 4.2 Bayesian calibration suggests that SOC is lost at higher rates in tropical than in temperate soils These results align with a statistical analysis of SOC stocks across depth at the 505 same experimental sites (Laub et al., 2022), which found limited differences between cropped and bare plots, and with another recent tropical long-term experiment (Cardinael et al., 2022), which estimated that less than 1 t of carbon input ha-1 per year came from maize roots. Overall, the considerably higher simulated SOM turnover rates in this study compared to those in temperate regions (Gurung input in the soil system for the storage of SOC. These results align with a statistical analysis of SOC stocks across depth at the 505 same experimental sites (Laub et al., 2022), which found limited differences between cropped and bare plots, and with another recent tropical long-term experiment (Cardinael et al., 2022), which estimated that less than 1 t of carbon input ha-1 per year came from maize roots. Overall, the considerably higher simulated SOM turnover rates in this study compared to those in temperate regions (Gurung Overall, the considerably higher simulated SOM turnover rates in this study compared to those in temperate regions (Gurung et al., 2020) suggest that the lower SOM stabilization potential of highly weathered minerals in tropical soils (Doetterl et al., 510 2015) is not adequately accounted for in DayCent. DayCent only includes an effect of clay content. Possibly, the effect of weathered minerals is large for both slow and passive SOM pools (increased turnover by 21 and 23 in this study, respectively) and is not adequately represented by texture alone. It was recently shown that the Millenial model, which has measurable pools, could better simulate the global SOC distribution than the CENTURY model, which has conceptual pools (Abramoff et al., 2022). Recent attempts to move DayCent in that direction have also shown some promising results (e.g. Dangal et al., 515 et al., 2020) suggest that the lower SOM stabilization potential of highly weathered minerals in tropical soils (Doetterl et al., 510 2015) is not adequately accounted for in DayCent. DayCent only includes an effect of clay content. Possibly, the effect of weathered minerals is large for both slow and passive SOM pools (increased turnover by 21 and 23 in this study, respectively) and is not adequately represented by texture alone. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 2022). Furthermore, it is possible that hand tillage twice a year and potential erosion also played a role in the high SOC losses that informed these fast turnover rates. Because DayCent does not include erosion and erosion was not measured in the experiments, the exact role of these processes is hard to determine. Therefore, the different calibrations of the DayCent model in our study and in Gurung et al. (2020), suggest that the calibration developed in this study should only be used for soils of similar mineralogy and under a similar climate and for a similar maize cropping system. 520 4.2 Bayesian calibration suggests that SOC is lost at higher rates in tropical than in temperate soils It was recently shown that the Millenial model, which has measurable pools, could better simulate the global SOC distribution than the CENTURY model, which has conceptual pools (Abramoff et al., 2022). Recent attempts to move DayCent in that direction have also shown some promising results (e.g. Dangal et al., 515 et al., 2020) suggest that the lower SOM stabilization potential of highly weathered minerals in tropical soils (Doetterl et al., 510 2015) is not adequately accounted for in DayCent. DayCent only includes an effect of clay content. Possibly, the effect of weathered minerals is large for both slow and passive SOM pools (increased turnover by 21 and 23 in this study, respectively) and is not adequately represented by texture alone. It was recently shown that the Millenial model, which has measurable pools, could better simulate the global SOC distribution than the CENTURY model, which has conceptual pools (Abramoff et al., 2022). Recent attempts to move DayCent in that direction have also shown some promising results (e.g. Dangal et al., 515 24 4.3 Maize crop module is more universally applicable than soil carbon module Although the mean yields per treatment were well represented and the provisioning of N via organic resource mineralization was well captured, as demonstrated by a good agreement between the mean simulated and measured yields in the -N treatments (Fig. 4), the improvement in the simulated maize grain yield through the joint Bayesian model calibration of SOM pools and the maize crop highlights the interconnectedness of both. Thus, even if the crop parameterization is correct, as indicated by 525 the low difference between initial and calibrated maize parameters, too low SOM pool turnover rates, by underestimating mineralization of N from soil, results in too high demand of maize for mineral N addition to produce a suitable yield. This can be seen in the poor simulation of the low input treatments of Sidada before calibration and their improvement after calibration (Fig A5). Interestingly, in contrast to SOC turnover rates, the values of the maize crop parameters in this study were close to the set of default parameter values of DayCent, especially the maximum production capacity (prdx(1)). This suggests that 530 the traits of the maize are universally represented in DayCent, whereas the turnover of the SOM is not. In this context, the changes in the values for optimal and maximum temperature for maize growth (ppdf(1) and ppdf(2)) could be attributed to the adaptation of local maize species to the higher temperatures in Kenya. For example, Yang et al. (2021) conducted region- specific Bayesian model calibration of DayCent maize growing parameters and found ppdf(1) to very between 26 and 32 °C. to the set of default parameter values of DayCent, especially the maximum production capacity (prdx(1)). This suggests that 530 the traits of the maize are universally represented in DayCent, whereas the turnover of the SOM is not. In this context, the changes in the values for optimal and maximum temperature for maize growth (ppdf(1) and ppdf(2)) could be attributed to the adaptation of local maize species to the higher temperatures in Kenya. For example, Yang et al. (2021) conducted region- specific Bayesian model calibration of DayCent maize growing parameters and found ppdf(1) to very between 26 and 32 °C. Therefore, the different optimal temperatures (33° C) and highest temperatures (40° C) for the calibration of maize to US 535 conditions by Necpálová et al. (2015) do not contradict the range of parameters of our study. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 4.3 Maize crop module is more universally applicable than soil carbon module An interesting observation is that the model bias for the mean yield of maize appears to be treatment specific (i.e., the mean yields of +N treatments of farmyard manure at 4 t C ha-1 yr-1 at all sites and of Tithonia at the same rate in all but Sidada, were underpredicted by DayCent), while the bias for SOC stocks was mostly site specific (i.e., SOC loss in Aludeka Sidada, were underpredicted by DayCent), while the bias for SOC stocks was mostly site specific (i.e., SOC loss in Aludeka was underpredicted, while overpredicted in Embu). As discussed above, the site bias of SOC is likely due to the fact that soil 540 texture alone is insufficient to explain the mineralogy-driven storage potential of SOC (e.g. Reichenbach et al., 2021; Mainka et al., 2022). On the other hand, our sensitivity test to mineral N input suggests that the yield bias at high N is due to DayCent’s inability to capture yield increases above 100-150 kg N per ha and season (Fig. A6); the +N treatments of Tithonia, Calliandra and farmyard manure at 4 t C ha-1 yr-1 supplied on average >250 kg N per ha and season. Another reason may be that DayCent does not include other potential beneficial effects of treatments, such as increased pH from farmyard manure application (Xiao 545 was underpredicted, while overpredicted in Embu). As discussed above, the site bias of SOC is likely due to the fact that soil 540 texture alone is insufficient to explain the mineralogy-driven storage potential of SOC (e.g. Reichenbach et al., 2021; Mainka et al., 2022). On the other hand, our sensitivity test to mineral N input suggests that the yield bias at high N is due to DayCent’s inability to capture yield increases above 100-150 kg N per ha and season (Fig. A6); the +N treatments of Tithonia, Calliandra and farmyard manure at 4 t C ha-1 yr-1 supplied on average >250 kg N per ha and season. Another reason may be that DayCent does not include other potential beneficial effects of treatments, such as increased pH from farmyard manure application (Xiao 545 et al., 2021; Mtangadura et al., 2017) and higher nutrient retention and water infiltration of treatments that maintained SOC stocks compared to those that showed a decline of SOC. 25 4.4 N2O emissions and global warming potential However, the strong differences in the yield-scaled GWP between treatments, such as a 72, 32, 63 and 14 % lower yield-scaled GWP in the FYM 1.2+N treatment compared to the control-N treatment, show that the yield-scaled GWP is highly relevant in practical terms. Low-emission and negative emission technology. All treatments had a positive absolute GWP, which is consistent with the reported losses of 570 SOC in long-term maize cultivation in SSA (Sommer et al., 2018; Laub et al., 2022). It is also consistent with the postulate that closing SSA yield gaps will boost absolute N2O emissions (Leitner et al., 2020). However, the strong differences in the yield-scaled GWP between treatments, such as a 72, 32, 63 and 14 % lower yield-scaled GWP in the FYM 1.2+N treatment compared to the control-N treatment, show that the yield-scaled GWP is highly relevant in practical terms. Low-emission and negative emission technology. All treatments had a positive absolute GWP, which is consistent with the reported losses of 570 SOC in long-term maize cultivation in SSA (Sommer et al., 2018; Laub et al., 2022). It is also consistent with the postulate that closing SSA yield gaps will boost absolute N2O emissions (Leitner et al., 2020). However, the strong differences in the yield-scaled GWP between treatments, such as a 72, 32, 63 and 14 % lower yield-scaled GWP in the FYM 1.2+N treatment compared to the control-N treatment, show that the yield-scaled GWP is highly relevant in practical terms. Low-emission and high-yield ISFM treatments, such as FYM 1.2+N, producing between 2 and 4 t of yield per season at emissions between 0.2 575 and 1 kg CO2 equivalent per kg of yield, are a suitable mitigation practice compared to standard practice, control with little or no inputs of organic or chemical fertilizer. 4.4 N2O emissions and global warming potential In general, the poor match between daily observed and measured N2O emissions (Fig. A10) illustrates the difficulty of simulat- ing the timing of microbial processes, in which nitrate (NO3-) is converted to N2 and N2O gasses, with intermediate complexity 550 models such as DayCent. Several recent studies show that poor simulation of N2O emissions by process models is common (Zhang and Yu, 2021; Wang et al., 2020), which is attributed, among other factors, to a poor representation of soil hydraulics. For example, Sommer et al. (2016) found mainly overestimated N2O emissions in Kenya. Gaillard et al. (2018) reported that there is a bias in simulated N2O emissions by most models, postulating an underestimation of strong N2O flushes. This also seems to be the case in our study, at least for treatments with high N inputs such as farmyard manure +N. Without resorting to 555 more intricate water flow equations, it could be difficult to make further improvements to the simulation of soil water content and N2O emissions, particularly in terms of accurately predicting daily and hourly events. DayCent is driven by inputs that are only resolved at daily time steps and fed with pedotransfer functions that are poorly represented in the tropics (Van Looy et al., 2017). However, the fact that cumulative N2O emissions were better captured than daily emissions and that there was no seems to be the case in our study, at least for treatments with high N inputs such as farmyard manure +N. Without resorting to 555 more intricate water flow equations, it could be difficult to make further improvements to the simulation of soil water content and N2O emissions, particularly in terms of accurately predicting daily and hourly events. DayCent is driven by inputs that are only resolved at daily time steps and fed with pedotransfer functions that are poorly represented in the tropics (Van Looy et al., 2017). However, the fact that cumulative N2O emissions were better captured than daily emissions and that there was no systematic under- or over-prediction of cumulative N2O emissions, does suggest that simulated N2O emissions were generally 560 reasonably well predicted with this current DayCent calibration. This is important for the predictions of the GWP. Because the simulation of SOC change showed low bias, we can conclude that this part of the GWP is well represented. 4.4 N2O emissions and global warming potential Depending on the site and treatment, N2O emissions contributed between 80% (Aludeka) and up to 20% of the GWP (other sites; Fig. 8). However, the larger confidence intervals of the measured compared to the simulated cumulative N2O emissions suggest systematic under- or over-prediction of cumulative N2O emissions, does suggest that simulated N2O emissions were generally 560 reasonably well predicted with this current DayCent calibration. This is important for the predictions of the GWP. Because the simulation of SOC change showed low bias, we can conclude that this part of the GWP is well represented. Depending on the site and treatment, N2O emissions contributed between 80% (Aludeka) and up to 20% of the GWP (other sites; Fig. 8). However, the larger confidence intervals of the measured compared to the simulated cumulative N2O emissions suggest that the DayCent model cannot fully represent the variability. Although DayCent’s simulation of N2O emissions is superior to 565 using emission factors (dos Reis Martins et al., 2022), simulating N2O emissions remains challenging due to the complexity of the processes involved and the high temporal and spatial variability. Despite this uncertainty, which has not yet been resolved, our analysis of yield-scaled and area-based GWP showed the i f h i f f O d li l h h ISFM i h i i b that the DayCent model cannot fully represent the variability. Although DayCent’s simulation of N2O emissions is superior to 565 using emission factors (dos Reis Martins et al., 2022), simulating N2O emissions remains challenging due to the complexity of the processes involved and the high temporal and spatial variability. Despite this uncertainty, which has not yet been resolved, our analysis of yield-scaled and area-based GWP showed the importance of the unit of reference. Our modeling results show that ISFM with maize monocropping cannot be seen as a Despite this uncertainty, which has not yet been resolved, our analysis of yield-scaled and area-based GWP showed the importance of the unit of reference. Our modeling results show that ISFM with maize monocropping cannot be seen as a negative emission technology. All treatments had a positive absolute GWP, which is consistent with the reported losses of 570 SOC in long-term maize cultivation in SSA (Sommer et al., 2018; Laub et al., 2022). It is also consistent with the postulate that closing SSA yield gaps will boost absolute N2O emissions (Leitner et al., 2020). https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 4.5 DayCent suitable to upscale simulations of "real" ISFM, but not sensitive for very high N inputs In the absence of major pests (which in the trials were controlled), the yearly variation in precipitation and temperature should be responsible for the differences, and if these are not well represented, we cannot trust that we can apply DayCent outside of the climatic range that it was calibrated for. 600 the long-term effect of relevant ISFM treatments on soil fertility, yield, and greenhouse gas emissions as well as their trade- 595 offs. Since year-to-year variations of yield were not captured very well, it is questionable how well the current calibration could represent scenarios of climate change, where temperature and precipitation patterns will become more erratic. In the absence of major pests (which in the trials were controlled), the yearly variation in precipitation and temperature should be responsible for the differences, and if these are not well represented, we cannot trust that we can apply DayCent outside of the climatic range that it was calibrated for. 600 4.5 DayCent suitable to upscale simulations of "real" ISFM, but not sensitive for very high N inputs Because mean yields were represented well by the calibrated version of DayCent, it can be used to upscale to national levels and predict the potential of ISFM to lower yield gaps. However, the levelling off of mean yields at very high N loads (Fig. A6) indicates that it may not be suitable to estimate the maximum achievable yields (e.g., Ittersum et al., 2016) and it should 585 be restricted to predictions for medium input levels of N. Given that the historical rates of N fertilizer application in Kenya were less than 50 kg of N ha-1 (World-Bank, 2021a), the model seems suitable to simulate the effect of applying real ISFM, which aims at maximum N use efficiency (Vanlauwe et al., 2010), with N rates considerably below the maximum N rates of the simulated field trials (e.g., 80 kg N per season; Mutuku et al., 2020). The prediction of mean yields worked well for Calliandra and farmyard manure treatments at 1.2 and 4 t C ha-1 in the -N treatment and was also acceptable for CT+N, i.e. all 590 treatments that supply N at the desired rate for ISFM. Hence, at these N-levels, simulated mean yields are likely representative of the ISFM yield potential. The poorer performance of sawdust is less relevant for upscaling, because this is a treatment for scientific understanding (testing if SOC really forms more efficient from lignin rich materials; Palm et al., 2001b). In summary, the mean yield potential and the change in the SOC stocks were well represented, so the calibration seems suitable for assessing the long-term effect of relevant ISFM treatments on soil fertility, yield, and greenhouse gas emissions as well as their trade- 595 offs. Since year-to-year variations of yield were not captured very well, it is questionable how well the current calibration could represent scenarios of climate change, where temperature and precipitation patterns will become more erratic. In the absence of major pests (which in the trials were controlled), the yearly variation in precipitation and temperature should be responsible for the differences, and if these are not well represented, we cannot trust that we can apply DayCent outside of the climatic range that it was calibrated for. 600 and predict the potential of ISFM to lower yield gaps. However, the levelling off of mean yields at very high N loads (Fig. 4.4 N2O emissions and global warming potential The difference in the yield-scaled GWP ranges of +N treatments that existed between western Kenyan sites (Aludeka and Sidada; 0.1 to 0.5 kg CO2 equivalent per kg of yield) compared to central Kenyan sites (Embu and Machanga; 0.8 to 1.1 kg CO2 equivalent per kg of yield), furthermore, show that N fertilizer should only high-yield ISFM treatments, such as FYM 1.2+N, producing between 2 and 4 t of yield per season at emissions between 0.2 575 and 1 kg CO2 equivalent per kg of yield, are a suitable mitigation practice compared to standard practice, control with little or no inputs of organic or chemical fertilizer. The difference in the yield-scaled GWP ranges of +N treatments that existed between western Kenyan sites (Aludeka and Sidada; 0.1 to 0.5 kg CO2 equivalent per kg of yield) compared to central Kenyan sites (Embu and Machanga; 0.8 to 1.1 kg CO2 equivalent per kg of yield), furthermore, show that N fertilizer should only be applied to responsive soils (Sileshi et al., 2022) to avoid high N2O emissions at minimal yield. Consequently, sustainable 580 intensification and mitigation of greenhouse gases can go hand in hand. be applied to responsive soils (Sileshi et al., 2022) to avoid high N2O emissions at minimal yield. Consequently, sustainable 580 intensification and mitigation of greenhouse gases can go hand in hand. 26 4.5 DayCent suitable to upscale simulations of "real" ISFM, but not sensitive for very high N inputs A6) indicates that it may not be suitable to estimate the maximum achievable yields (e.g., Ittersum et al., 2016) and it should 585 be restricted to predictions for medium input levels of N. Given that the historical rates of N fertilizer application in Kenya were less than 50 kg of N ha-1 (World-Bank, 2021a), the model seems suitable to simulate the effect of applying real ISFM, which aims at maximum N use efficiency (Vanlauwe et al., 2010), with N rates considerably below the maximum N rates of the simulated field trials (e.g., 80 kg N per season; Mutuku et al., 2020). The prediction of mean yields worked well for A6) indicates that it may not be suitable to estimate the maximum achievable yields (e.g., Ittersum et al., 2016) and it should 585 be restricted to predictions for medium input levels of N. Given that the historical rates of N fertilizer application in Kenya were less than 50 kg of N ha-1 (World-Bank, 2021a), the model seems suitable to simulate the effect of applying real ISFM, which aims at maximum N use efficiency (Vanlauwe et al., 2010), with N rates considerably below the maximum N rates of the simulated field trials (e.g., 80 kg N per season; Mutuku et al., 2020). The prediction of mean yields worked well for Calliandra and farmyard manure treatments at 1.2 and 4 t C ha-1 in the -N treatment and was also acceptable for CT+N, i.e. all 590 treatments that supply N at the desired rate for ISFM. Hence, at these N-levels, simulated mean yields are likely representative of the ISFM yield potential. The poorer performance of sawdust is less relevant for upscaling, because this is a treatment for scientific understanding (testing if SOC really forms more efficient from lignin rich materials; Palm et al., 2001b). In summary, the mean yield potential and the change in the SOC stocks were well represented, so the calibration seems suitable for assessing the long-term effect of relevant ISFM treatments on soil fertility, yield, and greenhouse gas emissions as well as their trade- 595 offs. Since year-to-year variations of yield were not captured very well, it is questionable how well the current calibration could represent scenarios of climate change, where temperature and precipitation patterns will become more erratic. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Code availability. An earlier version of DayCent is freely available under https://www.nrel.colostate.edu/projects/century/century-downloads. 615 php. For the latest version, we suggest to contact DayCent developers directly, who in our case kindly provided the latest DayCent version. Code availability. An earlier version of DayCent is freely available under https://www.nrel.colostate.edu/projects/century/century-downloads. 615 php. For the latest version, we suggest to contact DayCent developers directly, who in our case kindly provided the latest DayCent version. Data availability. The data sets used for the calibration of this study are available under the IITA data repository. For SOC: https://doi.org/ 10.25502/wdh5-6c13/d. For yields and biomass: https://doi.org/10.25502/be9y-xh75/d. Data availability. The data sets used for the calibration of this study are available under the IITA data repository. For SOC: https://doi.org/ 10.25502/wdh5-6c13/d. For yields and biomass: https://doi.org/10.25502/be9y-xh75/d. Author contributions. JS, MN and ML designed the modeling exercise. ML summarized the data, conducted the modeling exercise and prepared the original draft. MWMM, DM, RY, SMN and WW managed and maintained the long-term trials. ML, SMN, MN, WW, MvdB, 620 MC and JS were involved in the various sampling campaigns. MC, MN, BV and JS acquired funding for the long-term trials. All coauthors contributed in writing and editing of the final submitted article. Author contributions. JS, MN and ML designed the modeling exercise. ML summarized the data, conducted the modeling exercise and prepared the original draft. MWMM, DM, RY, SMN and WW managed and maintained the long-term trials. ML, SMN, MN, WW, MvdB, 620 MC and JS were involved in the various sampling campaigns. MC, MN, BV and JS acquired funding for the long-term trials. All coauthors contributed in writing and editing of the final submitted article. 5 Conclusions In this study, we demonstrated the effectiveness of jointly calibrating the SOM and plant modules of DayCent to simulate maize productivity under integrated soil fertility management (ISFM) in Kenya, using a Bayesian calibration. The calibration successfully constrained the most sensitive model parameters, which were identified by a global sensitivity analysis. Although successfully constrained the most sensitive model parameters, which were identified by a global sensitivity analysis. Although the default DayCent maize plant parameterization represented the tropical conditions in Kenya well (i.e., the highest probabil- 605 ity posterior parameter values were close to the default parameterization), the highest probability posterior SOM turnover rates were two and eight times faster than the default for the passive and slow SOM pool, respectively. This indicates that there is the potential for large losses of SOC in highly weathered tropical soils. However, SOM turnover was subject to high uncertainty, showing that the current module structure suboptimally captures SOM dynamics in highly weathered tropical soils. Never- theless, our leave-one-site out cross validation showed that the calibration-derived parameter set is robust for upscaling of the 610 model to larger areas in Kenya. At the same time, mean maize grain yields were much better represented than the year-to-year variability of yields, posing the question whether the effects of climate change can be adequately captured by DayCent. Finally, the default DayCent maize plant parameterization represented the tropical conditions in Kenya well (i.e., the highest probabil- 605 ity posterior parameter values were close to the default parameterization), the highest probability posterior SOM turnover rates were two and eight times faster than the default for the passive and slow SOM pool, respectively. This indicates that there is the potential for large losses of SOC in highly weathered tropical soils. However, SOM turnover was subject to high uncertainty, showing that the current module structure suboptimally captures SOM dynamics in highly weathered tropical soils. Never- theless, our leave-one-site out cross validation showed that the calibration-derived parameter set is robust for upscaling of the 610 model to larger areas in Kenya. At the same time, mean maize grain yields were much better represented than the year-to-year variability of yields, posing the question whether the effects of climate change can be adequately captured by DayCent. Finally, while no ISFM treatment was predicted to act as an absolute sink of greenhouse gases, yield-scaled emissions were lowest in treatments with high and intermediate yields. 27 27 Acknowledgements. This study was supported by funds from the European Union‘s Horizon2020 framework (LANDMARC; Grant agree- ment ID 869367) the Swiss National Science Foundation (SNSF; grant number 172940) and by the DSCATT project “Agricultural Inten- 625 sification and Dynamics of Soil Carbon Sequestration in Tropical and Temperate Farming Systems” (N° AF 1802-001, N° FT C002181), supported by the Agropolis Foundation (“Programme d’Investissement d’Avenir” Labex Agro, ANR-10-LABX-0001-01) and by the TOTAL Foundation within a patronage agreement. We further acknowledge funding and technical support from the Tropical Soil Biology and Fertility Institute of CIAT (TSBF-CIAT), the International Institute of Tropical Agriculture (ITTA), and ETH Zurich in maintaining the experiments throughout many years. The AI language model "Writefull for Overleaf" has been used to improve the grammar of the manuscript. 630 Competing interests. All authors declare that they have no conflict of interest. Acknowledgements. This study was supported by funds from the European Union‘s Horizon2020 framework (LANDMARC; Grant agree- ment ID 869367) the Swiss National Science Foundation (SNSF; grant number 172940) and by the DSCATT project “Agricultural Inten- 625 sification and Dynamics of Soil Carbon Sequestration in Tropical and Temperate Farming Systems” (N° AF 1802-001, N° FT C002181), supported by the Agropolis Foundation (“Programme d’Investissement d’Avenir” Labex Agro, ANR-10-LABX-0001-01) and by the TOTAL Foundation within a patronage agreement. We further acknowledge funding and technical support from the Tropical Soil Biology and Fertility Institute of CIAT (TSBF-CIAT), the International Institute of Tropical Agriculture (ITTA), and ETH Zurich in maintaining the experiments throughout many years. The AI language model "Writefull for Overleaf" has been used to improve the grammar of the manuscript. 630 Acknowledgements. This study was supported by funds from the European Union s Horizon2020 framework (LANDMARC; Grant agree ment ID 869367) the Swiss National Science Foundation (SNSF; grant number 172940) and by the DSCATT project “Agricultural Inten- 625 sification and Dynamics of Soil Carbon Sequestration in Tropical and Temperate Farming Systems” (N° AF 1802-001, N° FT C002181), supported by the Agropolis Foundation (“Programme d’Investissement d’Avenir” Labex Agro, ANR-10-LABX-0001-01) and by the TOTAL Foundation within a patronage agreement. We further acknowledge funding and technical support from the Tropical Soil Biology and Fertility Institute of CIAT (TSBF-CIAT), the International Institute of Tropical Agriculture (ITTA), and ETH Zurich in maintaining the experiments 28 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Appendix A: Appendix A1 Site and organic resource characteristics Table A1. Locations, soil properties and climatic conditions of the study sites. Soil properties are given for the 0 - 15 cm depth layer. Coordinates are given in the WGS 84 reference system. Soil characteristics Embu Machanga Sidada Aludeka Latitude -0.517 -0.793 0.143 0.574 Longitude 37.459 37.664 34.422 34.191 Initial soil C (%) 2.9 0.3 1.54 0.83 Initial N (%) 0.3 0.02 0.12 0.08 Initail bulk density (g cm-3) 1.26 1.51 1.3 1.45 pH (H2O) 5.43 5.27 5.4 5.49 Clay (%) 59.8 13.2 55.7 13.4 Soil type (FAO, 1998) Humic Nitisol Ferric Alisol Humic Ferralsol Acrisol Altitude (m)* 1380 1022 1420 1180 Annual rainfall (mm)* 1175 795 1730 1660 Mean annual temperature (◦C) 20.1 23.7 22.6 24.4 Months of long rainy season 3 - 8 3 - 8 3 - 9 3 - 9 Months of short rainy season 10 - 01 10 - 01 10 - 01 10 - 01 *Means calculated based on measured data from 2005 to 2020 A1 Site and organic resource characteristics Table A1. Locations, soil properties and climatic conditions of the study sites. Soil properties are given for the 0 - 15 cm depth layer. Coordinates are given in the WGS 84 reference system. Table A1. Locations, soil properties and climatic conditions of the study sites. Soil properties are given for the 0 - 15 cm depth layer. Coordinates are given in the WGS 84 reference system. Soil characteristics Embu Machanga Sidada Aludeka Latitude -0.517 -0.793 0.143 0.574 Longitude 37.459 37.664 34.422 34.191 Initial soil C (%) 2.9 0.3 1.54 0.83 Initial N (%) 0.3 0.02 0.12 0.08 Initail bulk density (g cm-3) 1.26 1.51 1.3 1.45 pH (H2O) 5.43 5.27 5.4 5.49 Clay (%) 59.8 13.2 55.7 13.4 Soil type (FAO, 1998) Humic Nitisol Ferric Alisol Humic Ferralsol Acrisol Altitude (m)* 1380 1022 1420 1180 Annual rainfall (mm)* 1175 795 1730 1660 Mean annual temperature (◦C) 20.1 23.7 22.6 24.4 Months of long rainy season 3 - 8 3 - 8 3 - 9 3 - 9 Months of short rainy season 10 - 01 10 - 01 10 - 01 10 - 01 *Means calculated based on measured data from 2005 to 2020 29 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table A3. DayCent model parameters (and feasible ranges) of parameters which were not included in the Bayesian model calibration due to a Sobol total sensitivity index < 1%. Range Initial Lower Upper Model Parameter Description width Units value value value file frtc(2) C allocated to roots at time frtc(3) without stress small fraction of NPP 0.20 0.15 0.25 crop.100 frtc(4) Max. increase in C going to roots under stress small fraction of NPP 0.10 0.08 0.12 crop.100 frtc(5) Max. increase in C going to roots under stress (maturity) small fraction of NPP 0.10 0.08 0.12 crop.100 biomax AGB at which min.and max. C/E ratios of plant increases small g biomass m-2 700.00 525.00 875.00 crop.100 pramx(1,2) Max. aboveground C/N ratio with biomass > biomax small C/N ratio 125.00 93.75 156.25 crop.100 prbmn(1,1) For computing min. C/N ratio for belowground matter small C/N ratio 45.00 33.75 56.25 crop.100 efrgrn(1) Fraction of above ground N which goes to grain. small fraction 0.75 0.56 0.94 crop.100 flig(1,1) Intercept for annual rainfall effect on lignin content small fraction of lignin 0.12 0.09 0.15 crop.100 ppdf(3) Right curve shape for temperature effect on growth curve very small unitless 1.00 0.90 1.10 crop.100 ppdf(4) Right curve shape for temperature effect on growth curve very small unitless 2.50 2.25 2.75 crop.100 favail(1) Fraction of N available per day to plants moderate fraction of N 0.15 0.07 0.22 crop.100 (aneref(1)-anaref(2)) Rain/ET ratio below which, no effect of anaerobiosis small unitless 1.00 0.50 1.50 fix.100 aneref(2) Rain/ET ratio with max. anaerobiosis effect moderate unitless 3.00 1.50 4.50 fix.100 damr(1,1)&(2,1) Fraction of surface N and soil N absorbed by residue large fraction of N 0.02 0.01 0.06 fix.100 damrmn(1) Min. C/N ratio allowed in residue after direct absorption moderate C/N 15.00 7.50 22.50 fix.100 dec1(2) Max. structural litter turnover small g g-1 yr-1 4.90 3.68 6.12 fix.100 dec2(2) Max. metabolic litter turnover small g g-1 yr-1 18.50 13.88 23.12 fix.100 dec3(2) Max. active pool turnover small g g-1 yr-1 7.30 5.47 9.12 fix.100 (decX(2)/decX(1)) Ratio soil to surface turnover (newly defined parameter) small unitless 1.25 0.94 1.56 fix.100 fwloss(1) Scaling factor; interception & evaporation by biomass moderate unitless 1.00 0.50 1.50 fix.100 fwloss(2) Scaling factor; bare soil precipitation evaporation moderate unitless 1.00 0.50 1.50 fix.100 fwloss(3) Scaling factor; transpiration water loss moderate unitless 1.00 0.50 1.50 fix.100 pabres Residue amount which results in max. direct N absorption moderate g C m-2 100.00 50.00 150.00 fix.100 teff(2) Y location of temperature inflection point (decomposition) large unitless 11.75 3.92 35.25 fix.100 teff(3) Step size of temperature effect on decomposition moderate unitless 29.70 14.85 44.55 fix.100 teff(4) Inflection point slope of temperature effect (decomposition) very large unitless 0.25 0.02 2.46 fix.100 varat11&12(1,1) Max. C/N ratio for material entering active pool small C/N 20.00 15.00 25.00 fix.100 varat11&12(2,1) Min. C/N ratio for material entering active pool small C/N 3.00 2.25 3.75 fix.100 varat21&22(1,1) Max. C/N ratio for material entering slow pool small C/N 20.00 15.00 25.00 fix.100 varat3(1,1) Max. C/N ratio for material entering passive pool small C/N 13.00 9.75 16.25 fix.100 varat3(2,1) Min. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table A2. Mean measured chemical characteristics (and 95% confidence intervals) of organic resources applied at all sites. Measurements were available from Embu and Machanga from 2002 to 2004, all sites from 2005 to 2007 and in 2018. Significant differences in residue properties were found between the different organic resources, but not between sites and years. Same letters within the same row indicate the absence of significant differences for that property (p < 0.05). Abbreviations: n.c. = not classified * according to Palm et al. (2001a). Measured property Tithonia Calliandra Maize stover Sawdust Farmyard manure C (g kg-1) 345b (333-357) 396c (383-409) 397c (386-408) 433d (416-449) 234a (213-255) N (g kg-1) 33.2d (28.9-38.2) 32.5d (28.3-37.3) 7.2b (6.5-8) 2.5a (2.1-2.8) 18.1c (15-21.8) C/N ratio 12.4a (10.8-14.1) 13.6a (11.9-15.5) 58.7b (52.8-65.2) 199.1c (174.1-227.7) 12.3a (9.9-15.4) P (g kg-1) 2.3d (1.8-2.9) 1.1c (0.8-1.5) 0.4b (0.3-0.6) 0.1a (0-0.2) 3.1d (2.3-3.9) K (g kg-1) 37.2c (21.2-65.2) 8.7b (5-15.3) 9b (6-13.5) 2.8a (1.6-4.9) 19.4bc (7.8-48.6) Lignin (g kg-1) 90ab (62-117) 105b (77-133) 48a (37-60) 172c (144-199) 198c (154-242) Polyphenols (g kg-1) 19c (14.9-24.3) 108.7d (85.3-138.6) 11.3b (9.5-13.6) 4.9a (3.8-6.2) 7.8ab (5.2-11.5) Lignin/N ratio 2.6a (1.8-3.7) 3.1ab (2.2-4.3) 6.2c (4.8-8) 58.3d (41.1-82.8) 6.9bc (3.9-12.3) Quality / turnover rate* High / fast High / slow Low / fast Low / slow n.c. Class* 1 2 3 4 n.c. kg N in 4.0 t C ha-1 yr-1, -N [+N] 323 [563] 295 [535] 68 [308] 20 [260] 324 [564] kg N in 1.2 t C ha-1 yr-1, -N [+N] 97 [337] 88 [328] 20 [260] 6 [246] 97 [337] 30 C/N ratio for material entering passive pool small C/N 6.00 4.50 7.50 fix.100 drain Fraction of excess water lost by drainage fraction of H2O 0.80 0.20 0.90 site.100 dmp_st Damping factor for calculating soil temperature by layer large unitless 0.00 0.00 0.01 sitepar.in N2Oadjust_(max-min) Proportion of nitrified N that is lost as N2O (difference) large fraction of N 0.00 0.00 0.01 sitepar.in Ncoeff Min water/temperature limitation coefficient (nitrification) large unitless 0.03 0.01 0.09 sitepar.in dmpflux The damping factor for soil water flux large unitless 0.00 0.00 0.00 sitepar.in astlig_TD lignin fraction content of organic matter small g g-1 biomass 0.09 0.07 0.11 omad.100 astrec(1)_TD C/N ratio of added organic matter very small C/N ratio 12.40 11.16 13.64 omad.100 astlig_CC lignin fraction content of organic matter small g g-1 biomass 0.10 0.08 0.13 omad.100 astrec(1)_CC C/N ratio of added organic matter very small C/N ratio 13.60 12.24 14.96 omad.100 astlig_MS lignin fraction content of organic matter small g g-1 biomass 0.05 0.04 0.06 omad.100 astrec(1)_MS C/N ratio of added organic matter very small C/N ratio 58.70 52.83 64.57 omad.100 astlig_SD lignin fraction content of organic matter small g g-1 biomass 0.17 0.13 0.21 omad.100 astrec(1)_SD C/N ratio of added organic matter very small C/N ratio 199.10 179.19 219.01 omad.100 astlig_FYM lignin fraction content of organic matter small g g-1 biomass 0.20 0.15 0.25 omad.100 astrec(1)_FYM C/N ratio of added organic matter small C/N ratio 12.30 11.07 13.53 omad.100 Table A3. DayCent model parameters (and feasible ranges) of parameters which were not included in the Bayesian model calibration due to a Sobol total sensitivity index < 1%. 31 A1 Posterior, when allowing even larger ranges Figure A1. Prior compared to posterior parameter distribution resulting from increasing the ranges of the uncertainty based Bayesian cali- bration. Dashed vertical lines represent the default parameter sets. The posterior distributions are based on all four sites combined. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. A1 Posterior, when allowing even larger ranges A1 Posterior, when allowing even larger ranges A1 Posterior, when allowing even larger ranges Figure A1. Prior compared to posterior parameter distribution resulting from increasing the ranges of the uncertainty based Bayesian cali- bration. Dashed vertical lines represent the default parameter sets. The posterior distributions are based on all four sites combined. Figure A1. Prior compared to posterior parameter distribution resulting from increasing the ranges of the uncertainty based Bayesian cali- bration. Dashed vertical lines represent the default parameter sets. The posterior distributions are based on all four sites combined. 32 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. x himax 27 30 2.0 3.0 0.2 0.6 1.0 0.10 0.25 0.3 0.6 0.9 0.40 0.55 27 30 0.072 x Density ppdf.1. 0.047 0.45*** x Density ppdf.2. 42 46 2.0 3.0 −0.30*** 0.63*** −0.06 x Density prdx.1. −0.048 −0.029 −0.01 −0.012 x Density clteff.1.2..4. 5 15 30 0.2 0.6 1.0 −0.0053 −0.00054 −0.12 0.083 0.014 x Density aneref.3. −0.19** 0.14 * 0.033 0.14 * −0.033 −0.059 x Density dec4 0.020 0.035 0.10 0.25 0.13 . −0.11 −0.037 −0.14 * −0.30*** −0.14 . −0.068 x Density dec5.2. 0.35*** 0.026 −0.054 0.24*** 0.067 0.034 −0.072 −0.096 x Density fwloss.4. 0.80 1.05 0.3 0.6 0.9 −0.048 −0.047 −0.041 0.036 −0.43*** −0.09 −0.17 * −0.20** 0.012 x Density pmco2.1..2. 0.40 0.55 0.095 0.0017 42 46 −0.011 −0.011 5 15 30 −0.24*** 0.01 0.020 0.035 −0.11 −0.0027 0.80 1.05 0.043 0.072 0.4 0.7 0.4 0.7 x Density ps1co2.1..2.....rsplig gure A2. Correlation of parameters from the posterior parameter sets. The posterior distributions are based on all four sites combined. Figure A2. Correlation of parameters from the posterior parameter sets. The posterior distributions are based on all four sites combined. 33 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. A2 Barplots of SOC and comparing measured and simulated mean yield −N 0 t C −N 1.2 t C −N 4 t C +N 0 t C +N 1.2 t C +N 4 t C Aludeka Embu Machanga Sidada Control Calliandra Farmyard manure Maize stover Sawdust Tithonia Calliandra Farmyard manure Maize stover Sawdust Tithonia Control Calliandra Farmyard manure Maize stover Sawdust Tithonia Calliandra Farmyard manure Maize stover Sawdust Tithonia −30 −20 −10 0 −30 −20 −10 0 −30 −20 −10 0 −30 −20 −10 0 Difference of SOC stocks in 2021 compared to start year (t ha−1 ) measured simulated Figure A3. Barplots of simulated and measured change of SOC stocks until 2021 from cross-validation, at the four study sites for the different organic resource and chemical nitrogen fertilizer treatments. Error bars represent 95% confidence intervals based on BC (simulations) and variance (measurements). A2 Barplots of SOC and comparing measured and simulated mean yield A2 Barplots of SOC and comparing measured and simulated mean yield Figure A3. Barplots of simulated and measured change of SOC stocks until 2021 from cross-validation, at the four study sites for the different organic resource and chemical nitrogen fertilizer treatments. Error bars represent 95% confidence intervals based on BC (simulations) and variance (measurements). 34 34 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0 t C Control 1.2 t C Calliandra 1.2 t C Farmyard manure 1.2 t C Maize stover 1.2 t C Sawdust 1.2 t C Tithonia 4 t C Calliandra 4 t C Farmyard manure 4 t C Maize stover 4 t C Sawdust 4 t C Tithonia −N Aludeka +N Aludeka −N Embu +N Embu −N Machanga +N Machanga −N Sidada +N Sidada 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 Maize grain yield (t ha−1) measured simulated 0 t C Control 1.2 t C Calliandra 1.2 t C Farmyard manure 1.2 t C Maize stover 1.2 t C Sawdust 1.2 t C Tithonia 4 t C Calliandra 4 t C Farmyard manure 4 t C Maize stover 4 t C Sawdust 4 t C Tithonia −N Aludeka +N Aludeka −N Embu +N Embu −N Machanga +N Machanga −N Sidada +N Sidada 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 Maize grain yield (t ha−1) measured simulated Figure A5. Measured and simulated maize grain yields over time, at the four study sites for the different organic resource and chemical nitrogen fertilizer treatments for the uncalibrated (top) and calibrated DayCent model (bottom). https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Machanga Sidada Aludeka Embu 0.0 2.5 5.0 7.5 0.0 2.5 5.0 7.5 2 4 6 2 4 6 Measured mean yield (t ha−1 ) Simulated mean yield (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Machanga Sidada Aludeka Embu 0 5 10 15 0 5 10 15 5 10 5 10 Measured mean AGB (t ha−1 ) Simulated mean AGB (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Figure A4. Mean simulated versus mean measured yield and aboveground biomass (AGB) from the leave-one-site-out cross-validation. Error bars represent the standard deviation of measured and simulated values over all years. Machanga Sidada Aludeka Embu 0 5 10 15 0 5 10 15 5 10 5 10 Measured mean AGB (t ha−1 ) Simulated mean AGB (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N 2 6 Simulated mean yield (t ha−1 ) Simulated mean AGB (t ha−1 ) Figure A4. Mean simulated versus mean measured yield and aboveground biomass (AGB) from the leave-one-site-out cross-validation. Error bars represent the standard deviation of measured and simulated values over all years. 35 0 t C Control 1.2 t C Calliandra 1.2 t C Farmyard manure 1.2 t C Maize stover 1.2 t C Sawdust 1.2 t C Tithonia 4 t C Calliandra 4 t C Farmyard manure 4 t C Maize stover 4 t C Sawdust 4 t C Tithonia −N Aludeka +N Aludeka −N Embu +N Embu −N Machanga +N Machanga −N Sidada +N Sidada 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 2005 2010 2015 2020 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 Maize grain yield (t ha−1) measured simulated Figure A5. Measured and simulated maize grain yields over time, at the four study sites for the different organic resource and chemical nitrogen fertilizer treatments for the uncalibrated (top) and calibrated DayCent model (bottom). 36 A3 Site specific sensitivities of yield to N fertilizer 5 Aludeka Embu Machanga Sidada 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 2 4 6 Added mineral N in kg/ha/season Mean yield in t/ha Figure A6. N response curve by site using the calibrated DayCent parameters. This was done to explain why DayCent insensitive at high N levels. N was given in 50/50 split application, at the two real dates. A3 Site specific sensitivities of yield to N fertilizer 635 Aludeka Embu Machanga Sidada 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 2 4 6 Added mineral N in kg/ha/season Mean yield in t/ha Figure A6. N response curve by site using the calibrated DayCent parameters. This was done to explain why DayCent insensitive at high N Figure A6. N response curve by site using the calibrated DayCent parameters. This was done to explain why DayCent insensitive at high N levels. N was given in 50/50 split application, at the two real dates. 37 37 37 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. A5 Calibration improvement by Site y = c(−3.361) + c(−0.2327) ⋅x, r2 = 0.0277 EF: −8.888; RMSE: 10.36 SB(%): 65; NU(%): 25; LC(%): 10 y = c(1.449) + c(0.09625) ⋅x, r2 = 0.104 EF: −0.38; RMSE: 11.116 SB(%): 35; NU(%): 0; LC(%): 65 y = c(−1.046) + c(0.1284) ⋅x, r2 = 0.0354 EF: −1.236; RMSE: 9.028 SB(%): 46; NU(%): 11; LC(%): 43 y = c(−1.601) + c(−0.03131) ⋅x, r2 = 0.00404 EF: −2.033; RMSE: 15.099 SB(%): 57; NU(%): 10; LC(%): 33 Aludeka Embu Machanga Sidada −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 Measured loss of SOC stocks (t ha−1 in 0−20 cm) Simulated loss of SOC stocks (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N y = c(0.1423) + c(0.3214) ⋅x, r2 = 0.501 EF: −0.043; RMSE: 3.589 SB(%): 46; NU(%): 6; LC(%): 48 y = c(7.449) + c(0.621) ⋅x, r2 = 0.582 EF: 0.351; RMSE: 6.55 SB(%): 35; NU(%): 0; LC(%): 64 y = c(4.315) + c(0.4998) ⋅x, r2 = 0.658 EF: 0.598; RMSE: 4.035 SB(%): 6; NU(%): 10; LC(%): 85 y = c(3.468) + c(0.7397) ⋅x, r2 = 0.715 EF: 0.705; RMSE: 4.748 SB(%): 3; NU(%): 0; LC(%): 96 Aludeka Embu Machanga Sidada −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 Measured loss of SOC stocks (t ha−1 in 0−20 cm) Simulated loss of SOC stocks (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Figure A8. Simulated compared to measured changes in SOC stocks since experiment start for the default DayCent parameter set (top) vs the calibrated parameter set by leave-one-site-out cross-validation (bottom). Grey bands denote the 95% confidence intervals of measured (horizontal) values and the 95% credibility intervals of posterior distribution (vertical). https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. A4 SOC stocks A4 SOC stocks y = c(11.78) + c(0.9024) ⋅x, r2 = 0.842 EF: 0.685; RMSE: 11.711 SB(%): 49; NU(%): 1; LC(%): 50 0 25 50 75 100 0 25 50 75 100 Measured SOC stock (t ha−1 in 0−20 cm) Simulated SOC stock (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N y = c(11.78) + c(0.9024) ⋅x, r2 = 0.842 EF: 0.685; RMSE: 11.711 SB(%): 49; NU(%): 1; LC(%): 50 0 25 50 75 100 0 25 50 75 100 Measured SOC stock (t ha−1 in 0−20 cm) Simulated SOC stock (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N y = c(2.882) + c(0.8843) ⋅x, r2 = 0.954 EF: 0.945; RMSE: 5.031 SB(%): 7; NU(%): 9; LC(%): 84 46.7 % are actually in 95% CI 0 25 50 75 100 0 25 50 75 100 Measured SOC stock (t ha−1 in 0−20 cm) Simulated SOC stock (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Figure A7. SOC: 1st DayCent outputs initial (left) vs calibrated by leave-one-site-out cross-validation (right). Grey bands denote the 95% confidence intervals of measured (horizontal) and posterior simulated (vertical) values. y = c(2.882) + c(0.8843) ⋅x, r2 = 0.954 EF: 0.945; RMSE: 5.031 SB(%): 7; NU(%): 9; LC(%): 84 46.7 % are actually in 95% CI 0 25 50 75 100 0 25 50 75 100 Measured SOC stock (t ha−1 in 0−20 cm) Simulated SOC stock (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Simulated SOC stock (t ha−1 in 0−20 cm) Figure A7. SOC: 1st DayCent outputs initial (left) vs calibrated by leave-one-site-out cross-validation (right). Grey bands denote the 95% confidence intervals of measured (horizontal) and posterior simulated (vertical) values. 38 y = c(−3.361) + c(−0.2327) ⋅x, r2 = 0.0277 EF: −8.888; RMSE: 10.36 SB(%): 65; NU(%): 25; LC(%): 10 y = c(1.449) + c(0.09625) ⋅x, r2 = 0.104 EF: −0.38; RMSE: 11.116 SB(%): 35; NU(%): 0; LC(%): 65 y = c(−1.046) + c(0.1284) ⋅x, r2 = 0.0354 EF: −1.236; RMSE: 9.028 SB(%): 46; NU(%): 11; LC(%): 43 y = c(−1.601) + c(−0.03131) ⋅x, r2 = 0.00404 EF: −2.033; RMSE: 15.099 SB(%): 57; NU(%): 10; LC(%): 33 Aludeka Embu Machanga Sidada −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 Measured loss of SOC stocks (t ha−1 in 0−20 cm) Simulated loss of SOC stocks (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Calliandra Control Simulated loss of SOC stocks (t ha−1 in 0−20 cm) y = c(0.1423) + c(0.3214) ⋅x, r2 = 0.501 EF: −0.043; RMSE: 3.589 SB(%): 46; NU(%): 6; LC(%): 48 y = c(7.449) + c(0.621) ⋅x, r2 = 0.582 EF: 0.351; RMSE: 6.55 SB(%): 35; NU(%): 0; LC(%): 64 y = c(4.315) + c(0.4998) ⋅x, r2 = 0.658 EF: 0.598; RMSE: 4.035 SB(%): 6; NU(%): 10; LC(%): 85 y = c(3.468) + c(0.7397) ⋅x, r2 = 0.715 EF: 0.705; RMSE: 4.748 SB(%): 3; NU(%): 0; LC(%): 96 Aludeka Embu Machanga Sidada −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 −10 0 10 20 30 Measured loss of SOC stocks (t ha−1 in 0−20 cm) Simulated loss of SOC stocks (t ha−1 in 0−20 cm) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Figure A8. Simulated compared to measured changes in SOC stocks since experiment start for the default DayCent parameter set (top) vs the calibrated parameter set by leave-one-site-out cross-validation (bottom). Grey bands denote the 95% confidence intervals of measured (horizontal) values and the 95% credibility intervals of posterior distribution (vertical). Figure A8. Simulated compared to measured changes in SOC stocks since experiment start for the default DayCent parameter set (top) vs the calibrated parameter set by leave-one-site-out cross-validation (bottom). Grey bands denote the 95% confidence intervals of measured (horizontal) values and the 95% credibility intervals of posterior distribution (vertical). 39 https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. y = c(0.7133) + c(0.809) ⋅x, r2 = 0.491 EF: 0.281; RMSE: 1.771 SB(%): 1; NU(%): 29; LC(%): 71 y = c(1.953) + c(0.2664) ⋅x, r2 = 0.144 EF: 0.039; RMSE: 1.905 SB(%): 0; NU(%): 11; LC(%): 89 y = c(1.342) + c(0.4954) ⋅x, r2 = 0.339 EF: −0.023; RMSE: 1.341 SB(%): 28; NU(%): 7; LC(%): 65 y = c(1.388) + c(0.5738) ⋅x, r2 = 0.274 EF: −0.143; RMSE: 2.262 SB(%): 8; NU(%): 29; LC(%): 64 Aludeka Embu Machanga Sidada 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 Measured yield (t ha−1 ) Simulated yield (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N y = c(0.8786) + c(0.5927) ⋅x, r2 = 0.467 EF: 0.406; RMSE: 1.609 SB(%): 5; NU(%): 6; LC(%): 90 y = c(1.604) + c(0.3262) ⋅x, r2 = 0.275 EF: 0.251; RMSE: 1.681 SB(%): 2; NU(%): 1; LC(%): 97 y = c(0.9263) + c(0.3525) ⋅x, r2 = 0.387 EF: 0.376; RMSE: 1.047 SB(%): 1; NU(%): 0; LC(%): 98 y = c(2.441) + c(0.4596) ⋅x, r2 = 0.302 EF: 0.218; RMSE: 1.871 SB(%): 0; NU(%): 10; LC(%): 89 Aludeka Embu Machanga Sidada 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 Measured yield (t ha−1 ) Simulated yield (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Figure A9. Simulated compared to measured yield for the default DayCent parameter set (top) vs the calibrated parameter set by leave-one- site-out cross-validation (bottom). Grey bands denote the 95% confidence intervals of measured (horizontal) values and the 95% credibility intervals of posterior distribution (vertical). References L., and Sanderman, J.: Improving Soil Car- bon Estimates by Linking Conceptual Pools Against Measurable Carbon Fractions in the DAYCENT Model Version 4.5, Journal of Advances in Modeling Earth Systems, 14, e2021MS002 622, https://doi.org/10.1029/2021MS002622, _eprint: 660 https://onlinelibrary.wiley.com/doi/pdf/10.1029/2021MS002622, 2022. 660 4.5, Journal of Advances in Modeling Earth Systems, 14, e2021MS002 622, https://doi.org/10.1029/2021MS002622, _eprint: 660 https://onlinelibrary.wiley.com/doi/pdf/10.1029/2021MS002622, 2022. 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Chivenge, P., Vanlauwe, B., Gentile, R., Wangechi, H., Mugendi, D., Kessel, C. v., and Six, J.: Organic Chivenge, P., Vanlauwe, B., Gentile, R., Wangechi, H., Mugendi, D., Kessel, C. v., and Six, J.: Organic and Mineral Input Management to E h C P d i i i C l K A J l 101 1266 1275 h //d i /10 2134/ j2008 0188 2009 650 Chivenge, P., Vanlauwe, B., Gentile, R., Wangechi, H., Mugendi, D., Kessel, C. v., and Six, J.: Organic and Mineral Input Management to Enhance Crop Productivity in Central Kenya, Agronomy Journal, 101, 1266–1275, https://doi.org/10.2134/agronj2008.0188x, 2009. 650 Enhance Crop Productivity in Central Kenya, Agronomy Journal, 101, 1266–1275, https://doi.org/10.2134/agronj2008.0188x, 2009. y = c(0.7133) + c(0.809) ⋅x, r2 = 0.491 EF: 0.281; RMSE: 1.771 SB(%): 1; NU(%): 29; LC(%): 71 y = c(1.953) + c(0.2664) ⋅x, r2 = 0.144 EF: 0.039; RMSE: 1.905 SB(%): 0; NU(%): 11; LC(%): 89 y = c(1.342) + c(0.4954) ⋅x, r2 = 0.339 EF: −0.023; RMSE: 1.341 SB(%): 28; NU(%): 7; LC(%): 65 y = c(1.388) + c(0.5738) ⋅x, r2 = 0.274 EF: −0.143; RMSE: 2.262 SB(%): 8; NU(%): 29; LC(%): 64 Aludeka Embu Machanga Sidada 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 Measured yield (t ha−1 ) Simulated yield (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Simulated yield (t ha−1 ) c(0.8786) + c(0.5927) ⋅x, r2 = 0.467 0.406; RMSE: 1.609 %): 5; NU(%): 6; LC(%): 90 y = c(1.604) + c(0.3262) ⋅x, r2 = 0.275 EF: 0.251; RMSE: 1.681 SB(%): 2; NU(%): 1; LC(%): 97 y = c(0.9263) + c(0.3525) ⋅x, r2 = 0.387 EF: 0.376; RMSE: 1.047 SB(%): 1; NU(%): 0; LC(%): 98 y = c(2.441) + c(0.4596) ⋅x, r2 = 0.302 EF: 0.218; RMSE: 1.871 SB(%): 0; NU(%): 10; LC(%): 89 Aludeka Embu Machanga Sidada 5 10 0 5 10 0 5 10 0 5 10 Measured yield (t ha−1 ) Calliandra Control Farmyard manure Maize stover Sawdust Tithonia −N +N Simulated yield (t ha−1 ) Figure A9. Simulated compared to measured yield for the default DayCent parameter set (top) vs the calibrated parameter set by leave-one- site-out cross-validation (bottom). Grey bands denote the 95% confidence intervals of measured (horizontal) values and the 95% credibility intervals of posterior distribution (vertical). 40 A6 N2O emissions Figure A10. Temporal development of measured (black) vs simulated (red) N2O emissions by site. The black error bars represent the 95% confidence intervals due to spatial replication error, the red error bars represent the 95% credibility intervals of simulated N2O emissions resulting from parameter distribution of the posterior parameter set. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. A6 N2O emissions A6 N2O emissions Figure A10. Temporal development of measured (black) vs simulated (red) N2O emissions by site. 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Zhai, R., Tao, F., Lall, U., and Elliott, J.: Africa Would Need to Import More Maize in the Future Even Un- der 1.5°C Warming Scenario, Earth’s Future, 9, e2020EF001 574, https://doi.org/10.1029/2020EF001574, _eprint: https://agupubs.onlinelibrary.wiley.com/doi/pdf/10.1029/2020EF001574, 2021. 46 Zhang, Y. and Yu, Q.: Does agroecosystem model improvement increase simulation accuracy for agricultural N2O emissions?, Agricultural 825 and Forest Meteorology, 297, 108 281, https://doi.org/10.1016/j.agrformet.2020.108281, 2021. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-1738 Preprint. Discussion started: 21 August 2023 c⃝Author(s) 2023. CC BY 4.0 License. Zhang, Y. and Yu, Q.: Does agroecosystem model improvement increase simulation accuracy for agricultural N2O emissions?, Agricultural 825 and Forest Meteorology, 297, 108 281, https://doi.org/10.1016/j.agrformet.2020.108281, 2021. 47 47
https://openalex.org/W4288378088
https://www.aanda.org/articles/aa/pdf/2019/04/aa33777-18.pdf
English
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<i>Planck</i>’s Dusty GEMS
Astronomy & astrophysics
2,019
cc-by
13,765
1. Introduction can be associated with low-excitation gas seen in [CII] absorp- tion, as also found observationally in the Milky Way (Gerin et al. 2015) and at high redshift in the Garnet (PLCK_G045.1+61.1, Nesvadba et al. 2016), a source whose [CI] properties we also discuss here. Lines of [CI], [CII], and CO are therefore comple- mentary probes of the gas in high-redshift galaxies. Numerous observations in recent years have firmly established that the vigorous star-formation episodes in massive, dusty star- burst galaxies at redshifts z ≥2, which form most of the stellar populations in these galaxies within a few hundred million years, are fueled by massive reservoirs of dense molecular gas (e.g., Tacconi et al. 2008; Ivison et al. 2011; Riechers et al. 2013, see Solomon & Vanden Bout 2005 and Carilli & Walter 2013 for reviews). The physical and kinematic properties of this gas, such as densities and mass surface densities, temperatures, and bulk and turbulent motion, are critical for understanding the regula- tion of and upper limits imposed on the vigorous star formation up to the highest star-formation rates found in the universe. Atomic carbon, specifically, is probed through two bright transitions, 3P1–3P0, ([CI] 1–0), and 3P2–3P1 ([CI] 2–1) at rest- frame frequencies of 492.1607 GHz and 809.3435 GHz, respec- tively, which are conveniently redshifted into the millimeter and lower sub-millimeter regime for redshifts z ∼2−4. With upper level energies of Eup,10 = 24.2 K and Eup,21 = 62.5 K, and critical densities of about 103 cm−3, they are easily excited over large ranges in gas density and temperature, from fairly diffuse gas (Phillips & Huggins 1981; Gerin & Phillips 2000; Goldsmith et al. 2012) to gas within dense molecular clouds (Papadopoulos et al. 2004). This makes them useful global probes of the cold neutral medium in very high-redshift galaxies. Perhaps most importantly, both [CI] lines remain optically thin even in very dusty, vigorous starburst galaxies, which is a clear advantage for mass estimates ensuring that mass scales linearly with line lumi- nosity. However, other systematic uncertainties remain, such as those related to the carbon abundance, with uncertainties of fac- tors of a few (e.g., Alaghband-Zadeh et al. 2013). Observations of CO, in contrast, are notoriously plagued by uncertainties related to most of the gas being hidden within optically thick clouds. ⋆Based on observations obtained with the 30-m telescope and the Plateau de Bure interferometer of IRAM under program IDs 082-12, D05-12, D09-12, 094-13, 223-13, 108-14, and 217-14. Astronomy & Astrophysics Astronomy & Astrophysics A&A 624, A23 (2019) https://doi.org/10.1051/0004-6361/201833777 c⃝N. P. H. Nesvadba et al. 2019 Open Access article, published by EDP Sciences, 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. ABSTRACT The bright 3P1–3P0 ([CI] 1–0) and 3P2–3P1 ([CI] 2–1) lines of atomic carbon are becoming more and more widely employed as tracers of the cold neutral gas in high-redshift galaxies. Here we present observations of these lines in the 11 galaxies of the set of Planck’s Dusty GEMS, the brightest gravitationally lensed galaxies on the extragalactic submillimeter sky probed by the Planck satellite. We have [CI] 1–0 and [CI] 2–1 measurements for seven and eight of these galaxies, respectively, including four galaxies where both lines have been measured. We use our observations to constrain the gas excitation mechanism, excitation temperatures, optical depths, atomic carbon and molecular gas masses, and carbon abundances. Ratios of LCI/LFIR are similar to those found in the local universe, and suggest that the total cooling budget through atomic carbon has not significantly changed in the last 12 Gyr. Both lines are optically thin and trace 1−6 × 107 M⊙of atomic carbon. Carbon abundances, XCI, are between 2.5 and 4 × 10−5, for an ultra-luminous infrared galaxy (ULIRG) CO-to-H2 conversion factor of αCO = 0.8 M⊙/ [K km s−1 pc2]. Ratios of molecular gas masses derived from [CI] 1–0 and CO agree within the measurement uncertainties for five galaxies, and agree to better than a factor of two for another two with [CI] 1–0 measurements, after carefully taking CO excitation into account. This does not support the idea that intense, high-redshift starburst galaxies host large quantities of “CO-dark” gas. These results support the common assumptions underlying most molecular gas mass estimates made for massive, dusty, high-redshift starburst galaxies, although the good agreement between the masses obtained with both tracers cannot be taken as independent confirmation of either αCO or XCI. Key words. galaxies: high-redshift – galaxies: formation – galaxies: starburst – galaxies: abundances Planck’s Dusty GEMS VII. Atomic carbon and molecular gas in dusty starburst galaxies at z = 2 to 4⋆ N. P. H. Nesvadba1, R. Cañameras2, R. Kneissl3,4, S. Koenig5, C. Yang3, E. Le Floc’h6, A. Omont7, and D. Scott8 (Affiliations can be found after the references) Received 4 July 2018 / Accepted 20 December 2018 Received 4 July 2018 / Accepted 20 December 2018 1. Introduction The results of the analysis of the CO lines, and of detailed radiative transfer and photodissotiation region (PDR) models of the CO and [CI] lines, are presented in a companion paper (C18). Here we mainly focus on the physical and empirical properties that can be derived analytically and from [CI] alone, or that use [CI] for empirical constraints. This paper is organized as follows. In Sect. 2 we present the details of our [CI] observations and data reduction. In Sect. 3 we describe our analysis and how we corrected for gravitational lensing, including the possibility of differential lensing between gas and dust, which we constrain explicitly using sub-arcsecond interferometry. In Sect. 4 we use the [CI] lines to constrain the contribution of atomic carbon to line cooling in order to investi- gate the heating mechanism, [CI] excitation temperatures, opti- cal depths, abundances, and masses of atomic carbon. We also determine the gas distribution and starburst mode from the [CI] line fluxes and line ratios. In Sect. 5 we discuss the implications of our analysis for H2 gas mass estimates and the CO-to-H2 con- version factor. We summarize our results in Sect. 6. g A number of studies have therefore targeted atomic carbon in high-redshift galaxies, ranging from the ground-work laid by Brown & Vanden Bout (1992), Barvainis et al. (1997), and Weiß et al. (2005a), for example, to more recent studies, in particular of strongly gravitationally lensed starburst galaxies selected in the infrared (Walter et al. 2011; Alaghband-Zadeh et al. 2013; Bothwell et al. 2017; Yang et al. 2017; Andreani et al. 2018). Such studies found luminous line emission in either one or both lines. They also showed that atomic carbon can remain opti- cally thin out to the highest gas-mass surface densities and star- formation rates, and is consistent with high carbon abundances of a few times 10−5, similar to those found in low-redshift galax- ies (Gerin & Phillips 2000; Weiß et al. 2001; Israel & Baas 2002, 2003). This suggests that they have high metallicities akin to the solar value, with little difference between starburst galax- ies and quasars (Alaghband-Zadeh et al. 2013). The [CI] lines, combined with other far-infrared (FIR) and millimeter emission lines, also provide interesting constraints on the gas density and strength of UV radiation fields within star-forming regions (e.g., Kaufman et al. 1999; Le Petit et al. 2. Observations and data reduction The data we present here were obtained as part of several b i ith EMIR t th 30 t l f IRAM ith Here we present new observations of [CI] 1–0 and [CI] 2– 1 in a small set of 11 of the brightest gravitationally lensed sub-millimeter galaxies on the sky observed with the Planck all-sky survey (Planck Collaboration Int. XXVII 2015; Planck Collaboration Int. XXXIX 2016), Planck’s Dusty GEMS. This sample is smaller than those found with other infrared-to- millimeter surveys of high-redshift galaxies (Vieira et al. 2013; Wardlow et al. 2013), which are excellent data sets in their own right, but the GEMS are particularly bright dust continuum emit- ters, reflecting the high completeness limit of Planck at ∼600 mJy. Data were taken under a range of atmospheric conditions. For the 3 mm observations, precipitable water vapor columns (pwv) were mostly between 1 mm and 8 mm. A small part of the observ- ing time suffered from even higher pwv; including these scans did not improve the signal-to-noise ratios (S/Ns) in the final com- bined data sets, and so these scans were discarded. Observations at 1.3 mm were carried out when the pwv was 1 mm or less. g g p y All GEMS have spectroscopically confirmed redshifts of z = 2.2−3.6, and apparent FIR luminosities between 5 and 27 × 1013 µ L⊙(Cañameras et al. 2015), mainly powered by star formation and boosted by gravitational lensing from fore- ground clusters or massive individual galaxies by luminosity- averaged factors, µ ∼10−30 (Cañameras et al. 2017a, 2018a,b, C18 hereafter). Environments probed by these galaxies range from intense, maximally star-forming clumps (Cañameras et al. 2017b, 2018b) to diffuse gas probed by [CII] absorption (Nesvadba et al. 2016), as observed with ALMA and IRAM interferometry. Contamination from AGNs is very weak, con- tributing ≤10% (Cañameras et al. 2015) of the IR luminosity. We used the FTS and WILMA backends with Wobbler switching throws of 60′′, which is significantly larger than the diameter of our most extended sources, about 10′′. To point the telescope we used blind offsets from radio-loud quasars at dis- tances of a few degrees from our targets. We performed a point- ing approximately once every 2 h, and refocused the telescope every 3–4 h, and at sunrise and sunset. Individual scans were 30 s in duration, and we obtained a calibration after every 6 min of observing. 1. Introduction This adds considerable uncertainty when generalizing the results of these observations to the overall molecular gas component in galaxies, without falling back on empirical relationships whose use cannot always be justified easily for each individual galaxy and analysis. Thanks to the new generation of wide-band millimeter and sub-millimeter receivers, and sensitive interferometers like ALMA and IRAM’s NOEMA, we are now able to study these gaseous reservoirs in galaxies in the early universe at an inter- esting level of detail, extending and complementing the classi- cal CO emission-line studies through observations of additional tracers. This includes the fine-structure lines of atomic or singly ionized carbon, [CI] and [CII], which are excellent tracers of the cold neutral gas in galaxies, and various other tracers of denser gas. Beuther et al. (2016) argue that [CI] is the best tracer of the cold neutral medium because [CII] can also be associated with ionized gas whereas CO emission only probes fairly dense molecular gas, and misses more diffuse gas that is however seen in [CI]. Goldsmith et al. (2012) even argue that [CI] emission A23, page 1 of 11 A&A 624, A23 (2019) The [CI] fine structure lines also arise from physically rel- atively simple systems, meaning that many of their physical characteristics can be calculated directly from their observed properties and measured brightness temperatures or line fluxes; for example, masses and abundances, or their contribution to the cooling budget. Several studies also suggest that they are less sensitive than CO to variations in metallicity (which can lead to significant reservoirs of so-called “CO-dark” gas, e.g., Wolfire et al. 2010; Bolatto et al. 2013; Rémy-Ruyer et al. 2015; Balashev et al. 2017), abundance ratios, for example due to enhanced cosmic ray fluxes (Roueff& Flower 1992; Papadopoulos et al. 2004; Bisbas et al. 2015, 2017), X-ray heat- ing from AGN (Meijerink et al. 2007), or molecule destruction in shocks (e.g., Krips et al. 2016). Furthermore, Papadopoulos et al. (2004) and Papadopoulos & Greve (2004) established the [CI] 1–0 line as a tracer of gas in high-redshift galaxies that seems to be well mixed with the molecular gas. the goal of constraining the global gas properties of these galax- ies using several CO transitions and the two [CI] lines. In total, we observed 48 CO lines and 15 [CI] lines in 11 galaxies. 1. Introduction 2006), and can serve as a discriminant between the “starburst” and “disk” modes of star formation, where the latter is characterized by a larger fraction of diffuse gas (Geach & Papadopoulos 2012). Throughout the paper, we adopt the flat ΛCDM cos- mology from Planck Collaboration XVI (2014), with H0 = 68 km s−1 Mpc−1, Ωm = 0.31, and ΩΛ = 1−Ωm. For example, at z = 3.0 this implies a luminosity distance of 26.0 Gpc, and a projected physical scale of 7.9 kpc arcsec−1. 2. Observations and data reduction We obtained deep spectroscopy of several bright millimeter emission lines, including the [CI] lines presented here, with the wide-band millimeter receiver EMIR at the 30-m telescope of IRAM in several runs between November 2012 and February 2015. In total, we obtained between 81 and 171 min of on-source observing time per source. Individual observing dates and inte- gration times, tuning frequencies, and rms noise values are given in Table 1 for each source and emission line. The analysis of the CO lines is presented in C18. Depending on the redshift of each source, the [CI] 1–0 line either falls into the 3 mm or 2 mm band, and the [CI] 2– 1 line either into the 2 mm or the 1.3 mm band. In two sources, PLCK_G113.7+61.0 and PLCK_G138.6+62.0, the [CI] 1–0 line was used to confirm the spectroscopic redshift previously esti- mated from a blind line search in the 3 mm band (Cañameras et al. 2015). In most cases, the [CI] lines were observed with dual-band observations, that is, in parallel to other bright mil- limeter emission lines. Here we present new observations of [CI] 1–0 and [CI] 2– 1 in a small set of 11 of the brightest gravitationally lensed sub-millimeter galaxies on the sky observed with the Planck all-sky survey (Planck Collaboration Int. XXVII 2015; Planck Collaboration Int. XXXIX 2016), Planck’s Dusty GEMS. This sample is smaller than those found with other infrared-to- millimeter surveys of high-redshift galaxies (Vieira et al. 2013; Wardlow et al. 2013), which are excellent data sets in their own right, but the GEMS are particularly bright dust continuum emit- ters, reflecting the high completeness limit of Planck at ∼600 mJy. All GEMS have spectroscopically confirmed redshifts of z = 2.2−3.6, and apparent FIR luminosities between 5 and 27 × 1013 µ L⊙(Cañameras et al. 2015), mainly powered by star formation and boosted by gravitational lensing from fore- ground clusters or massive individual galaxies by luminosity- averaged factors, µ ∼10−30 (Cañameras et al. 2017a, 2018a,b, C18 hereafter). Environments probed by these galaxies range from intense, maximally star-forming clumps (Cañameras et al. 2017b, 2018b) to diffuse gas probed by [CII] absorption (Nesvadba et al. 2016), as observed with ALMA and IRAM interferometry. Contamination from AGNs is very weak, con- tributing ≤10% (Cañameras et al. 2015) of the IR luminosity. 2. Observations and data reduction We list the source name, right ascension and declination, redshift, luminosity distance, observed FIR luminosity, transition, tuning fre- quency, date of our observations, time spent on the target, and root mean square of the resulting spectrum. Dots indicate lines outside of the atmospheric windows. Notes. We list the source name, right ascension and declination, redshift, luminosity distance, observed FIR luminosity, transition, tuning fre- quency, date of our observations, time spent on the target, and root mean square of the resulting spectrum. Dots indicate lines outside of the atmospheric windows. Lline = 1.04 × 10−3 S line ∆v νrest (1 + z)−1 D2 L, (1) (1) resolutions of 0.195 and 2 MHz, respectively, and 16 GHz and 8 GHz of bandwidth, respectively, with horizontal and vertical polarizations observed in parallel. We typically rebinned the data tomoreappropriatespectralresolutionsbetween30and50 km s−1 (see Figs. 2 and 3). where S line∆v is the velocity-integrated line flux given in Jy km s−1, νrest the frequency in the rest-frame in GHz, z is the redshift, DL is the luminosity distance in megaparsecs, and Lline is given in solar luminosities. An alternative way to express line luminosities is by setting where S line∆v is the velocity-integrated line flux given in Jy km s−1, νrest the frequency in the rest-frame in GHz, z is the redshift, DL is the luminosity distance in megaparsecs, and Lline is given in solar luminosities. An alternative way to express line luminosities is by setting All data were calibrated at the telescope and reduced with the CLASS package of the GILDAS software of IRAM (Gildas Team 2013). We inspected all individual scans by eye and used simple first-order polynomials to correct the baselines, after carefully masking the spectral range expected to be covered by the bright emission lines. For scans with strong “platforming” in their FTS spectra, we used the rou- tine FtsPlatformingCorrection5.class kindly provided by C. Kramer to obtain individual spectral scans with reasonably flat baselines. We used the values given on the EMIR website1 to approximate the antennae efficiency and to translate the mea- sured brightness temperatures into flux density units (Jy). L′ line = 3.26 × 107 S line∆v ν−2 obs D2 L (1 + z)−3, (2) (2) where νobs is the observed frequency. The resulting luminosities, L′, are proportional to the brightness temperature and are used, for example, to calculate gas masses. L′ is given in K km s−1 pc2. 1 http://www.iram.es/IRAMES/mainWiki/ Iram30mEfficiencies 2. Observations and data reduction p g g p In total, we measured [CI] 1–0 in seven galaxies, and [CI] 2–1 in eight. This includes four galaxies where we measured both [CI] lines (Fig. 1). Individual line profiles are shown in Fig. 2 for [CI] 1–0 and in Fig. 3 for [CI] 2–1. CO(7–6) has a rest fre- quency of 806.6518 GHz, only 2.7 GHz (or about 1000 km s−1) to the red from the [CI] 2–1 line at 809.3435 GHz. The figures showingthe[CI]2–1linesthereforealsocovertheCO(7–6)linein all cases except one. In PLCK_G244.8+54.9, [CI] 2–1 falls right at the band edge; it can be measured with a reliable calibration, unlike CO(7–6). We refer to C18 for a detailed discussion of the line properties of CO(7–6) and other CO lines. 2. Observations and data reduction The FTS and WILMA backends have intrinsic The data we present here were obtained as part of several observing runs with EMIR at the 30-m telescope of IRAM, with A23, page 2 of 11 N. P. H. Nesvadba et al.: Planck’s Dusty GEMS: Atomic gas probed through [CI] Table 1. Targets and details of our [CI] observations. Source RA Dec Redshift DL µgas LFIR Trans. ν0 Date ToT rms (J2000) (J2000) (Gpc) (1013 L⊙) (GHz) (mm/dd/yy) (min) (mK) PLCK_G045.1+61.1 15:02:36.04 +29:20:51 3.43 29.86 8.4 ± 0.1 1–0 111.035 02/03/14 81 1.17 PLCK_G045.1+61.1 2–1 . . . . . . . . . . . . PLCK_G080.2+49.8 15:44:32.40 +50:23:46 2.60 21.79 4.6 ± 0.1 1–0 136.749 02/03/14 122 2.35 PLCK_G080.2+49.8 2–1 224.400 02/03/14 162 0.82 PLCK_G092.5+42.9 16:09:17.76 +60:45:21 3.26 28.61 24.8 ± 02 1–0 115.639 04/19/14 & 23/02/15 314 1.25 PLCK_G092.5+42.9 2–1 . . . . . . . . . . . . PLCK_G102.1+53.6 14:29:17.98 +51:29:09 2.92 24.99 7.9 ± 0.1 1–0 . . . . . . . . . . . . PLCK_G102.1+53.6 2–1 206.267 02/19,21,23/15 81 0.5 PLCK_G113.7+61.0 13:23:02.88 +55:36:01 2.41 19.88 9.9 ± 0.2 1–0 144.160 02/21/15 202 0.3 PLCK_G113.7+61.0 2–1 236.666 02/19/15 150 1.2 PLCK_G138.6+62.0 12:02:07.68 +53:34:40 2.44 20.20 9.0 ± 0.1 1–0 143.677 07/06/13 80 0.8 PLCK_G138.6+62.0 2–1 231.300 02/19/15 102 2.0 PLCK_G145.2+50.9 10:53:2.56 +60:51:49 3.55 31.76 21.8 ± 0.2 1–0 108.167 05/06/14 120 1.3 PLCK_G145.2+50.9 2–1 . . . . . . . . . . . . PLCK_G165.7+67.0 11:27:14.60 +42:28:25 2.24 18.18 10.3 ± 0.1 1–0 152.070 01/31/14,02/01-04/14 172 0.4 PLCK_G165.7+67.0 2–1 245.500 02/19-20/15 126 0.7 PLCK_G200.6+46.1 09:32:23.67 +27:25:00 2.97 25.14 5.7 ± 0.1 1–0 . . . . . . . . . . . . PLCK_G200.6+46.1 2–1 206.276 02/21/15 162 1.3 PLCK_G231.3+72.2 11:39:21.60 +20:24:53 2.86 24.00 7.5 ± 0.1 1–0 . . . . . . . . . . . . PLCK_G231.3+72.2 2–1 209.100 02/20/15 120 0.911 PLCK_G244.8+54.9 10:53:53.04 +05:56:21 3.01 25.47 26.5 ± 0.2 1–0 . . . . . . . . . . . . PLCK_G244.8+54.9 2–1 205.200 02/19/15 120 2.08 Notes. We list the source name, right ascension and declination, redshift, luminosity distance, observed FIR luminosity, transition, tuning fre quency, date of our observations, time spent on the target, and root mean square of the resulting spectrum. Dots indicate lines outside of the atmospheric windows. Table 1. Targets and details of our [CI] observations. Notes. 3. Line measurements [CI] 1–0 line of the GEMS for which [CI] 2–1 and CO(7–6) fall outside the atmospheric windows. Blue curves indicate the single- component Gaussian fits to the [CI] 1–0 line. the brightest CO lines (C18). The results of our line fits are listed in Table 2, and are not corrected for gravitational lensing. Luminosity-weighted magnification factors are given in Table 1 of C18. tion factors derived from the CO line emission, i.e., assuming that both CO and [CI] come from gas clouds with similar spa- tial distributions. We thus neglect a potential contribution from faint, very extended diffuse gas outside the bright star-forming regions themselves. This assumption can be tested indirectly by comparing the line profiles, which indeed do not show signifi- cant differences when integrated over entire sources. The values we used for this paper are listed in Table 3. We constructed detailed gravitational lens models for all GEMS (Cañameras et al. 2017a, 2018b, and in prep.), which we derived with the publicly available Lenstool package (Jullo & Kneib 2009). Lenstool models the lensing potentials as pseudo- isothermal ellipsoids and derives the properties of these ellip- soids by calculating the expected position of multiple gravita- tionally lensed arclets behind the lensing structure. We used the HST/WFC3 imaging recently presented by Frye et al. (2019), for the five sources where it was available, and ground-based CFHT imaging with 0.8′′–1.0′′ resolution otherwise. Residuals between observed and modeled positions of arclets are smaller than the size of the PSF in all models. We can also use the different estimates for the dust and gas masses to roughly constrain the impact of differential lensing on the various lines in the sub-millimeter and millimeter regimes, finding rather moderate typical differences of about 25%, with- out any dramatic outliers. This is also to be expected, given that the dust and the mid-J CO and [CI] 2–1 lines should mainly originate from gas and dust in actively star-forming regions (Cañameras et al. 2015, and noting that the GEMS do not show evidence of luminous AGNs). From the detailed lensing models (constrained from the WFC3 morphologies), sub-arcsecond millimeter dust, CO emission-line maps, and the kinematic properties of the gas in each source (thereby taking into account the source morphology and differential lensing), we calculate luminosity-weighted aver- age magnification factors separately for the gas and the dust, finding values between 6 and 30 (see Table 1 of C18). 3. Line measurements We detected all targeted [CI] 1–0 and [CI] 2–1 lines, that is, all [CI] lines from galaxies in this sample that fall into the atmo- spheric windows, with line fluxes between 3.4 and 21 Jy km s−1 and FWHM line widths between 220 and 640 km s−1. We used the CLASS function line for an initial line fit after subtracting the baseline (Gildas Team 2013), which we then confirmed with the mpfit routine using IDL (Markwardt 2009). Within the limits imposed by different S/Ns, our fit results are consistent with those obtained for CO by C18. The line profiles are generally smooth enough to be well fit- ted with single Gaussian profiles (Figs. 1–3). In three of the four galaxies where both [CI] 1–0 and [CI] 2–1 had been measured, the profiles of both lines are similar within the S/N of the present data, and the line centers are at similar redshifts for both transi- tions (see Fig. 1). In the fourth, PLCK_G113.7+61.0, [CI]1–0, and [CI]2–1 are offset by 700 km s−1, comparable to the FWHM of the [CI] 1–0 line (639 ± 100, Table 2), and perhaps indicat- ing velocity structure within the galaxy. In PLCK_G045.1+61.1, PLCK_G092.5+42.9, and PLCK_G244.8+54.9, the S/Ns are not high enough to fit two separate line components, as done for We followed Solomon & Vanden Bout (2005), for example, to calculate emission-line luminosities, Lline and L′ line, from these fluxes by setting A23, page 3 of 11 A&A 624, A23 (2019) Fig. 1. [CI] 2–1 and CO(7–6) (top panel) and [CI] 1–0 (bottom panel) spectra of the four GEMS for which we observed both lines. The blue curve shows the single-component Gaussian fit to the [CI] lines. Upper panel: CO(7–6), which is redshifted relative to [CI] 2–1, and is discussed in detail in C18. Fig. 1. [CI] 2–1 and CO(7–6) (top panel) and [CI] 1–0 (bottom panel) spectra of the four GEMS for which we observed both lines. The blue curve shows the single-component Gaussian fit to the [CI] lines. Upper panel: CO(7–6), which is redshifted relative to [CI] 2–1, and is discussed in detail in C18. Fig. 2. [CI] 1–0 line of the GEMS for which [CI] 2–1 and CO(7–6) fall outside the atmospheric windows. Blue curves indicate the single- component Gaussian fits to the [CI] 1–0 line. Fig. 2. A23, page 4 of 11 3. Line measurements Deriving average magnifications for the dust and gas from pixel-by-pixel reconstructions of the source-plane image suggests uncertainties from differential lensing of about 25%. We can also constrain the likely impact of differential lens- ing directly from the present data. As we show in Sect. 4.1, the cooling budget from [CI] relative to the FIR luminosity and to CO(4–3) is within a factor of two of that found in other sam- ples of nearby and high-redshift galaxies, including gravitation- ally lensed and unlensed galaxies (Figs. 4 and 5), respectively. In the absence of a systematic conspiracy with other astrophysical quantities, this suggests that differential lensing does not intro- duce larger uncertainties than other effects. Moreover, integrated mass estimates from CO(1–0) and [CI] 1–0 are very similar, as we show in Sect. 5. g PLCK_G138.6+62.0 is the only galaxy for which we do not have spatially resolved millimeter or sub-millimeter morpholo- gies, so we adopt an empirically estimated factor µ = 20 in this case (for details see Cañameras et al. 2015). For the [CI] line we are most concerned with here, we use the magnifica- For the same reason, we consider it unlikely that we have missed a dominant component of CO-dark, [CI] 1–0 -emitting gas that is strongly gravitationally lensed and has significant N. P. H. Nesvadba et al.: Planck’s Dusty GEMS: Atomic gas probed through [CI] Fig. 3. [CI] 2–1 and CO(7–6) lines of the GEMS for which [CI] 1–0 falls outside the atmospheric windows. For PLCK_G244.8+54.9, [CI] 2–1 falls right at the edge of the band, so that [CI] 2–1 can be reliably measured, but CO(7–6) cannot. Blue curves indicate the single-component Gaussian fits to the [CI] 2–1 line. Fig. 3. [CI] 2–1 and CO(7–6) lines of the GEMS for which [CI] 1–0 falls outside the atmospheric windows. For PLCK_G244.8+54.9, [CI] 2–1 falls right at the edge of the band, so that [CI] 2–1 can be reliably measured, but CO(7–6) cannot. Blue curves indicate the single-component Gaussian fits to the [CI] 2–1 line. Table 2. [CI] line properties. 3. Line measurements Source Line νobs Redshift FWHM µ TK µ I[CI] µ L′ µ L (GHz) (km s−1) (mK) (Jy km s−1) (1011 K km s−1 pc2) (108 L⊙) PLCK_G045.1+61.1 [CI] 1–0 111.133 ± 0.024 3.4280 ± 0.0002 589 ± 145 2.3 8.4 ± 1.7 2.3 ± 0.5 8.6 ± 1.7 PLCK_G080.2+49.8 [CI] 1–0 136.767 ± 0.008 2.5985 ± 0.0002 242 ± 61 3.8 6.2 ± 0.9 1.1 ± 0.2 4.0 ± 0.7 PLCK_G080.2+49.8 [CI] 2–1 224.847 ± 0.025 2.5995 ± 0.0003 312 ± 24 2.3 5.8 ± 1.0 0.37 ± 0.08 6.2 ± 1.2 PLCK_G092.5+42.9 [CI] 1–0 115.641 ± 0.016 3.2559 ± 0.0006 475 ± 128 4.5 13.5 ± 2.6 3.3 ± 0.6 12.8 ± 2 PLCK_G102.1+53.6 [CI] 2–1 206.608 ± 0.006 2.9173 ± 0.0001 220 ± 21 2.3 4.0 ± 0.4 0.3 ± 0.3 5.2 ± 0.5 PLCK_G113.7+61.0 [CI] 1–0 144.019 ± 0.020 2.41730 ± 0.0003 639 ± 100 2.3 10.2 ± 1.2 1.5 ± 0.2 5.9 ± 0.7 PLCK_G113.7+61.0 [CI] 2–1 237.395 ± 0.002 2.40927 ± 0.0001 504 ± 10 2.9 9.2 ± 1.0 0.5 ± 0.05 8.6 ± 0.9 PLCK_G138.6+62.0 [CI] 1–0 142.974 ± 0.020 2.4423 ± 0.0003 575 ± 86 2.4 9.5 ± 1.5 1.5 ± 0.2 5.6 ± 0.9 PLCK_G138.6+62.0 [CI] 2–1 235.129 ± 0.003 2.4421 ± 0.0001 526 ± 5 4.5 18.8 ± 0.2 1.1 ± 0.01 18.0 ± 0.1 PLCK_G145.2+50.9 [CI] 1–0 108.204 ± 0.009 3.5485 ± 0.0003 405 ± 63 5.8 14.8 ± 3.5 4.2 ± 1.0 16.0 ± 3.8 PLCK_G165.7+67.0 [CI] 1–0 152.079 ± 0.009 2.2362 ± 0.0001 629 ± 46 2.6 11.1 ± 2.8 1.5 ± 0.4 5.6 ± 1.4 PLCK_G165.7+67.0 [CI] 2–1 250.059 ± 0.025 2.2366 ± 0.0002 418 ± 6 2.6 8.3 ± 0.2 0.4 ± 0.09 6.8 ± 0.1 PLCK_G200.6+46.1 [CI] 2–1 203.697 ± 0.018 2.97326 ± 0.0001 412 ± 5 2.5 8.3 ± 0.1 0.7 ± 0.01 11.1 ± 0.1 PLCK_G231.3+72.2 [CI] 2–1 209.729 ± 0.015 2.85899 ± 0.0001 319 ± 18 2.7 6.9 ± 0.4 0.5 ± 0.03 8.7 ± 0.5 PLCK_G244.8+54.9 [CI] 2–1 202.113 ± 0.002 3.00440 ± 0.0001 586 ± 5 4.5 21.0 ± 0.2 1.7 ± 0.02 28.6 ± 0.3 Notes. We list the source name, the line, observed frequency, redshift, full-width at half maximum of the line, main-beam brightness temperature, integrated line flux, and the line luminosity in units proportional to brightness temperature and energy, respectively. 3. Line measurements Here we complement and extend these analyses by focusing on the constraints that can be derived solely from the atomic carbon lines, as well as several empirical constraints on the gas masses and the distribution of interstellar gas in the GEMS. A thorough analysis of the gas excitation using the radia- tive transfer code RADEX and PDR model of Kaufman et al. (1999) has already been presented by C18, using [CI] as well as multiple CO lines, generally between J = 3−2 and J = 7−6, and even above J = 9−8 for two galaxies. These latter authors find that the gas in the GEMS is characterized by luminosity- weighted average gas densities of n ∼104−5 cm−3, and radiation fields of 102−4 G0; these are in the range of other starburst galax- ies at low and high redshift. Here we complement and extend these analyses by focusing on the constraints that can be derived solely from the atomic carbon lines, as well as several empirical constraints on the gas masses and the distribution of interstellar gas in the GEMS. 3. Line measurements We give observed values here, where µ indicates the gravitational magnification factor. Luminosity-weighted average gravitational correction factors are given in Table 1 of C18, and are repeated in our Table 3 for convenience. Notes. We list the source name, the line, observed frequency, redshift, full-width at half maximum of the line, main-beam brightness temperature, integrated line flux, and the line luminosity in units proportional to brightness temperature and energy, respectively. We give observed values here, where µ indicates the gravitational magnification factor. Luminosity-weighted average gravitational correction factors are given in Table 1 of C18, and are repeated in our Table 3 for convenience. other, with other emission lines (in particular CO), and with the FIR continuum, all provide interesting diagnostic constraints. transversal positional or velocity offsets from the molecular clouds. However, this does not imply that CO and [CI] are exactly co-spatial (e.g., Offner et al. 2014). On scales of a few hundred parsecs or less, smaller than the area that is being magnified by the gravitational lens, and in directions roughly along the line of sight, or perpendicular to the magnification direction, the diffuse and dense gas may or may not be well mixed, without impact on differential lensing. This would be the case for a clumpy interstel- lar medium for example. Sizes of the order of 100 pc are consis- tent with the Jeans-length, for example, in dense fragmenting gas disks of high-z galaxies including the GEMS (Hodge et al. 2018; Cañameras et al. 2017b; Swinbank et al. 2011, C18). other, with other emission lines (in particular CO), and with the FIR continuum, all provide interesting diagnostic constraints. A thorough analysis of the gas excitation using the radia- tive transfer code RADEX and PDR model of Kaufman et al. (1999) has already been presented by C18, using [CI] as well as multiple CO lines, generally between J = 3−2 and J = 7−6, and even above J = 9−8 for two galaxies. These latter authors find that the gas in the GEMS is characterized by luminosity- weighted average gas densities of n ∼104−5 cm−3, and radiation fields of 102−4 G0; these are in the range of other starburst galax- ies at low and high redshift. 4.1. Atomic line cooling Blue, green, and orange dots indicate the samples of low-redshift star-forming galaxies from Kamenetzky et al. (2016), and the high-redshift samples of grav- itationally lensed sources from the SPT (Bothwell et al. 2017) and the submillimeter galaxies of Alaghband-Zadeh et al. (2013), respectively. The blue line shows the average relationship derived from the compar- ison samples. The blue hatched region shows a range of a factor of ±2 around this average. The typical measurement uncertainty is shown in the lower-right corner. In nearby ULIRGs, Rosenberg et al. (2015) find that the com- bined ratio of the two [CI] lines is LCI10+21/LFIR = 1 −5 × 10−5 in most galaxies, except for the lowest FIR luminosities in the LIRG regime, where ratios can reach about 1×10−4. The GEMS for which both lines have been measured have combined ratios LCI10+21/LFIR = 1.2 and 2.6 × 10−5, which are in the lower range found in the nearby universe; we find similar values when using the [CI] 2–1 / [CI] 1–0 ratios of the GEMS as a fiducial correc- tion factor of the missing second line in the samples of Bothwell et al. (2017) and Alaghband-Zadeh et al. (2013). Riechers et al. (2013) found LCI10+21/LFIR = 2−5×10−5 in a luminous starburst at z = 6.3. Finding similar line-to-continuum flux ratios in high- and low-redshift galaxies imposes at least loose upper limits on the importance of global changes in the gas-heating processes in high-redshift galaxies due to cosmic rays (Bisbas et al. 2017) or X-rays (Meijerink et al. 2007). If, on top of the heating from UV photons, bolometrically significant additional heating mech- anisms like X-rays, cosmic rays, or shocks were present (which predominantly boost the line, but not the continuum luminosities at long wavelengths), we would expect these ratios to be system- atically greater in high-redshift galaxies. Our results suggest that this is not the case. Given the scatter in the relationships, how- ever, this does not imply that such mechanisms are not present; they simply cannot dominate the overall gas heating budgets. This suggests, at least for the small sample sizes and S/Ns obtained for current samples of high-redshift galaxies, that the contribution of atomic gas to the overall cooling budget of the galaxies has remained approximately constant since about 1 Gyr after the Big Bang, and has at most slightly increased with cosmic time. 4.1. Atomic line cooling With the line fluxes measured in Sect. 3, we can derive luminos- ity ratios between the [CI] 2–1 and [CI] 1–0 lines for the four galaxies, in cases where both lines have been measured. We find ratios of LCI2−1/CI1−0 = 1.2–3.3 (Table 2). Given the wide range in gas conditions probed by the [CI] lines, their ratios with each We can use the [CI] line luminosities, LCI, and the FIR luminosi- ties from Cañameras et al. (2015) integrated over a wavelength range of 8−1000 µm to estimate the total cooling from atomic gas in the GEMS. Using the luminosities L[CI1−0 and L[CI]2−1 A23, page 5 of 11 A23, page 5 of 11 A&A 624, A23 (2019) Fig. 4. Cooling budget through [CI]1–0 line emission. [CI]1–0 luminosity (left panel), LCI1−0, and ratio with FIR luminosity, LCI1−0/LFIR (right panel), as a function of FIR luminosity, LFIR. The red stars are the GEMS. Blue, pink, green, and yellow symbols indicate the samples of low- redshift spiral galaxies from Kamenetzky et al. (2016), the main sequence galaxies at z ∼1.2 from Valentino et al. (2018), the high-redshift samples of gravitationally lensed sources from the South Pole Telescope (Bothwell et al. 2017) and the submillimeter galaxies of Alaghband- Zadeh et al. (2013), respectively. The blue line shows the average relationships derived by Valentino et al. (2018) for their main sequence galaxies and comparison samples, and the red line shows an equivalent relationship with a slope of unity. Blue hatched bands show a range of ±2 around these averages. Typical error bars of our measurements are shown in the lower- and upper-right corners of the two panels, respectively. Fig. 4. Cooling budget through [CI]1–0 line emission. [CI]1–0 luminosity (left panel), LCI1−0, and ratio with FIR luminosity, LCI1−0/LFIR (right panel), as a function of FIR luminosity, LFIR. The red stars are the GEMS. Blue, pink, green, and yellow symbols indicate the samples of low- redshift spiral galaxies from Kamenetzky et al. (2016), the main sequence galaxies at z ∼1.2 from Valentino et al. (2018), the high-redshift samples of gravitationally lensed sources from the South Pole Telescope (Bothwell et al. 2017) and the submillimeter galaxies of Alaghband- Zadeh et al. (2013), respectively. The blue line shows the average relationships derived by Valentino et al. (2018) for their main sequence galaxies and comparison samples, and the red line shows an equivalent relationship with a slope of unity. 4.1. Atomic line cooling Blue hatched bands show a range of ±2 around these averages. Typical error bars of our measurements are shown in the lower- and upper-right corners of the two panels, respectively. Fig. 5. Ratio of LCI1−0 to LCO(4−3) as a function of the cooling bud- get through [CI] 1–0. The red stars are the GEMS. Blue, green, and orange dots indicate the samples of low-redshift star-forming galaxies from Kamenetzky et al. (2016), and the high-redshift samples of grav- itationally lensed sources from the SPT (Bothwell et al. 2017) and the submillimeter galaxies of Alaghband-Zadeh et al. (2013), respectively. The blue line shows the average relationship derived from the compar- ison samples. The blue hatched region shows a range of a factor of ±2 around this average. The typical measurement uncertainty is shown in the lower-right corner. listed in Table 2, we find LCI1−0/LFIR and LCI2−1/LFIR ratios of 5 to 20×10−6. We adopted the measured values and did not correct for differences in dust and gas magnifications, which would have changed our results by at most about 25%. Values for individual galaxies are given in Table 6. g g Bothwell et al. (2017) found LCI1−0/LFIR = 7.7±2.4×10−6 in their sample of 13 gravitationally lensed galaxies at z ∼4 from the SPT survey. Walter et al. (2011) measured LCI1−0/LFIR = 1−15×10−6, albeit using FIR luminosities that were derived prior to the launch of the Herschel satellite, from the flux density at 850 µm and a fiducial dust temperature of T = 35 K. Alaghband- Zadeh et al. (2013) found LCI1−0/LFIR = 2.6±0.5×10−5 for their newly observed sources, and LCI1−0/LFIR = 8 × 10−6 for sources culled from the literature (which have considerable overlap with the sample of Walter et al. 2011). Valentino et al. (2018) find about half the value, L[CI]1−0/LFIR = 1.4 × 10−5 for a set of main sequence galaxies at z = 1.2. The GEMS therefore fall within the wide range previously found in other high-redshift galaxies. This can also be seen from Fig. 4, where we plot LCI and LCI/LFIR as a function of LFIR. The GEMS follow similar trends to the samples of low- and high-redshift galaxies. All fall within a factor of two of the best-fit relations. Fig. 5. Ratio of LCI1−0 to LCO(4−3) as a function of the cooling bud- get through [CI] 1–0. The red stars are the GEMS. 4.1. Atomic line cooling In nearby galaxies, the two [CI] lines contribute together about 1.5% to the total gas cooling rate (Rosenberg et al. 2015). 4.2. Heating mechanism and AGN contamination 4.3. Star-formation mode Here Tex is the excitation temperature of the gas in kelvin, K, assuming LTE, and Tmb is the rest-frame peak intensity of the line in main beam brightness temperature, and is also given in kelvin. A similar expression can be given for [CI] 2–1: Here Tex is the excitation temperature of the gas in kelvin, K, assuming LTE, and Tmb is the rest-frame peak intensity of the line in main beam brightness temperature, and is also given in kelvin. A similar expression can be given for [CI] 2–1: Greve et al. (2012) and Papadopoulos & Geach (2012) pro- posed that the ratio of the line luminosities of CO(4–3) and [CI] 1–0 can be used to qualitatively infer the relative amount of dense molecular and more diffuse gas. They associate higher ratios of dense molecular to diffuse gas with starburst galax- ies, and galaxies with a more balanced ratio of dense and dif- fuse gas with quiescently star-forming (disk-like) galaxies. From observations of nearby galaxies, they infer an average ratio of rCO(4−3)/CI10 = 4.55 ± 1.5 for starburst (ULIRG) environments, and rCO(4−3)/CI10 = 0.45 −1.3 for disk galaxies. In the GEMS, the corresponding ratios are between 2.6 and 5.8. For galaxies where we have a direct measurement of [CI] 1–0, we find ratios of 2.9−3.3, and 2.6−5.8 for the galaxies without [CI] 1–0 mea- surements (where we used the [CI] 2–1 measurement corrected for an average [CI] 2–1/[CI] 1–0 ratio instead). These results are all in the starburst regime, as also expected from the high star- formation rate densities found by Cañameras et al. (2017b). τ[CI]2−1 = −ln (1 −Tmb,[CI]2−1 (e38.8/Tex −1)/38.8). (4) (4) The excitation temperature in kelvin can be found from the ratio of line luminosities, L′ CI2−1/L′ CI1−0, by setting Tex = hν21/kB ln N10 N21 g21 g10 !−1 = 38.8 ln(2.11/R) [K], (5) (5) where R is the ratio between the integrated luminosities, L′, of the [CI] 2–1 and [CI] 1–0 lines, kB is the Boltzmann constant, h is Planck’sconstant,ν21 therest-framefrequencyofthe[CI]2–1line, N10 and N21 are the column densities of the [CI] 1–0 and 2–1 lines, respectively, and g21 and g10 are the corresponding Gaunt factors. 4.2. Heating mechanism and AGN contamination For convenience, we also list the magnification factors for the gas, µgas, taken from Table 1 of C18, which we have used to correct these measurements for gravitational lensing. Walter et al. 2011). In other words, the line luminosity remains proportional to the total mass. Since we have both the [CI] 2–1 and [CI] 1–0 line measured in four GEMS, we can test this assumption directly. We saw in Sect. 4.2 that the gas in the present galaxies is pre- dominantly heated by UV photons and we can therefore follow Schneider et al. (2003) and Walter et al. (2011) who derived the optical depth, τ, of the [CI] 1–0 emitting gas in PDRs by setting τ[CI]1−0 = −ln (1 −Tmb,[CI]1−0 (e23.6/Tex −1)/23.6). (3) (3) 4.3. Star-formation mode y We note that using [CI] 2–1 instead of [CI] 1–0 can lead to uncertainties of factors of two to three, and additionally for some galaxies we used CO(3–2) instead of CO(4–3) because the latter falls outside the atmospheric window (see Table 5). From the CO spectral line energy diagrams shown by C18, we know that this might bias the line ratios of the GEMS about 25% low compared to estimates with CO(4–3). Since we only aim at loosely classi- fying the GEMS between two groups that differ by an order of magnitude on average, and do not use the precise value of these line ratios, we find that our conclusions are not compromised by these additional systematic uncertainties. With the luminosities and main-beam brightness tempera- tures given in Table 2, we find excitation temperatures, Tex = 21−37 K. This is consistent with previous work (Jiao et al. 2017) and systematically lower than the dust temperatures found by Cañameras et al. (2015), which are between 33 and 50 K for the same galaxies; this might indicate that [CI] has a signifi- cant extended component (see also Sect. 5), or that the dust and atomic gas are not in thermal equilibrium. Both lines are optically thin in the GEMS, and compara- ble to those in other high-redshift galaxies (Walter et al. 2011; Alaghband-Zadeh et al. 2013). The corresponding optical depths of the [CI] 1–0 line are between τ10 = 0.01 and 0.14, and for the [CI] 2–1 line are generally between τ21 = 0.01 and 0.06. In PLCK_G244.8+54.9 we find τ21 = 0.55 for a fiducial tem- perature of Tex = 20 K. This temperature is likely too low for a galaxy with such highly excited gas (C18). For Tex = 40 K, we would find a more typical value of τ21 = 0.13. Results for indi- vidual galaxies are listed in Table 4; in galaxies where only one 4.2. Heating mechanism and AGN contamination The ratio of the [CI] 2–1 and the [CI] 1–0 line fluxes can inform us about the presence of X-ray heating from AGNs. A23, page 6 of 11 N. P. H. Nesvadba et al.: Planck’s Dusty GEMS: Atomic gas probed through [CI] Table 3. Intrinsic masses of atomic carbon, MCI, and molecular gas, MH2, estimated from [CI] 1–0 in the seven galaxies where we observed this line. Radiative-transfer models of gas heating from UV photons imply an upper limit to the [CI] 2–1/[CI] 1–0 ratio, which cannot be exceeded without the presence of a harder incident radiation field like that from an AGN. Meijerink et al. (2007) calculated line ratios for gas heated by UV and X-ray photons, as expected for regions of intense star formation and circum-nuclear environ- ments within AGN host galaxies, respectively. For a wide range in gas density between about 10 and 106 cm−3, they predict that X-ray heating will produce line ratios between [CI] 2–1 and [CI] 1–0 of L21/L10 ≳3.5. Ratios lower than this are a clear indica- tion of UV heating in gas with typical densities of a few times 102−4 cm−3 as in the GEMS (C18). Source MCI MH2 µgas (107 M⊙) (1010 M⊙) PLCK_G045.1+61.1 1.9 13.5 15.5 PLCK_G080.2+49.8 0.8 6.2 15.9 PLCK_G092.5+42.9 3.7 25.8 12.0 PLCK_G113.7+61.0 2.0 14.6 9.7 PLCK_G138.6+62.0 1.0 6.8 20. PLCK_G145.2+50.9 6.3 43.7 8.9 PLCK_G165.7+67.0 0.8 5.8 24.1 Notes. For convenience, we also list the magnification factors for the gas, µgas, taken from Table 1 of C18, which we have used to correct these measurements for gravitational lensing. In Table 4 we give the luminosity ratios for the four galax- ies where we measured both [CI] lines. In PLCK_G080.2+49.8, PLCK_G113.7+61.0, and PLCK_G165.7+67.0 we find very similar ratios of between L[CI21/LCI10 = 1.2 ± 0.3 and LCI21/LCI10 = 1.5 ± 0.2. However, the ratio in the fourth source, PLCK_G138.6+62.0, is significantly higher: LCI21/LCI10 = 3.3± 0.1. This source therefore falls near the regime where an AGN could have an impact, although it is still within the range expected for intensely star-forming systems. Finding little evi- dence for AGN X-ray heating from the line ratios confirms our previous results from the mid-to-far-infrared spectral energy dis- tributions, which also suggest that AGNs are weak compared to the UV radiation from young stellar populations (Cañameras et al. 2015), or absent. Notes. 4.5. Mass of atomic carbon and carbon abundances A major advantage of using optically thin lines for mass esti- mates is that the line luminosity is proportional to the mass of the tracer. We follow Walter et al. (2011) and Weiß et al. (2005b) in estimating the mass of atomic carbon by setting Using the total molecular gas mass estimates of C18, and assuming, for the sake of this specific analysis, that αCO,ULIRG is the perfect choice for these targets (we discuss this choice more broadly in the following section), we find carbon abundances of between 2.3 and 4.0 × 10−5 (Table 6). MCI = 5.71 × 10−4 Q(Tex) 1/5eT1/Tex L′ CI10 [M⊙], (6) MCI = 5.71 × 10−4 Q(Tex) 1/5eT1/Tex L′ CI10 [M⊙], (6) These abundance estimates are consistent with the canonical value proposed by Weiß et al. (2005a), and derived for M82. They are also consistent with previous work by Alaghband- Zadeh et al. (2013) and Danielson et al. (2011) for gravitationally lensed, dusty starburst galaxies at similar redshifts; these authors found values of 3−4 × 10−5, comparable to what we find here. Several recent analyses, however, come to different conclusions. For example, Bothwell et al. (2017) found a high average car- bon abundance of XCI = 7.3 × 10−5 in a sample of 13 strongly lensed dusty starburst galaxies from the SPT survey at z ∼4, when adopting αCO,ULIRG, whereas Valentino et al. (2018) very recently found significantly lower values in a sample of main sequence disk galaxies at z = 1.2, adopting a higher CO-to- H2 conversion factor, which is presumably more appropriate for main sequence galaxies. We continue the discussion of the car- bon abundances after deriving molecular gas masses from the [CI] 1–0 luminosities in the following section. where Tex is the excitation temperature, Q(Tex) the partition function Q(Tex) = 1.0 + 3e−T1/Tex + 5e−T2/Tex , and L′ CI10 are the measured luminosities of [CI] 1–0. We use the measured excitation temperature, Tex, for the four galaxies where we observed both [CI] line fluxes (Table 4). The quantities T1 = 23.6 K and T2 = 62.5 K correspond to the energies above the ground state for [CI] 1–0 and [CI] 2–1, respectively. Results are listed in Table 6. Overall, we find that atomic carbon masses are between 8 × 106 M⊙and 5 × 107 M⊙after correcting for the gravitational magnification given in Table 3. 4.4. Excitation temperatures and optical depth One of the main advantages in using [CI] instead of CO lines as a tracer of mass is that they should remain optically thin out to the high volume-averaged column densities typically encoun- tered in rapidly star-forming dusty high-redshift galaxies (e.g., A23, page 7 of 11 A23, page 7 of 11 A&A 624, A23 (2019) Table 4. Ratios of line luminosities to the FIR luminosity of [CI] 1–0 and [CI] 2–1, and luminosity ratios of [CI] 2–1 and [CI] 1–0. Source LCI10/LFIR LCI21/LFIR LCI21/LCI10 L′ CI21/L′ CI10 Tex τCI1−0 τCI2−1 µMCI (×10−6) (×10−6) (K) (108 M⊙) PLCK_G045.1+61.1 10.2 ± 2.0 ... ... ... 20a 0.04a ... 3.0 ± 0.5 PLCK_G080.2+49.8 8.7 ± 1.5 13.5 ± 2.6 1.6 ± 0.2 0.33 ± 0.1 22.3+3.75 −3.5 0.14 0.05 1.3 ± 0.3 PLCK_G092.5+42.9 5.2 ± 0.8 ... ... ... 20a 0.07a ... 4.4 ± 0.3 PLCK_G102.1+53.6 ... 6.6 ± 0.6 ... ... ... ... 0.02a ... PLCK_G113.7+61.0 6.0 ± 0.7 8.6 ± 0.9 1.5 ± 0.2 0.33 ± 0.2 21.0+8.7 −7.3 0.03 0.02 2.0 ± 0.4 PLCK_G138.6+62.0 6.1 ± 1.0 20.0 ± 0.1 3.3 ± 0.1 0.73 ± 0.2 36.7+10.5 −8.3 0.01 0.01 1.9 ± 0.3 PLCK_G145.2+50.9 7.3 ± 1.7 ... ... ... 20a 0.02a ... 5.6 ± 1.0 PLCK_G165.7+67.0 5.4 ± 1.3 6.6 ± 0.1 1.2 ± 0.3 0.27 ± 0.1 18.7+3.38 −3.5 0.02 0.01 2.0 ± 0.3 PLCK_G200.6+46.1 ... 19.5 ± 0.2 ... ... ... ... 0.01a ... PLCK_G231.3+72.2 ... 11.6 ± 0.7 ... ... ... ... 0.06a ... PLCK_G244.8+54.9 ... 10.8 ± 0.1 ... ... ... ... 0.55a ... Notes. Excitation temperature and optical depths of [CI] 1–0 and [CI] 2–1 are also given, as well as the mass of atomic carbon (not corrected for gravitational magnification µ) for galaxies with [CI] 1–0 observation. (a)For galaxies without either [CI] 1–0 or [CI] 2–1 measurements, we adopted a fiducial excitation temperature of Tex = 20 K. of line luminosities to the FIR luminosity of [CI] 1–0 and [CI] 2–1, and luminosity ratios of [CI] 2–1 and [CI] 1–0. Notes. Excitation temperature and optical depths of [CI] 1–0 and [CI] 2–1 are also given, as well as the mass of atomic carbon (not corrected for gravitational magnification µ) for galaxies with [CI] 1–0 observation. (a)For galaxies without either [CI] 1–0 or [CI] 2–1 measurements, we adopted a fiducial excitation temperature of Tex = 20 K. 4.4. Excitation temperatures and optical depth derive carbon mass estimates for the GEMS that have only [CI] 2–1 measurements. [CI] line falls into the atmospheric windows, we adopt a fiducial excitation temperature of Tex = 20 K, consistent with the aver- age of three of the GEMS. Similar temperatures are found for the lower-excitation component traced by CO lines by Yang et al. (2017) and C18. By using the lowest representative temperature measurement, we bias the optical depth of the lines high, since the gas becomes optically thicker with decreasing temperature. Had we adopted Tex = 37 K instead (the highest excitation tem- perature measured amongst the GEMS), we would have obtained optical depths that were approximately 80% lower. Combining our mass estimates of atomic carbon with the molecular gas mass estimates derived from CO by C18 allows us to estimate a carbon abundance, XCI = X[CI]/X[H2] = MC/6MH2. Obviously, a CO mass estimate must be chosen for this calculation that in itself does not depend on carbon abun- dance. We are using the ULIRG conversion factor, αCO,ULIRG = 0.8 M⊙/ [K km s−1 pc2], which satisfies this criterion. Solomon et al. (1997) derived this value from dynamical mass estimates of nearbyULIRGs,supposingthatthemoleculargasmassisequalto the difference between dynamical and stellar mass. For the same reason, αCO,ULIRG also naturally accounts for He. 4.5. Mass of atomic carbon and carbon abundances In principle, both lines of [CI] can be used as mass tracers. Weiß et al. (2005b) give an equivalent equation to Eq. (6) for [CI] 2–1. However, in practice, estimates based on [CI] 2–1 are much more sensitive to the excitation temperature. Whereas the mass estimate derived from [CI] 1–0 changes by only about 1% for a temperature range between 20 and 50 K, mass estimates from [CI] 2–1 change by more than a factor of three. Since the excita- tion temperature in the four galaxies with [CI] 1–0 and [CI] 2–1 measurements does not correlate with the dust temperature, to estimate robust excitation temperatures from [CI] 2–1, we would need to observe both lines, in which case we would estimate the atomic carbon mass directly from [CI] 1–0. We therefore do not A23, page 8 of 11 5. Molecular gas mass estimates from [CI] and CO We set XCI = 3 × 10−5, the standard value that has also been commonly adopted in previous work (e.g., Walter et al. 2011), and A10 = 7.93×10−8,similartopreviousauthors.For Q10 weadopted 0.49,themedian Q10 valueusedbyPapadopoulosetal.(2004)and also used previously by Alaghband-Zadeh et al. (2013). With the flux measurements listed in Table 2, between 3 and 21 Jy km s−1, and the redshifts listed in the same table, we find total molecular gas mass estimates, MH2,CI between 10 and 40 × 1011 µ−1 M⊙for the seven galaxies for which [CI] 1–0 has been measured. Results for individual sources are listed in Table 6. In Table 6, we also compare with molecular gas mass esti- mates derived from CO line emission, for a CO-to-H2 conver- sion factor of αCO,ULIRG = 0.8 M⊙/ [K km s−1 pc2]. We follow C18, who derived gas masses from the measured CO(4–3) or CO(3–2) luminosities, depending on which line falls into the atmospheric windows, and taking CO(3–2) when both lines are available. These luminosities, L′, were corrected by ratios of R32 = L′ CO(3−2)/L′ CO(1−0) = 0.4 and R43 = L′ CO(4−3)/L′ CO(1−0) = 0.3 (C18) to extrapolate to L′ CO(1−0). C18 derived these average line ratios by comparing with the CO(1–0) mass estimates of Harrington et al. (2018), which are available for four GEMS, for αCO,ULIRG = 0.8 M⊙/ [K km s−1 pc2]. In the following we use these ratios to adopt a common procedure for our entire sample, including galaxies with and without CO(1–0) measurements. Collecting large enough sets of emission lines of individual high-redshift galaxies to study their gas excitation in detail is often very challenging. To obtain these mass estimates, it was critical in our case to accurately take into account gas excitation when extrapolating from mid-J CO line luminosities to the lumi- nosities of CO(1–0), since the line ratios are lower than others in the literature by factors of 1.5–2 (for details see C18). Had we used the higher values of for example Spilker et al. (2014) or Danielson et al. (2011), we would have been led to believe that we had found considerably higher molecular gas masses from [CI] 1–0 than from the mid-J CO lines. Figures 4 and 5 show that the GEMS as an ensemble have somewhat lower ratios of LCI10/LCO43, and somewhat higher ratios of LCI10/LFIR compared to other populations of high- and low-redshift galaxies, which is consistent with this finding. 5. Molecular gas mass estimates from [CI] and CO Weiß et al. (2005b), Papadopoulos et al. (2004), and Wagg et al. (2006) were among the first to propose the use of [CI] emission-line measurements to estimate total molecular gas masses for high-redshift galaxies. The main motivation was that these lines are bright and optically thin, and that for a given carbon N. P. H. Nesvadba et al.: Planck’s Dusty GEMS: Atomic gas probed through [CI] in carbon abundance measurements, and in molecular gas mass estimates from [CI], is still αCO. abundance and excitation parameter, Q10, a simple scaling between [CI] 1–0 line flux and total gas mass can be given, as follows: The total atomic carbon mass estimates, however, are inde- pendent of the chosen αCO. Therefore, significantly increas- ing αCO for galaxies like the GEMS would also imply that we should adopt equally low carbon abundances. We argued in C15 that the metallicities in the GEMS are probably already solar or greater, relying on gas-to-dust ratios as previously estimated by Magdis et al. (2011). Solar or greater gas-phase metallici- ties in massive, dusty starburst galaxies at redshifts z ∼2−3 are also suggested by studies of warm ionized gas in these galaxies (Takata et al. 2006; Nesvadba et al. 2007), as well as by the abundances found in the photospheres of the domi- nant stellar populations in massive low-redshift galaxies, which probe the metallicities at the time when these stars were formed; they are solar or super-solar (Gallazzi et al. 2005), and also do not favor unusually low carbon abundances in the GEMS and other, similar high-redshift galaxies. This makes a much lower XCI and a higher αCO than the ULIRG-value implausible, at least for this type of high-redshift galaxy. For bluer, lower- mass, and less intensely star-forming galaxies, this is proba- bly different, and overall, the range of αCO is probably set by a range of parameters, including in particular metallicity (e.g., Bolatto et al. 2013). MH2,[CI] = 1380 × D2 L (1 + z) A−1 10,−7 X−1 CI,−5 Q−1 10 ICI [M⊙], (7) (7) where DL is the luminosity distance in units of gigaparsecs, z is redshift, and ICI is the integrated line flux of [CI] 1–0 in Jy km s−1. The Einstein A coefficient for [CI] 1–0, A10, is given in units of 10−7 s−1, and the carbon abundance, XCI, is in units of 10−5. 5. Molecular gas mass estimates from [CI] and CO This may indicate that their gas is perhaps somewhat denser or more highly excited than in other galaxies at the same redshifts, as also shown by C18, and as would be consistent with targeting particularly bright galaxies on the sub-millimeter sky. Despite these indications, they fall well within the scatter of the overall population. With these line ratios, we find excellent agreement between the masses derived from [CI] and CO for all GEMS for which [CI] 1–0 observations are available. Amongst the four sources for which CO(1–0) measurements are available, three have consistent mass estimates from CO(1–0) and [CI] 1–0 within the measurement uncertainties. Only one source, PLCK_113.7+61.1, has a somewhat higher mass estimate from [CI] 1–0 than from CO(1–0), with a ratio MH2,CI10/MH2,CO10 = 1.4 ± 0.1. For the overall sample, and using molecular gas mass estimates derived from CO(4–3) or CO(3–2), five of seven sources have consistent mass estimates (within 2σ), and two sources have somewhat larger mass estimates from [CI] 1–0 than from CO: MH2,CI10/MH2,CO10 = 1.7 ± 0.2. Individual results are listed in Table 6. p p If our results are applicable to more general populations of massive, dusty, high-redshift starburst galaxies, then this would imply that most of the discrepancy seen in mass estimates from [CI] and CO could be dominated by the diversity in average gas excitation of these galaxies. For example, similar effects could be at play for other samples of vigorous starburst galaxies show- ing enhanced carbon abundances, like those found with the SPT (Bothwell et al. 2017). The origin of this diversity may either be differences in the excitation process itself, or in the relative con- tribution of high and low-excitation gas (e.g., Ivison et al. 2010; Harrisetal.2010;Yangetal.2017,C18).Eventhelargestratiosof MH2,CI to MH2,CO amongst the GEMS, namely MH2,CI/MH2,CO = 1.7 ± 0.2, could reflect differences in gas excitation rather than additional gas components not seen in CO(1–0). For example, Papadopoulos & Greve (2004) state a range of a factor of three of plausible excitation parameters Q10 for molecular gas mass esti- mates from [CI] 1–0. Finding consistent mass estimates with two independent tracers is certainly an encouraging result, and may serve as a val- idation of applying low-redshift calibrations to (at least this type of) high-redshift galaxies. 5. Molecular gas mass estimates from [CI] and CO This result also confirms that using low, average CO-to-H2 conversion factors akin to the used fac- tor αCO,ULIRG = 0.8 M⊙/ [K km s−1 pc2] (Solomon et al. 1997), average carbon abundance of about XCI = 3 × 10−5 (Weiß et al. 2005a), and excitation parameter Q10 ∼0.5 (Papadopoulos et al. 2004), is an adequate, internally consistent choice of parameters. However, this result should be interpreted with some caution. In particular, it cannot be used as a justification for any particu- lar choice of XCI or αCO, since both are degenerate, as inserting the expression of carbon abundance explicitly into Eq. (7) imme- diately shows. Consequently, the largest systematic uncertainty Finding consistent mass estimates with two independent tracers is certainly an encouraging result, and may serve as a val- idation of applying low-redshift calibrations to (at least this type of) high-redshift galaxies. This result also confirms that using low, average CO-to-H2 conversion factors akin to the used fac- tor αCO,ULIRG = 0.8 M⊙/ [K km s−1 pc2] (Solomon et al. 1997), average carbon abundance of about XCI = 3 × 10−5 (Weiß et al. 2005a), and excitation parameter Q10 ∼0.5 (Papadopoulos et al. 2004), is an adequate, internally consistent choice of parameters. However, this result should be interpreted with some caution. In particular, it cannot be used as a justification for any particu- lar choice of XCI or αCO, since both are degenerate, as inserting the expression of carbon abundance explicitly into Eq. (7) imme- diately shows. Consequently, the largest systematic uncertainty Regardless of these concerns, our results do suggest that CO(1–0) and [CI] 1–0 are probing similar gas reservoirs within the GEMS, and that the impact of differential lensing does not A23, page 9 of 11 A&A 624, A23 (2019) Table 5. Diagnostic line ratios. 5. Molecular gas mass estimates from [CI] and CO Source CO transition µL′ CO a µL′ CI10 L′ CO/L′ CI10 (1011 K km s−1 pc2) (1011 K km s−1 pc2) PLCK_G045.1+61.1 4–3 7.5 ± 0.9 2.3 ± 0.5 3.3 ± 0.8 PLCK_G080.2+49.8 3–2 2.9 ± 0.2 1.1 ± 0.2 2.6 ± 0.5 PLCK_G092.5+52.9 4–3 10.9 ± 0.7 3.3 ± 0.6 3.3 ± 0.6 PLCK_G102.1+53.6 3–2 2.2 ± 0.8 0.7b 3.1 PLCK_G113.7+61.0 4–3 3.7 ± 0.3 1.5 ± 0.2 2.5 ± 0.4 PLCK_G138.6+62.0 4–3 4.9 ± 0.3 1.5 ± 0.2 3.3 ± 0.5 PLCK_G145.2+50.9 4–3 12.2 ± 2.4 4.2 ± 1.0 2.9 ± 0.9 PLCK_G165.7+67.0 4–3 4.6 ± 0.3 1.5 ± 0.4 3.1 ± 0.8 PLCK_G200.6+46.1 3–2 6.0 ± 0.6 1.3b 4.6 PLCK_G231.3+72.2 3–2 5.5 ± 0.8 0.9b 6.4 PLCK_G244.8+54.9 4–3 7.0 ± 0.7 3.1b 2.3 Notes. (a)Taken from C18. (b)Estimated from [CI] 2–1, assuming a ratio ICI1−0/ICI2−1 = 1.8, the average of the values of the four galaxies where we cover both lines. Error bars include the measurement uncertainties, and are only given for galaxies for which [CI] 1–0 has actually been measured. Table 6. Molecular gas mass estimates derived from [CI] 1–0, and from CO. Notes. (a)Taken from C18. (b)Estimated from [CI] 2–1, assuming a ratio ICI1−0/ICI2−1 = 1.8, the average of the values of the four galaxies where we cover both lines. Error bars include the measurement uncertainties, and are only given for galaxies for which [CI] 1–0 has actually been measured. Table 6. Molecular gas mass estimates derived from [CI] 1–0, and from CO. Table 6. Molecular gas mass estimates derived from [CI] 1–0, and from CO. Source µ MH2,CIa µ MH2,CO43,extrb MH2,CI/MH2,CO10c MCI/MH2,CO43,extr XCI,CO10 XCI,CO43,extr (1011 M⊙) (1011 M⊙) (×10−5) (×10−5) PLCK_G045.1+61.1 20.9 ± 4.7 19.9 ± 2.4 ... 1.0 ± 0.3 ... 2.5 ± 0.5 PLCK_G080.2+49.8 9.8 ± 0.8 5.7 ± 0.4 ... 1.7 ± 0.2 ... 3.8 ± 0.9 PLCK_G092.5+52.9 30.9 ± 2.3 24.8 ± 1.6 1.1 ± 0.4 1.2 ± 0.1 2.7 ± 0.9 3.0 ± 0.3 PLCK_G113.7+61.0 14.2 ± 0.7 11.6 ± 0.9 1.4 ± 0.1 1.2 ± 0.1 3.2 ± 1.0 2.9 ± 0.6 PLCK_G138.6+62.0 13.4 ± 0.9 14.6 ± 0.9 1.1 ± 0.4 0.9 ± 0.1 2.6 ± 0.7 2.2 ± 0.4 PLCK_G145.2+50.9 38.9 ± 0.5 23.3 ± 0.9 ... 1.7 ± 0.1 ... 4.0 ± 0.7 PLCK_G165.7+67.0 13.5 ± 0.4 15.0 ± 0.4 0.95 ± 0.1 0.9 ± 0.1 2.3 ± 0.5 2.2 ± 0.3 PLCK_G244.8+54.9 ... 6. Summary and conclusions We have presented an analysis of the [CI] 1–0 and [CI] 2–1 emission lines in Planck’s Dusty GEMS, a small sample of 11 of the brightest high-redshift galaxies on the sub-millimeter sky observed with the Planck satellite. We have detailed lens models derived with Lenstool from sub-arcsecond interferometry for all galaxies (Cañameras et al. 2018b), and can therefore explicitly account for source morphology and differential lensing between dense gas and dust (finding that it does not play a major role). We detect all [CI] lines from those galaxies where these lines fall into the atmospheric windows. In total, we measured the [CI] 1–0 line in seven, and the [CI] 2–1 line in eight galaxies, measur- ing both lines for four galaxies. Our main results are as follows. – The GEMS have [CI] line fluxes between 4 and 21 Jy km s−1, 5 5 We have presented an analysis of the [CI] 1–0 and [CI] 2–1 emission lines in Planck’s Dusty GEMS, a small sample of 11 of the brightest high-redshift galaxies on the sub-millimeter sky observed with the Planck satellite. We have detailed lens models derived with Lenstool from sub-arcsecond interferometry for all galaxies (Cañameras et al. 2018b), and can therefore explicitly account for source morphology and differential lensing between dense gas and dust (finding that it does not play a major role). – The intrinsic masses of atomic carbon are between 0.8 and 6.3 × 107 M⊙, corresponding to atomic carbon abundances between XCI = 2 × 10−5 and 4 × 10−5. This is comparable to theusuallyadoptedvalueof3×10−5 initiallyderivedforM82, and several other samples of high-redshift galaxies. However, recent studies have also found either higher (Bothwell et al. 2017) or lower values (Valentino et al. 2018), in either case within a factor of about two. – H2 gas mass estimates from [CI] 1–0 (and adopting a car- bon abundance of 3 × 10−5), correspond to those measured from CO within the measurement uncertainties for five of seven galaxies for which [CI] 1–0 has been measured, and within factors 1.7 for the other two. 5. Molecular gas mass estimates from [CI] and CO 14.0 ± 1.4 ... ... ... ... Notes. Ratios of mass estimates and carbon abundance, XCI, for the different molecular gas mass estimates from CO. (a)For XCI = 3 × 10−5, and αCO,ULIRG = 0.8 M⊙/ [K km s−1 pc2]. (b)Using the average luminosity ratios of the GEMS, R32 = L′ CO(3−2)/L′ CO(1−0) = 0.4 and L′ CO(4−3)/L′ CO(1−0) = 0.3 (C18) to extrapolate to L′ CO(1−0). (c)CO(1–0) is taken from Harrington et al. (2018), for αCO,ULIRG = 0.8 M⊙/ [K km s−1 pc2]. Notes. Ratios of mass estimates and carbon abundance, XCI, for the different molecular gas mass estimates from CO. (a)For XCI = 3 × 10−5, and αCO,ULIRG = 0.8 M⊙/ [K km s−1 pc2]. (b)Using the average luminosity ratios of the GEMS, R32 = L′ CO(3−2)/L′ CO(1−0) = 0.4 and L′ CO(4−3)/L′ CO(1−0) = 0.3 (C18) to extrapolate to L′ CO(1−0). (c)CO(1–0) is taken from Harrington et al. (2018), for αCO,ULIRG = 0.8 M⊙/ [K km s−1 pc2]. dominate the observed luminosity and mass estimates derived from either line. In particular, and while we do see multiple gas components with different excitation conditions in the GEMS (C18), we find no evidence that such galaxies have large frac- tions of “CO-dark” cold, neutral gas, that would not be seen in CO(1–0). 0.01−0.14) gas in star-forming regions dominated by UV heating, without major contribution from an AGN, and excitation temperatures of typically about Tex = 20 K, with one galaxy having Tex = 36 K. – The line ratios of [CI] 1–0 and CO(4–3) are between 2.3 and 3.5. Following Greve et al. (2012) and Papadopoulos & Geach (2012) we interpret this as a sign that these galax- ies are undergoing starbursts, not the more regular, longer- term star formation typical of disk galaxies at similar redshifts. N. P. H. Nesvadba et al.: Planck’s Dusty GEMS: Atomic gas probed through [CI] 2013, Nature, 496, 32 Rosenberg, M. J. F., van der Werf, P. 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Munchʼs “Scream” as a sense-giving object for the conceptual entity
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2023 2023 2023 Випуск 42 DOI: https://doi.org/10.26565/2073-4379-2023-42-02 УДК 81.23: 372.881.1 MUNCHʼS “SCREAM” AS A SENSE-GIVING OBJECT FOR THE CONCEPTUAL ENTITY Sergey Volkov PhD in Education, Associate Professor, Higher Institute of Languages of Tunis, Oriental Languages Department, University of Carthage (1003, Tunisia, Tunis, 14 Ibn Maja Avenue); e-mail: serguei.volkov@islt.ucar.tn; orcid: https://orcid.org/0000-0002-9682-4797 Sergey Volkov PhD in Education, Associate Professor, Higher Institute of Languages of Tunis, Oriental Languages Department, University of Carthage (1003, Tunisia, Tunis, 14 Ibn Maja Avenue); e-mail: serguei.volkov@islt.ucar.tn; orcid: https://orcid.org/0000-0002-9682-4797 A conceptual entity is a phenomenon that only exists in our thoughts as knowledge, linguistic, cultural, iconic, etc. All objects falling under this category are those that were created only by human intellect (power of eason or emotions). The article focuses on how a conceptual idea about the object chosen or given for in-depth discussion or study in an academic setting might be constructed and how the sense-giving potential of this object forms the conceptual entity. In this context, the object acts as a symbol for the idea and transforms into a sense-making tool that affects how linguistic or visual patterns manifest in the human mind. The articleʼs goal is to illustrate the problem of the expansion of every field of knowledge through the development of new concepts. This goal involves visual art as a reference point, notably Edvard Munchʼs Scream series becoming an object for idea generation. Practically, conceptualization strategies can make teaching a foreign language easier. The use of visual art images as nonverbal communication tools gives teachers a chance to improve studentsʼ discourse skills. The cognitive approach to understanding the scream image in the artwork enables us to connect it to symbolic qualities that heighten its emotional resonance and give it fundamentally indivisible characteristics. This article takes a cognitive approach to communication. Hence, teaching it involves developing mental representations of the conversational subject and its linguistic realizations. As the result, considering the object (image) as a conceptual phenomenon of visual culture gives the following sequence of causal modes of its cognition: stimulation – intentional stances and cognitive attitude – mentalization / presence of mental content – ideation / reasoning / presence of substantial content – conceptualization. All of these are either postures or processes in which a kind of object appears, depending on the psychodynamic or psychoemotional state of its meditator, more specifically: stimulus object – intentional object – mental object – ideative object – substantial object / semantic object – conceptual object. So, Munchʼs conception of the scream has ______________ © Volkov S., 2023 25 ISSN 2073-4379 Викладання мов у вищих навчальних закладах освіти ... It is appropriate to use the cognitive method to interpret the semantic content of the concept. According to this method, an individualʼs representations of his or her cognitions in his or her experiences are linked to the developed idea and lead to its conceptualization in the identity and, presumably, in the community. Understanding of a conceptualization modifying principle is made possible by an investigation of the change from the individualʼs thought to the concept represented in a verbal or visual sign. realizations, in the cognitive account. It is appropriate to use the cognitive method to interpret the semantic content of the concept. According to this method, an individualʼs representations of his or her cognitions in his or her experiences are linked to the developed idea and lead to its conceptualization in the identity and, presumably, in the community. Understanding of a conceptualization modifying principle is made possible by an investigation of the change from the individualʼs thought to the concept represented in a verbal or visual sign. Analysis of current research. The author of the present article addresses one aspect of the learning potential of the named principle in his work on realizing the task of awakening in a studentʼs mind, when breaking an artistʼs code, “the desire to think about an ideative or conceptual object that evokes categorical associations” [16: 70]. In distinguishing itself by the novelty of “fusing” natural human and visual languages based on a cognitive interpretation of the results of the analysis of the significant components of the “iconic” image of Munchʼs painting, the article only draws on relevant research in cognitive science and highlights actual positions in the parsing of this artifact. Firstly, thinking on this topic is consistent with cognitive theories that reveal aids for comprehending objects, as follows: 1) the theory of intentionality by Daniel C. Dennett with its intentional stance from which the patterns in human behaviour are describable and predictive [3]; 2) cognitive poetics as part of cognitive linguistics by Mark Turner and Reuven Tsur: “it attempts to find out how poetic language and form, or the criticʼs decisions, are constrained and shaped by human information processing” [14: 2]; 3) cognitive representation (or conceptual system) developed by Leonard Talmy [13], where the user of a language uses linguistic tools customized to encode and extrinsically communicate his or her cognitive representation. ISSN 2073-4379 Викладання мов у вищих навчальних закладах освіти ... not assumed a stable character, we are not inclined to consider the scream as a concept and leave it to us to designate it as a conceptual sense-given object. Future studies might focus on creating conceptual frameworks for the language learning process. Keywords: concept, conceptual object, idea, sense-giving, teaching, visual language. Problem statement. Though theoretically sense-giving could be classified as a noosphere category, either acquired culturally or specifically defined like imagineering, it will always be a characteristic of humans as an ultra-cultural species that is evolving in convergence at the genetic, intellectual, and cultural levels. It imparts activities to consciousness to determine the absolute magnitude of external objects, events, and experiences to improve mindfulness. Its cognitive “savor” (or adaptation to a changing environment) supports labile readiness to discover changes inside the object without altering its shape, to recognize the significance of those changes, and to apply it in achieving cognitions, realms of sense, including conceptual processing. It implies that sense is intended to be a definite entity that is embodied in the object (if human-made) and given (if natural). In both cases, the object chosen to symbolize the idea will be a sense-generating object that influenced how language or visual patterns are formed in the human mind. The question of “How is an idea given sense, and why do we need to conceptualize words or images?” arises in this conceivable scenario. The aim. This articleʼs specific goal is to illustrate the idea that every field of knowledge is expanded through the development of new concepts. The visual artwork, specifically Edvard Munchʼs Scream type, served as the reference point for this objective. It finally went beyond artistic culture and negotiated change, questioning its position in popular culture, and it became an object for the concept of visual language. In practical terms, teaching and learning aspects of cultural communication can be simplified by using techniques of conceptualizing objects, particularly dialoguing that covers mental activities and language skills acquisition as in thinking imaginatively together with linguistic performance. Visual art images as nonverbal communicative means provide the teacher with a way to enhance studentsʼ discourse skills. In this instance, communication can be considered a sequence of mental representations interacting with conceptual knowledge about an object, as well as their verbal 26 2023 Випуск 42 realizations, in the cognitive account. ISSN 2073-4379 Викладання мов у вищих навчальних закладах освіти ... Secondly, Munchʼs diaries, the memoirs of boswells, and writings of venerators of his artistry are of great value in understanding his artistic consciousness [5; 9; 11; 2; 8]. This includes the novel of the same title as the painting by the Polish writer and Munchʼs promoter Stanisław Przybyszewski, which conveys the artistʼs rueful feelings, and, accordingly, scholarly interest in the interaction between the painterʼs brush and the word-painterʼs word, in particular the work of L. Głuchowska [7]. The number of interpretations of Munchʼs painting symbol can be boundless. Here are some of them, as found in Pridauxʼs book [11]: 1) the image on the reverse, 2) the dilemma 27 ISSN 2073-4379 Викладання мов у вищих навчальних закладах освіти ... of modern man, a visualization of Nietzscheʼs cry, “God is dead, and we have nothing to replace him”, 3) the panic-chaos for the creative inspiration, 4) a scream of fear before the storm and thunder when nature prepares to speak to creatures who imagine themselves to be gods, etc. All opinions must form the basis of the concept, and primarily the authorʼs vision. Munchʼs words were: “… The Scream? I was being stretched to the limit – nature was screaming in my blood – I was at breaking point… I felt it all. After that, I gave up hope ever of being able to love again” [11: 168]. Moreover, he said: “We paint the souls” [11: 167]. of modern man, a visualization of Nietzscheʼs cry, “God is dead, and we have nothing to replace him”, 3) the panic-chaos for the creative inspiration, 4) a scream of fear before the storm and thunder when nature prepares to speak to creatures who imagine themselves to be gods, etc. All opinions must form the basis of the concept, and primarily the authorʼs vision. Munchʼs words were: “… The Scream? I was being stretched to the limit – nature was screaming in my blood – I was at breaking point… I felt it all. After that, I gave up hope ever of being able to love again” [11: 168]. Moreover, he said: “We paint the souls” [11: 167]. Thirdly, there are the works of researchers who link the creation of visual art with the mental side of human life and existence in social space. So, A. Випуск 42 psychodynamic or psychoemotional state of its meditator, more specifically: stimulus object – intentional object – mental object – ideative object – substantial object / semantic object – conceptual object. Each of these should be explained. j p Stimulus object: the object of affect (plaintive voice heard, shriek, scream) + the object of effect (cry sensed, cry or scream in reply). j y y p y Intentional object: the object to be set, willed, intent object. Mental object: the object of thought. Mental object: the object of thought. Mental object: the object of thought. Ideative object: the object of ideation. Ideative object: the object of ideation. Ideative object: the object of ideation. Substantial object / semantic object: a substantial object is a realistic object and a semantic object is an object that is interpreted according to semantic relations in a new environment. Conceptual object: the object identified and to be conceptualized (as a concept). Munchʼs impulse to create a conceptual image was stimulated by particular and probably adverse circumstances. The external stimulus was either “nature screaming” that was heard or a thought that burst out of his mind, and the latter seems more plausible. The internal impetus for the screaming picture lurked in his previous psychological experience and was first reflected in his writing and then developed during his work on the painting and its later versions. As he wrote in his sketchbooks, the first impression he had because of this excitement disappeared, and he could not write down what he saw either with painfully agitated feelings or in a joyful mood [8: 72]. In the attempt to recapture this first picture, i.e., the first impression, a new, altered image emerged in his mind, which was, hypothetically, in one way or another connected to the first image on the sensory level. The intentions changed and led to various transformations that are traced or can be visible, for example, in the autograph (the initial paintingʼs title idea was The Scream of Nature [8: 72]), in the paint, and in the moving of the image from version to version. But cognitive attitude, in our view, remained the same. Logically, a cognitive setting is sensitized when a perceived sound and, probably simultaneously or later, an object seen and associated with the sound (a mummy, in the case of Munch, by implication of [4: 8]) transform into a single visual-aural or “sounding” visual image. ISSN 2073-4379 Викладання мов у вищих навчальних закладах освіти ... Pitman as a specialist in Psychiatry acknowledges depicting “mental anguish” in the “dehumanized figure, into which viewers project their own neuroses” [10: 72]. A. Akhtar-Khavari concerns “cognitive and temporal dimensions of emotional experiences of fear” and considers art to “help us to think critically, creatively, ethically or politically about the concepts or ideologies within international environmental law” as well as “within the natural world” [1: 130]. Presentation of the main material. The interdependence between a concept and an idea is to be defined initially. They “are two terms relating to one and the same inner fact or psychological factor; the difference between them is that concept expresses the subjective aspect of this inner fact, and idea underlines the objective aspect” [12: 271]. The concept, then, appears to be a realistic idea taken from virtuality and adapted to human spirituality, mindset and self- expression, and culture in general. Furnishing know-how of the object virtuality representation or the object conceptualization (the scream) in the visual language and simultaneously in natural language symbols (or vice versa) correlates with the dynamics with which the idea of the object emerges in the artistʼs mind. Considering the object (image) as a conceptual phenomenon of visual culture gives the following chain of causal modes of its development, i.e., stimulation – intentional stances / transformations of intent / cognitive attitude (cognitive set) – mentalization / having mental content – ideation / sensing (reasoning) / having substantial content – conceptualization. All of these are either postures or processes in which a kind of object appears, depending on the 28 2023 Випуск 42 Випуск 42 It should not go unspoken that cognitive set is defined as being “the predetermined way an individual construes a situation, which is based on a group of concepts, related to the self and other things, that 29 ISSN 2073-4379 Викладання мов у вищих навчальних закладах освіти ... determines an individualʼs view of the world and influences his or her behavior” [15: 207]. The artistʼs cognitive set seems to be realized in the processes of mentalization and ideation of the image. Mentalization is thought as “the process by which we realize that having a mind mediates our experience of the world” [6: 3] and intrinsically constitutes “the ability to understand oneʼs own and othersʼ mental states, thereby comprehending oneʼs own and othersʼ intentions and affects” [15: 640]. During mentalization the inception object (here sound, loud vocalization) became, so to speak, “calibrated” and took on a familiar shape. determines an individualʼs view of the world and influences his or her behavior” [15: 207]. The artistʼs cognitive set seems to be realized in the processes of mentalization and ideation of the image. Mentalization is thought as “the process by which we realize that having a mind mediates our experience of the world” [6: 3] and intrinsically constitutes “the ability to understand oneʼs own and othersʼ mental states, thereby comprehending oneʼs own and othersʼ intentions and affects” [15: 640]. During mentalization the inception object (here sound, loud vocalization) became, so to speak, “calibrated” and took on a familiar shape. In the ideation process, the idea of the intended object itself is generated, and the substance of its concept is brought out by giving it essential features and framing it with additional strokes. In the latter case, the colour and the hues, which carry symbolic information, are significant: they help, as it were, to divine the image, and they participate in sensing the picture. In Munch, the dark blue is the city with its world, nature, or draped over it like a veil, and partly the robe of the screaming man (combined with darker tones – black, lurid). This fact may mean that the image contains the soul, but the soul and the spiritual essence of the world are separated. And this separation is indicated by the red (safety railing), that is, the image of God for a wight. It is a relic soul. Випуск 42 Випуск 42 health. Green is the colour of earthly, self-satisfied peace, one could also say, an earthly paradise. It affects people and causes boredom because it moves nowhere and has no echoes of joy, sadness, or passion; it demands nothing and calls nowhere. One might say that the colours represent the individualʼs cognitive dissonance, the clash of intellectual and emotional reactions in his mind. Further to the above, in Munchʼs painting, the information carrier is, first, an image with mouth open (symbolizing a scream – even if it is not a resonant scream), hands covering ears (symbolizing detachment from the world), and eyes widened madly (symbolizing the horror of the unknown); second, the itemʼs name The Scream. Conclusion. Because Munchʼs conception of the scream has not assumed a stable character, we are not inclined to consider the scream as a concept and leave it to us to designate it as a conceptual object given sense. The concept is a proffered identity for the object, authentic through the “interweaving” of its distinct values, creating a specific unit of knowledge components, including but not limited to linguistic, cultural, and iconic. This unit can be codified in a conceptual entity that has a certain link in the minds of the bearers of one culture with a single idea intelligible to all. In this case, it acquires a more generic designation. For example, we would call Munchʼs scream a concept if it referred to the conceptual entity “decomposition” or “dissolution” rather than to human anxiety or despair, loneliness, estrangement, pain, fear, dread, terror, etc. Decomposition means more. This is a decoupling of the intellect – rational and emotional – which manifests itself in the fact that man is aware of his emotions but, for reasons beyond his control, is mentally incapable of communicating his experiences to others, more generally, of communicating in a society. The willingness to do so is untenable. In fact, instead of screaming, this is blotting out the scream of reality. Scream as decomposition denotes the collapse after which life comes or death occurs, whether physically or spiritually. This categorial meaning establishes the datum point of the creator-owned design, the ultimate in the conceptualization of this object. An object conceptualization scheme can be developed based on the process described regarding the conceptualization of the visual object scream. Випуск 42 On Munchʼs canvas, the colour green is barely perceptible because of being lost in the blue, but the greenish cast in parts of the picture, most notably on the figureʼs face, speaks of a force that gives life, and faith. It is, however, a small force (the minute yellow strokes underline this), insufficient to overcome thoughts of the end (the bluish tint on the face and hands testify to this). We can assume, then, that in the cry, there is faith; though being weak, it has not abandoned the soul yet. The scream of the soul echoes an invocation, even if it seems to go nowhere. Therefore, the question remains whether there is salvation in Munchʼs scream. The external contrast of red with yellow (the sky at sunset) and sordid blue lines (the duster enveloping the earth on the other side of the road) lays stress on this conclusion. Yellow, a typically earthy colour, creates a sickly atmosphere, especially when intermixed with blue. When compared to the mental state of man, it is a colorful representation of madness. And this is exactly the impression one gets when he or she sees The Scream. On the contrary, red is reminiscent of a person who is convinced of his powers, and thus of extraordinary 30 2023 Випуск 42 Such a scheme should reflect cases capturing the stages of learning to analyse an object in depth to recognize different levels 31 ISSN 2073-4379 Викладання мов у вищих навчальних закладах освіти ... of meaning in it. The framework must fix the studentʼs experiences that initiated the choice of the object and developed a belief in its significance: 1) factors influencing the attitude change toward some object; 2) external triggers (the environment); 3) internal triggers (emotions; images); 4) idea-generating patterns (the characteristic of the object in the system). Using findings to construct conceptualization schemes for educational purposes is a prospect for further research. REFERENCES 1. Akhtar-Khavari, A. (2015). Fear and Ecological (In)Justice in Edvard Munchʼs the Scream of Nature. NAVEIÑ REET: Nordic Journal of Law and Social Research. Special Issue on Law and Art [online]. 2 (6), pp. 130–151. Available at: https://ssrn.com/abstract=2688781 [Accessed 01 March 2023]. 2. Bischoff, U. (2011). Edvard Munch. Cologne, Germany: Taschen GmbH. 3. Dennett, D.C. (1998). The Intentional Stance. 7th ed. Cambridge, Massachusetts, London, England: A Bradford Book, The MIT Press. First published in 1987. 4. Edvard Munch: Symbols and Images. (1978). Washington: National Gallery of Art. 5. Edvard Munchʼs Writings. emunch.no [online]. Available at: https://www.emunch.no/english.xhtml [Accessed 01 March 2023]. 6. Fonagy, P., Gergely, G., Jurist, E.L., Target, M. (2002). Affect regulation, mentalization and the development of the self. New York: Other Press. 7. Głuchowska, L. (2013). Munch, Przybyszewski and The Scream. Kunst og Kultur, 96 (4), pp. 182–193. DOI: https://www.idunn.no/doi/10.18261/ISSN1504-3029-2013-04-03. 8. Messer, T.M. (1985). Edvard Munch. New York: Harry N. Abrams, Inc., Publishers. 9. Munch, E. (2005). The Private Journals of Edvard Munch: We are flames which pour out of the earth. Holland, J.G. (Ed. and transl.); Høifødt, F. (Forward). Madison, Wisconsin: The University of Wisconsin Press. 10. Pitman, A. (2011). Edvard Munch (1863-1944) The Scream. The British Journal of Psychiatry, 198 (1), pp. 72–72. DOI: https://doi.org/10.1192/bjp.198.1.72. 11. Prideaux, S. (2012). Edvard Munch: Behind the Scream. 3d ed. New Haven and London: Yale University Press. 12. Roberti, F. (1962). Dictionary of Moral Theology. Westminster, Md.: Newman Press. 13. Talmy, L. (2000). Toward a Cognitive Semantics. Vol. I: Concept Structuring Systems. Cambridge, Massachusetts, London, England: 32 Випуск 42 A Bradford Book, The MIT Press. DOI: https://doi.org/10.7551/mitpress/6847.001.0001. A Bradford Book, The MIT Press. DOI: https://doi.org/10.7551/mitpress/6847.001.0001. p g p 14. Tsur, R. (2008). Toward a Theory of Cognitive Poetics. Brighton, Portland, Toronto: Sussex Academic Press. DOI: https://doi.org/10.1075/p&c.17.2.12fre. 15. VandenBos, G.R. (Ed.). (2015). APA Dictionary of Psychology. 2nd ed. Washington, DC: American Psychological Association. 16. Volkov, S.A. (2022). Ideation in its adaptation to the educational process. Research Result. Pedagogy and Psychology of Education, 8 (1), pp. 64–75. DOI: https://doi.org/10.18413/2313-8971-2022-8-1-0-6. The article was received by the editors 02.03.2023. The article was received by the editors 02.03.2023. The article was recommended for printing 22.03.2023. In cites: Volkov S. (2023). Munchʼs “Scream” as a sense-giving object for the conceptual entity. Teaching languages at higher educational establishments at the present stage. Intersubject relations. 42, pp. 23–33. DOI: https://doi.org/10.26565/2073-4379-2023-42-02. ISSN 2073-4379 Викладання мов у вищих навчальних закладах освіти ... комунікації. Використання образів візуального мистецтва як інструментів невербальної комунікації дає поштовх до розвитку дискурсивних навичок студентiв. Когнітивний підхід до осмислення образу крику в полотні Е. Мунка дозволяє виявити його символічні характеристики, які посилюють емоційний резонанс і надають принципово нового значення візуальному обʼєкту. Навчання комунікації здійснюється як побудова ланцюжка ментальних репрезентацій, що взаємодіють із концептуальними знаннями про предметність, а також їх вербальними реалізаціями. У результаті розгляд обʼєкта як концептуального феномену візуальної культури дає таку послідовність причинно-наслідкових модусів його осмислення: стимуляція – інтенціональна установка і когнітивне спрямування – менталізація / наявність ментального змісту – ідеація / осмислення / наявність предметного змісту – концептуалізація. Це процеси когнітивної трансформації певного обʼєкта, які залежать від психодинамічного або психоемоційного стану медитатора, а саме: обʼєкт – стимул – інтенційний обʼєкт – ментальний обʼєкт – ідеативний обʼєкт – предметний обʼєкт / семантичний обʼєкт – концептуальний обʼєкт. Уважаємо, що «крик» у Е. Мунка не набув сталого характеру концепта. Ми позначаємо його як концептуальний обʼєкт. Майбутні дослідження доцільно зосередити на розробці схем концептуалізації у навчанні мов. Ключовi слова: візуальна мова, ідея, концепт, концептуальний обʼєкт, навчання, смислоутворення. Ключовi слова: візуальна мова, ідея, концепт, концептуальний обʼєкт, навчання, смислоутворення. «КРИК» МУНКА ЯК ОБʼЄКТ, ЩО ПОРОДЖУЄ СЕНС КОНЦЕПТУАЛЬНОЇ СУТНОСТІ Сергiй Волков канд. пед. наук, доцент вiддiлення східних мов Вищого iнституту мов Тунicy Університету Карфагена (1003, Туніс, проспект Ібн Мажа, 14); e-mail: serguei.volkov@islt.ucar.tn; orcid: https://orcid.org/0000-0002-9682-4797 Сергiй Волков канд. пед. наук, доцент вiддiлення східних мов Вищого iнституту мов Тунicy Університету Карфагена (1003, Туніс, проспект Ібн Мажа, 14); e-mail: serguei.volkov@islt.ucar.tn; orcid: https://orcid.org/0000-0002-9682-4797 Концептуальна сутність – це явище, яке існує лише в наших думках як знаннєве, мовне, культурне, іконічне тощо. Всі обʼєкти, що підпадають під цю категорію, створені лише людським інтелектом, силою розуму або емоцій. Автор зосереджується на тому, як може бути сконструйоване концептуальне уявлення про обʼєкт, обраний або наданий для поглибленого обговорення чи дослідження в академічному середовищі, і як смислотворчий потенціал цього обʼєкта формує концептуальну одиницю. У цьому контексті обʼєкт виступає символом ідеї і перетворюється на інструмент смислотворення, що впливає на те, як лінгвістичні чи візуальні патерни проявляються у свідомості людини. Мета цієї статті – проілюструвати проблему розширення знаннєвого та комунікативного простору через розробку нових концептів. Автор розглядає візуальне мистецтво як джерело такого процесу, зокрема проаналізовано, як аналіз картини «Крик» Е. Мунка сприяє генерації ідей. Стратегії концептуалізації полегшують навчання міжкультурної 33 Як цитувати: Volkov S. Munchʼs “Scream” as a sense-giving object for the conceptual entity. Викладання мов у вищих навчальних закладах освіти на сучасному етапі. Міжпредметні звʼязки. 2023. Вип. 42. С. 25–35. DOI: https://doi.org/10.26565/2073-4379-2023-42-02. СПИСОК ВИКОРИСТАНИХ ДЖЕРЕЛ 1. Akhtar-Khavari A. Fear and Ecological (In)Justice in Edvard Munchʼs the Scream of Nature. NAVEIÑ REET: Nordic Journal of Law and Social Research. Special Issue on Law and Art [online]. 2015. 2 (6). P. 130–151. Available at: https://ssrn.com/abstract=2688781 [Accessed 01 March 2023]. 2. Bischoff U. Edvard Munch. Cologne, Germany: Taschen GmbH, 2011. 3. Dennett D.C. The Intentional Stance. 7th ed. Cambridge, Massachusetts, London, England: A Bradford Book, The MIT Press, 1998. 4. Edvard Munch: Symbols and Images. Washington: National Gallery of Art, 1978. 5. Edvard Munchʼs Writings. emunch.no [online]. Available at: https://www.emunch.no/english.xhtml [Accessed 01 March 2023]. 6. Fonagy P., Gergely G., Jurist E.L., Target M. Affect regulation, mentalization and the development of the self. New York: Other Press, 2002. 34 Випуск 42 2023 7. Głuchowska L. Munch, Przybyszewski and The Scream. Kunst og Kultur. 2013. 96 (4). P. 182–193. DOI: https://www.idunn.no/doi/10.18261/ISSN1504-3029-2013-04-03. https://www.idunn.no/doi/10.18261/ISSN1504-3029-2013-04-03. 8. Messer T.M. Edvard Munch. New York: Harry N. Abrams, Inc., Publishers, 1985. 9. Munch E. The Private journals of Edvard Munch: We are flames which pour out of the earth. Holland J.G. (Ed. and transl.); Høifødt F. (Forward). Madison, Wisconsin: The University of Wisconsin Press, 2005. 10. Pitman A. Edvard Munch (1863-1944) The Scream. The British Journal of Psychiatry. 2011. 198 (1). P. 72–72. DOI: https://doi.org/10.1192/bjp.198.1.72. 11. Prideaux S. Edvard Munch: Behind the Scream. New Haven and London: Yale University Press, 2012. 12. Roberti F. Dictionary of Moral Theology. Westminster, Md.: Newman Press, 1962. 13. Talmy L. Toward a Cognitive Semantics. Vol. I: Concept Structuring Systems. Cambridge, Massachusetts, London, England: A Bradford Book, The MIT Press, 2000. DOI: https://doi.org/10.7551/mitpress/6847.001.0001. 14. Tsur R. Toward a Theory of Cognitive Poetics. Brighton, Portland, Toronto: Sussex Academic Press, 2008. DOI: https://doi.org/10.1075/p&c.17.2.12fre. 15. VandenBos G.R. (Ed.). APA Dictionary of Psychology. 2nd ed. Washington, DC: American Psychological Association, 2015. 16. Volkov S.A. Ideation in its adaptation to the educational process. Research Result. Pedagogy and Psychology of Education. 2022. 8 (1). P. 64–75. DOI: https://doi.org/10.18413/2313-8971-2022-8-1-0-6. Стаття надійшла до редакції 02.03.2023. Стаття надійшла до редакції 02.03.2023. Стаття надійшла до редакції 02.03.2023. Статтю рекомендовано до друку 22.03.2023. Як цитувати: Volkov S. Munchʼs “Scream” as a sense-giving object for the conceptual entity. Викладання мов у вищих навчальних закладах освіти на сучасному етапі. Міжпредметні звʼязки. 2023. Вип. 42. С. 25–35. DOI: https://doi.org/10.26565/2073-4379-2023-42-02. 35 35
https://openalex.org/W2924756667
https://discovery.ucl.ac.uk/id/eprint/10088726/3/Reeves%20s12987-019-0158-1.pdf
English
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Region-specific blood-brain barrier transporter changes leads to increased sensitivity to amisulpride in Alzheimer’s disease
bioRxiv (Cold Spring Harbor Laboratory)
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Sekhar et al. Fluids Barriers CNS (2019) 16:38 https://doi.org/10.1186/s12987-019-0158-1 Sekhar et al. Fluids Barriers CNS (2019) 16:38 https://doi.org/10.1186/s12987-019-0158-1 Fluids and Barriers of the CNS Abstract Background:  Research into amisulpride use in Alzheimer’s disease (AD) implicates blood–brain barrier (BBB) dysfunc- tion in antipsychotic sensitivity. Research into BBB transporters has been mainly directed towards the ABC super- family, however, solute carrier (SLC) function in AD has not been widely studied. This study tests the hypothesis that transporters for organic cations contribute to the BBB delivery of the antipsychotics (amisulpride and haloperidol) and is disrupted in AD. Methods:  The accumulation of ­[3H]amisulpride (3.7–7.7 nM) and ­[3H]haloperidol (10 nM) in human (hCMEC/D3) and mouse (bEnd.3) brain endothelial cell lines was explored. Computational approaches examined molecular level interac- tions of both drugs with the SLC transporters [organic cation transporter 1 (OCT1), plasma membrane monoamine trans- porter (PMAT) and multi-drug and toxic compound extrusion proteins (MATE1)] and amisulpride with the ABC transporter (P-glycoprotein). The distribution of ­[3H]amisulpride in wildtype and 3×transgenic AD mice was examined using in situ brain perfusion experiments. Western blots determined transporter expression in mouse and human brain capillaries . Results:  In vitro BBB and in silico transporter studies indicated that ­[3H]amisulpride and ­[3H]haloperidol were trans- ported by the influx transporter, OCT1, and efflux transporters MATE1 and PMAT. Amisulpride did not have a strong interaction with OCTN1, OCTN2, P-gp, BCRP or MRP and could not be described as a substrate for these transporters. Amisulpride brain uptake was increased in AD mice compared to wildtype mice, but vascular space was unaffected. There were no measurable changes in the expression of MATE1, MATE2, PMAT OCT1, OCT2, OCT3, OCTN1, OCTN2 and P-gp in capillaries isolated from whole brain homogenates from the AD mice compared to wildtype mice. Although, PMAT and MATE1 expression was reduced in capillaries obtained from specific human brain regions (i.e. putamen and caudate) from AD cases (Braak stage V–VI) compared to age matched controls (Braak stage 0–II). Conclusions:  Together our research indicates that the increased sensitivity of individuals with Alzheimer’s to ami- sulpride is related to previously unreported changes in function and expression of SLC transporters at the BBB (in particular PMAT and MATE1). Dose adjustments may be required for drugs that are substrates of these transporters when prescribing for individuals with AD. Keywords:  Amisulpride, MATE1, PMAT, OCT1, Blood–brain barrier, Alzheimer’s Region‑specific blood–brain barrier transporter changes leads to increased sensitivity to amisulpride in Alzheimer’s disease Gayathri Nair Sekhar1, Alice L. Fleckney1, Sevda Tomova Boyanova1, Huzefa Rupawala1, Rachel Lo1, Hao Wang1, Doaa B. Farag1,2, Khondaker Miraz Rahman1, Martin Broadstock3,4, Suzanne Reeves5 and Sarah Ann Thomas1* Background *Correspondence: sarah.thomas@kcl.ac.uk 1 Faculty of Life Sciences and Medicine, School of Cancer and Pharmaceutical Sciences, King’s College London, Franklin‑Wilkins Building, 150 Stamford Street, Waterloo, London SE1 9NH, UK Full list of author information is available at the end of the article *Correspondence: sarah.thomas@kcl.ac.uk 1 Faculty of Life Sciences and Medicine, School of Cancer and Pharmaceutical Sciences, King’s College London, Franklin‑Wilkins Building, 150 Stamford Street, Waterloo, London SE1 9NH, UK Full list of author information is available at the end of the article Antipsychotic drugs are associated with significant harm in older people, particularly those with dementia who are more susceptible to antipsychotic drug related morbidity © The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 2 of 19 are involved. Haloperidol, a first generation antipsychotic drug which is highly selective for D2 receptors [24], was chosen as a clinically relevant comparator, as it is a posi- tively charged molecule at pH7.4 and a OCT1 substrate and inhibitor [25, 26] and likely to utilize similar trans- porters to amisulpride to cross the BBB. Although the drug is no longer used as a first line treatment for psy- chosis, due to its propensity to cause parkinsonism, it is used at very low doses in the treatment of delirium, which most commonly occurs in those aged over 65 years and with an underlying cognitive impairment (https​://www. nice.org.uk/, accessed 15.11.2019). The drug is also exten- sively used in palliative care and, as a result, is one of the 20 drugs on the WHO list of essential medications. (parkinsonism, postural hypotension, stroke) and mor- tality than other diagnostic groups [1, 2]. This has led to restrictions on the national health service (NHS) use of this class of drugs in the pharmacological management of psychosis and agitation in dementia. Emerging evidence from research into amisulpride use in older people with Alzheimer’s disease (AD) psychosis suggests that blood– brain barrier (BBB) dysfunction may be an important contributor to this heightened sensitivity [3, 4]. g y Amisulpride is a benzamide derivative, second gen- eration antipsychotic drug, used to treat schizophrenia [5] and a drug for which the optimal dose (400–800 mg/ day), blood concentration (100–319  ng/ml) and striatal dopamine D2/3 receptor occupancy range to avoid non- response and parkinsonism (40–70%) are well estab- lished in young adults with schizophrenia [6–8]. Despite being highly selective for dopamine D2/3 receptors [in vitro ­(Ki = 2.8  nM) and D3 ­(Ki = 3.2  nM)] amisulpride has a low propensity to induce parkinsonism, due to its poor BBB penetration and mesolimbic selectivity [9]. In an open treatment study which used amisulpride use in older people with AD psychosis and very late-onset (> 60 years) schizophrenia-like psychosis (VLOSLP), treat- ment response and parkinsonism occurred at very low doses (25–75 mg/day AD, 50–100 mg/day VLOSLP), and at correspondingly low blood drug concentrations (40– 100 ng/ml AD, 40–169 ng/ml VLOSLP) due to higher than anticipated striatal dopamine D2/3 receptor occupancies (caudate occupancy, steady state treatment, 50  mg/day amisulpride; 41–83% AD, 41–59% VLOSLP) [3, 10–12]. These findings strongly implicate age and AD-specific changes in central pharmacokinetics in antipsychotic drug sensitivity, particularly at the BBB, which controls drug entry through the expression of transporters [13]. Further- more, they suggest that amisulpride [14, 15] is a sufficiently sensitive tool with which to probe BBB functionality.h This study tested the hypothesis that there was an interaction between amisulpride and influx and/or efflux transporters at the BBB which was relevant from a phar- macodynamic perspective by: 1. identifying the transporter involved in the CNS dis- tribution of amisulpride and haloperidol, by examin- ing their kinetic characteristics and inhibitor sensi- tivity at the human and mouse BBB in vitro. 2. confirming the molecular level interactions of ami- sulpride and haloperidol with the selected BBB trans- porters using an in silico computational approach. 3. establishing whether amisulpride access to the CNS is increased in transgenic AD mice,  which harbour the human amyloid precursor protein (APP)-Swedish mutation (KM670/671NL), tau mutation (P301L), and presenilin-1 mutation (M146V) compared to wildtype mice. 4. investigating transporter expression in human (and mouse) brain endothelium from age-matched post- mortem AD and healthy aged controls. 5. Examining the type of medications prescribed to individuals with AD and age matched controls. p y The majority of research into BBB transporters has been directed towards the ABC superfamily, which are ATP- dependent efflux transporters such as P-glycoprotein (P-gp) [16], whose action is compromised in age [17], and more markedly so in AD [18–21]. It has been suggested that amisulpride is a weak P-gp substrate [22, 23], but the importance of P-gp relative to other transporters, espe- cially members of the SLC superfamily, remains unclear. Amisulpride is predominately positively charged (98.9%) at physiological pH (pKa 9.37), and is likely a substrate for the organic cation transporters (OCT) and organic cation transporters novel (OCTN); as observed using the immortalized human cerebral microvessel endothelial cell line (hCMEC/D3) [14]. However, it is also possible that other SLC transporters of organic cations, such as plasma membrane monoamine transporter (PMAT) and multi- drug and toxic compound extrusion proteins (MATEs), Overall BBB dysfunction in the AD process and its potential impact on drug delivery in particular on antip- sychotic medication will be explored (Fig. 1). The results can also be used to inform further studies [27]. Abstracts of this work have been presented [28, 29]. Materials [O-methyl-3H]amisulpride (MW374.8; specific activ- ity 77  Ci/mmol; 97% radiochemical purity) was triti- ated (TRQ41291 Quotient, UK). ­[3H(G)]haloperidol (MW375.9; specific activity, 20 Ci/mmol; 99% radiochemi- cal purity: cat# ART1729) was purchased from American Radiolabelled Chemicals Inc, St. Louis, Missouri, USA. ­[14C(U)]sucrose (MW359.48; specific activity 536  mCi/ Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 3 of 19 •hCMEC/D3 •bEnd.3 In vitro BBB models •Amisulpride •Haloperidol •Sucrose Test molecule •MTT assay •Transporter use •Extracellular space •Transporter protein expression EXPERIMENT Interacon with transporters In silico: Computaonal study to explore interacon of test molecules with selected transporters (OCT1, MATE1, PMAT and P-gp). In vitro: Octanol-saline paron coefficient to assess lipophilicity. In vitro BBB models (human and mouse) followed by in silico and physicochemical assessments. a In vitro BBB models (human and mouse) followed by in silico and physicochemical assessments. a Test molecule •Amisulpride •Haloperidol •Sucrose hCMEC/D3 •bEnd.3 BBB models p •Haloperidol •Sucrose molecule Extracellular space •Transporter protein expression Interacon with transporters •Wildtype (WT; n=6) •3xTg AD (n=4) GENOTYPE 2 groups 2 brain regions •12-13 mths (n=4). •16 mths(n=3). •24 mths (n=6). AGE WT 3 groups •Amisulpride accumulaon. •Vascular space. •Two brain regions. •Transporter total protein expression. EXPERIMENT Brain Perfusion Endpoints In silico: Computaonal study to explore interacon of test molecules with selected transporters (OCT1, MATE1, PMAT and P-gp). In vitro: Octanol-saline paron coefficient to assess lipophilicity. In vivo BBB studies in wild type and transgenic mice. b In silico: Computaonal study to explore interacon of test molecules with selected transporters (OCT1, MATE1, PMAT and P-gp). In vitro: Octanol-saline paron coefficient to assess lipophilicity. •Wildtype (WT; n=6) •3xTg AD (n=4) GENOTYPE 2 groups 2 brain regions •12-13 mths (n=4). •16 mths(n=3). •24 mths (n=6). AGE WT 3 groups •Amisulpride accumulaon. •Vascular space. •Two brain regions. •Transporter total protein expression. EXPERIMENT Brain Perfusion Endpoints In vivo BBB studies in wild type and transgenic mice. b In vivo BBB studies in wild type and transgenic mice. b •12-13 mths (n=4). •16 mths(n=3). •24 mths (n=6). • Healthy • AD PHENOTYPE HUMAN 2 groups Age matched •OCT1 •OCTN1 & 2 •MATE1 •MATE2 •PMAT EXPERIMENT Total and individual transporter protein expression Human ƒssue studies Medicaon history examined c Fig. 1  Flow charts to provide an overview of the experimental design for the in silico, in vitro and in vivo approaches. Experiments from the three approaches were performed in parallel Human ƒssue studies c • Healthy • AD Fig. Materials 1  Flow charts to provide an overview of the experimental design for the in silico, in vitro and in vivo approaches. Experiments from the three approaches were performed in parallel Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 4 of 19 mmol; 99% radiochemical purity: cat# MC266) was pur- chased from Moravek Biochemicals, USA. Amisulpride (MW369.5, > 98% purity) was purchased from Cayman Chemicals, UK (cat#71675-85-9). Haloperidol (MW375.9; > 98% purity) was purchased from Sigma-Aldrich, Dorset, UK (cat#H1512). Anti-SLC22A1 antibody (Cat#ab55916; RRID:AB_882579), Anti-SLC22A2 antibody (Cat#ab170871: RRID:AB_2751021, Anti-SLC22A3 antibody (Cat#ab183071; RRID:AB_2751016), Anti-SLC22A4 antibody (Cat#ab200641; RRID:AB_2751017), Anti-SLC22A5 anti- body (Cat#ab180757; RRID:AB_2751018), Anti-SLC47A1 antibody (Cat#ab104016: RRID:AB_10711136), Anti- SLC47A2 (Cat#ab174344: RRID:AB_2751019), SLC29A4 antibody (Cat#ab56554: RRID:AB_2190909), Goat Anti- Rabbit IgG H&L (cat#ab6721: RRID:AB_955447) and Rab- bit anti-mouse HRP (Cat#ab6728: RRID:AB_955440) were purchased from Abcam, UK. Anti-SLC47A1 (Cat#ab174344: RRID:AB_2751019) was purchased from Alomone Laboratories, Israel. Anti-SLC29A4 (Cat#: bs-4176R: RRID:AB_11108960) was purchased from Bioss antibodies, USA. Goat anti-rabbit (IgG)-HRP was purchased from (Cell Signalling. Cat#7074S:AB_2099233). Transferrin receptor monoclonal antibody was purchased from (Thermo Fisher Scientific, CAT# 13-6800 RRID: AB_2533029). Table  1 details the dilutions used. All consumables were purchased within the years 2012 to 2018 except the transferrin receptor antibody which was purchased in 2019. The predicted antibodies with their predicted molecular weight (MW) WB were all made up in PBS-T with 5% BSA. Actual band sizes may differ due to post translational modifications or cleavages. (Validation data is available from the Abcam website (https​://www.abcam​.com/nav/prima​ry-antib​odies​), cell signalling technology website (https​://www.cells​ignal​.co.uk) and Alonome labs website (https​://www.alomo​ne.com). Accessed 19.10.18. Validation data is available from the BIOSS USA antibodies website (http://www.bioss​usaan​tibod​ies.com). Accessed 4.12.18. Verification data is available from ThermoFisherScientific (https​://www.therm​ ofish​er.com). Accessed 20.11.2019 In vitro model of the BBB Cell cultureh The hCMEC/D3 (human) and bEnd.3 (mouse) are well- established models of the BBB [21, 30–32]. They are not listed on the misidentified cell line register (version 9 released 14th October 2018). Both lines require differ- ent mediums to grow to confluence and their BBB phe- notype has been confirmed using Western blots, confocal and transmission electron microscopy [32] (“Western Blot procedure” section). Functional expression of sev- eral transporters has been demonstrated by our group [32–34]. a. hCMEC/D3 cells (passages 27–35) were provided under a MTA and maintained in ­Clonetics® endothe- lial cell growth medium-2 MV Bullet Kit (Cat# CC-3162 Lonza, UK) containing the endothelial basal medium, the SingleQuotsTM growth factor kit, foe- Table 1  Primary and secondary antibodies used for protein expression studies Table 1  Primary and secondary antibodies used for protein expression studies Table 1  Primary and secondary antibodies used for protein expression studies The predicted antibodies with their predicted molecular weight (MW) WB were all made up in PBS-T with 5% BSA Actual band sizes may differ due to post Protein Primary antibody Secondary antibody Western blot (WB) OCT-1 (SLC22A1) Rabbit polyclonal anti-human and mouse (Abcam, Cat#ab55916; RRID:AB_882579), WB dilution—1:250 Goat anti-rabbit HRP (Abcam, cat#ab6721: RRID:AB_955447) dilution—1:1000 OCT-2 (SLC22A2) Rabbit monoclonal to human and mouse (Abcam, Cat#ab170871: RRID:AB_2751021), WB dilution—1:2000 Goat anti-rabbit HRP (Abcam, cat#ab6721: RRID:AB_955447) dilution—1:2000 OCT-3 (SLC22A3) Rabbit polyclonal to human and mouse (Abcam, Cat#ab183071; RRID:AB_2751016), WB dilution—1:600 Goat anti-rabbit HRP (Abcam, cat#ab6721: RRID:AB_955447) dilution—1:2000 OCTN1 (SLC22A4) Rabbit polyclonal to human and mouse (Abcam, Cat#ab200641; RRID:AB_2751017), WB dilution—1:1000 Goat anti-rabbit (IgG)-HRP (Cell Signalling. Cat#7074S:AB_2099233) dilution—1:1000 OCTN2 (SLC22A5) Rabbit polyclonal to human and mouse (Abcam, Cat#ab180757; RRID:AB_2751018), WB dilution—1:1000 Goat anti-rabbit (IgG)-HRP (Cell Signalling. Cat#7074S: AB_2099233) dilution—1:1000 MATE1 (SLC47A1) Rabbit polyclonal to human from Abcam (Cat#ab104016: RRID AB_10711136), WB dilution—1:500 Goat anti-rabbit HRP (Abcam, Cat#ab6721: RRID:AB_955447) dilution—1:2000 MATE1 (SLC47A1) Rabbit polyclonal to mouse from Alomone labs (Cat#ANT-131: RRID:AB_2751020), WB dilution—1:800 Goat anti-rabbit HRP (Abcam, Cat#ab6721: RRID:AB_955447) dilution—1:2000 MATE2 (SLC47A2) Rabbit polyclonal to human and mouse (Abcam, Cat#ab174344: RRID:AB_2751019), WB dilution—1:500 Goat anti-rabbit HRP (Abcam, Cat#ab6721: RRID:AB_955447) dilution—1:2000 PMAT (SLC29A4) Mouse monoclonal to human and rat (Abcam, Cat#ab56554: RRID:AB_2190909), WB dilution—1:500 Rabbit anti-mouse HRP (Abcam, Cat#ab6728: RRID:AB_955440) dilution 1:2000 PMAT (SLC29A4) Rabbit polyclonal to human, mouse and rat (Bioss Antibodies; Cat#:bs-4176R: RRID:AB_11108960), WB Dilution—1:800 for hCMEC/D3—1:650 for b.End3, 1:600 mouse capillaries Goat anti-rabbit (IgG)-HRP (Cell Signalling. Cat#7074S: RRID:AB_2099233) dilution—1:1000 TfR Transferrin receptor monoclonal antibody (Thermo Fisher Sci- entific, CAT# 13-6800: RRID: AB_2533029), WB Dilution—1:1000 Rabbit polyclonal Secondary to Mouse IgG—HRP (Abcam, CAT# ab6728 RRID: 955_440) WB dilution—1:1000 GAPDH Rabbit polyclonal to GAPDH (Abcam, Cat#ab9485: RRID:AB_307275), WB dilution 1: 2500 or 1:10,000 Cytotoxicity assay f y y y Cytotoxicity of amisulpride (0.1–20  μM) and eflornith- ine (250–500 μM) on both cell lines was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [32]. The results were expressed as a percentage of cell viability. This assay was also uti- lized on hCMEC/D3 cells (passage 32 and/or 33) for 2 h to assess toxicity of 1 μM famotidine, 2 μM famotidine, 2 μM lopinavir, 20 μM ergothioneine, 5 μM l-carnitine, 3 μM nifekalant hydrochloride and 1.5 μM ­[14C]sucrose. We have already published MTT assay results for eflor- nithine (250  μM) in hCMEC/D3 cells and haloperidol (40 μM), dexamethasone (200 μM), pentamidine (10 μM), ko143 (1  μM), MK571 (10  μM), amantadine (500  μM), corticosterone (50 μM), pheophorbide A (1 μM) and pra- zosin (100 μM) in both cell lines [32, 34]. No significant effect was observed except with prazosin on bEnd.3 cells. Transporter inhibition assay Transporter interaction was examined by incubating ­[3H] amisulpride and ­[14C]sucrose with potential inhibitors. These included unlabelled amisulpride and inhibitors/ substrates of OCT, OCTN, MATE, PMAT, P-gp, BCRP, and MRP at established concentrations (Additional file 1: Table S1). The MATE1 inhibitor, famotidine, was also uti- lized at a higher concentration of 2 μM. ATP depletion (Promega Enliten assay) was carried out by pre-incuba- tion with the glycolysis inhibitor, 10 mM 2-deoxy-d-glu- cose, for an hour before incubating the cells with ­[3H] amisulpride and ­[14C]sucrose. This assay had previously been shown to inhibit drug efflux by our group [32, 34]. Both lines were maintained at 37  °C/5% ­CO2 in an incubator with saturated humidity. Medium was changed every 2–3  days. Cells were split when they reached 80–90% confluency and were seeded onto 96-well plates (ThermoScientific, UK) at 20,000 cells/cm2 for b.End3 cells and 25,000  cells/cm2 for hCMEC/D3 cells. The hCMEC/D3 96-well plates were pre-coated with 0.1 mg/ ml rat tail collagen type1 (Gibco cat#A1048301).l Both lines were confluent in 4–5  days and were left for another 4  days to allow for further differentiation before experimentation. The medium was changed every 2–3 days. Drug accumulation assay Accumulation assays were performed on confluent cell monolayers grown in the centre 60 wells of 96-well plates. Each passage was regarded as one ‘n’. No sample calcula- tion was performed. The accumulation buffer (pH7.4) composition was 135 mM NaCl, 10 mM HEPES, 5.4 mM KCl, 1.5 mM ­CaCl2, 1.2 mM ­MgCl2, and 1.1 mM d-glu- cose and water. It also contained 3.7–7.7 nM [Omethyl- 3H]amisulpride and 9.4  µM ­[14C(U)]sucrose or 10  nM ­[3H]haloperidol and 3.8  μM ­[14C]sucrose. ­[14C]sucrose is a similar size to the test molecules and was used as an inert marker of extracellular space and membrane integ- rity. After the exposure period, buffer was aspirated and the wells washed with ice-cold ­PBS+ (Sigma-Aldrich, UK) to remove drug that was not taken up by cells and to stop further transport. 1% Triton X-100 (Sigma-Aldrich) was added and the plate was incubated for an hour at 37  °C to lyse the cells and to release accumulated ­[3H] drug. 100 µl from each of the wells was transferred to a vial and scintillation fluid (4 ml) added (Optiphase Hisafe 2, PerkinElmer, UK). Radioactivity was measured using a Packard Tri-Carb 2900TR liquid scintillation coun- ter (PerkinElmer, UK) and corrected for background. The remaining 100 µl in each well were used to perform a bicinchoninic acid (BCA) protein assay. A range of 2–30 μl mg−1 of protein was acceptable. All data for ­[3H] Secondary antibody Western blot (WB) They were grown in T-75 flasks (Fisher Scientific, cat# 15350591) using high glucose Dulbeccos Modified Eagles Medium (Sigma- Aldrich, UK, cat# D6429) supplemented with 10% FBS (vol/vol) and 1% penicillin–streptomycin (vol/ vol: Fisher Scientific cat#10003927). b. bEnd.3 cells (passages 17–25), isolated from the SV129 strain of mice and transformed with the Polyoma virus middle T-antigen, were purchased from ATCC​® and underwent authentication tests during the accessioning process (CRL-2299: RRID:CVCL0170) [36]. They were grown in T-75 flasks (Fisher Scientific, cat# 15350591) using high glucose Dulbeccos Modified Eagles Medium (Sigma- Aldrich, UK, cat# D6429) supplemented with 10% FBS (vol/vol) and 1% penicillin–streptomycin (vol/ vol: Fisher Scientific cat#10003927). Secondary antibody Western blot (WB) Secondary antibody Western blot (WB) Secondary antibody Western blot (WB) Goat anti-rabbit HRP (Abcam, cat#ab6721: RRID:AB_955447) dilution—1:2000 Goat anti-rabbit HRP (Abcam, Cat#ab6721: RRID:AB_955447) dilution—1:2000 Goat anti-rabbit HRP (Abcam, Cat#ab6721: RRID:AB_955447) dilution—1:2000 Rabbit anti-mouse HRP (Abcam, Cat#ab6728: RRID:AB_955440) dilution 1:2000 PMAT (SLC29A4) Rabbit polyclonal to human, mouse and rat (Bioss Antibodies; Cat#:bs-4176R: RRID:AB_11108960), WB Dilution—1:800 for hCMEC/D3—1:650 for b.End3, 1:600 mouse capillaries Goat anti-rabbit (IgG)-HRP (Cell Signalling. Cat#7074S: RRID:AB_2099233) dilution—1:1000 Rabbit polyclonal Secondary to Mouse IgG—HRP (Abcam, CAT# ab6728 RRID: 955_440) WB dilution—1:1000 Rabbit polyclonal Secondary to Mouse IgG—HRP (Abcam, CAT# ab6728 RRID: 955_440) WB dilution—1:1000 TfR Transferrin receptor monoclonal antibody (Thermo Fisher Sci- entific, CAT# 13-6800: RRID: AB_2533029), WB Dilution—1:1000 TfR Transferrin receptor monoclonal antibody (Thermo Fisher Sci- entific, CAT# 13-6800: RRID: AB_2533029), WB Dilution—1:1000 GAPDH Rabbit polyclonal to GAPDH (Abcam, Cat#ab9485: RRID:AB_307275), WB dilution 1: 2500 or 1:10,000 GAPDH Rabbit polyclonal to GAPDH (Abcam, Cat#ab9485: RRID:AB_307275), WB dilution 1: 2500 or 1:10,000 The predicted antibodies with their predicted molecular weight (MW) WB were all made up in PBS-T with 5% BSA. Actual band sizes may differ due to post translational modifications or cleavages. (Validation data is available from the Abcam website (https​://www.abcam​.com/nav/prima​ry-antib​odies​), cell signalling technology website (https​://www.cells​ignal​.co.uk) and Alonome labs website (https​://www.alomo​ne.com). Accessed 19.10.18. Validation data is available from the BIOSS USA antibodies website (http://www.bioss​usaan​tibod​ies.com). Accessed 4.12.18. Verification data is available from ThermoFisherScientific (https​://www.therm​ ofish​er.com). Accessed 20.11.2019 Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 5 of 19 tal bovine serum (FBS), penicillin–streptomycin and HEPES (Sigma-Aldrich, UK) [32, 34, 35]. tal bovine serum (FBS), penicillin–streptomycin and HEPES (Sigma-Aldrich, UK) [32, 34, 35]. amisulpride or ­[3H]haloperidol were expressed as a vol- ume of distribution ­(Vd) after correction for ­[14C]sucrose. The ­Vd was calculated from the sum of accumulated radi- oactivity (a sum of efflux and influx of the molecule) (dis- integrations per minute (dpm)/mg protein) over the ratio of dpm/μl of accumulation buffer. Outliers were identi- fied by examining the ­[14C]sucrose values. ­[14C]sucrose values are presented in the figures and tables. tal bovine serum (FBS), penicillin–streptomycin and HEPES (Sigma-Aldrich, UK) [32, 34, 35]. b. bEnd.3 cells (passages 17–25), isolated from the SV129 strain of mice and transformed with the Polyoma virus middle T-antigen, were purchased from ATCC​® and underwent authentication tests during the accessioning process (CRL-2299: RRID:CVCL0170) [36]. Lipophilicity Lipophilicity is a standard physicochemical measure which is very important in terms of understanding drug distribution across membranes including the BBB and can be expressed in the form of an octanol-saline parti- tion coefficient. The higher the lipophilicity the greater the ability of the molecule to cross the plasma membrane by passive diffusion. An octanol-saline partition coeffi- cient for ­[3H]amisulpride and ­[3H]haloperidol was deter- mined [37]. Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 6 of 19 In silico computational study water ad libitum. The experiment on each animal had to be performed within set time frames to allow the three age groups to be achieved. Experiments were performed between 9am and 5  pm. The experimenter was not blinded. p y Using in silico molecular docking, we tested the molecu- lar level interactions of amisulpride, haloperidol and pra- zosin (OCT1 and OCT3 substrate) with the transporters OCT1, PMAT and MATE1. Due to the unavailability of the crystal structures of these transporters, molecular models of OCT1, PMAT and MATE1 were developed using homology modelling with Swiss-model webserver using PDB codes 4PYP, 5Y50 and 4ZOW, respectively, as templates (Additional file 1). A further molecular dock- ing study was performed to explore the interaction of amisulpride, dexamethasone (P-gp substrate) and colchi- cine (P-gp substrate) in the binding site of the multidrug transporter ABCB1 (P-glycoprotein), and the pbd code 6FN1 [38] was used as the template. Molecular docking was performed using Dock Ligands (CDOCKER) pro- tocol from Discovery studio version 4.0. CDOCKER is an implementation of a CHARMm based docking tool where each orientation is subjected to simulated anneal- ing molecular dynamics. The binding sites were chosen after comparing the docking results of the ligands in all possible binding cavities within the transporters. The median life span of 3×TgAD mice has been reported to be 673  days (22  months) which is shorter than the 907 day (30 months) lifespan of C57BL/6J [41]. Adult male BALB/c mice (inbred strain) were purchased from Harlan UK Limited (Oxon, UK). All mice were anaesthetised (2 mg/kg i.p. medetomidine hydrochloride and 150 mg/kg i.p. ketamine) and heparinized (100 U i.p.) in a procedure room separate to the laboratory where the brain perfusion was performed. Advice was sought from the named veterinary surgeon regarding the anaesthetic. In situ brain perfusionf To assess differences in ­[3H]amisulpride and ­[14C]sucrose transport into the brain in ageing and in AD, wildtype (C57BL6/129 n = 17) and transgenic AD (3×Tg n = 6) mice were used. Perfusion (10  min; 5  ml/min) with a warmed (37 °C) and oxygenated (95% ­O2; 5% ­CO2) arti- ficial plasma was via a cannula in the left ventricle of the heart as previously described [42]. The artificial plasma consisted of a modified Krebs–Henseleit mammalian Ringer solution with the following constituents: 117 mM NaCl, 4.7  mM KCl, 2.5  mM ­CaCl2, 1.2  mM ­MgSO4, 24.8  mM ­NaHCO3, 1.2  mM ­KH2PO4, 10  mM glucose, and 1 g/l bovine serum albumin. With the start of perfu- sion the right atrium of the heart was sectioned to pre- vent the recirculation of the artificial plasma. At the end of perfusion the animal was decapitated and the brain removed. To determine the brain concentration of ­[3H] amisulpride, frontal and occipital cortex samples were taken and weighed using a Leica S4E microscope (Pur- chased 2005). Brain samples were then solubilized with 0.5  ml of Solvable (PerkinElmer, USA) and liquid scin- tillation fluid (3.5  ml; Lumasafe; PerkinElmer) added. Radioactivity in the samples were determined using the Tri-Carb2900TR scintillation counter (Purchased 2007). Animal model of AD All experiments were performed in accordance with the Animal Scientific Procedures Act (1986) and Amend- ment Regulations 2012 and with consideration to the ARRIVE guidelines. The study was approved by the King’s College London Animal Welfare and Ethical Review Body and performed under license: 70/7755. A total of 40 mice were used for this study. Mice were housed at King’s College London. C57BL6/129 mice (wild-type) and the triple transgenic AD model (3×TgAD) of C57 mice which harbour the human APP-Swedish mutation (KM670/671NL), tau mutation (P301L), and presenilin-1 mutation (M146V) were uti- lized at 12–13 (mid-age), 16 (old) or 24 (elderly) months old. The 3×TgAD is an established AD model displaying the temporal and spatial progression and mirroring the neuropathological development seen in AD [39]. These mice were bred and genotyped at King’s College London and were a gift. They had originally been supplied by the Jackson Laboratory (RRID:IMSR_JAX:008880). No sam- ple size calculation was performed and the study was not pre-registered. Previous experience suggested that even with the limited number of animals available there would still be sufficient power (95%) to detect significant differ- ences (p < 0.05) [40]. Welfare was assessed daily by animal technologists. Animals were identified by earmarks and housed together by age and genotype in guideline com- pliant cages. All animals were maintained under stand- ard temperature/lighting conditions and given food and Human brain microvasculature isolation Brain capillaries from frontal cortex, caudate nucleus, and putamen samples were isolated after homogenising 300 mg tissue and carrying out a dextran-based density- gradient centrifugation to produce a capillary-enriched pellet. The pellet was further lysed with 500  µl of ice-cold RIPA buffer with added protease inhibitors at 4 °C and then centrifuged at 8000×g for 15 min at 4 °C. The result- ing supernatant was taken for Western blot analysis to examine transporter expression. The presence of trans- ferrin receptor in the supernatant indicated that the method generated samples containing capillary endothe- lial cells. Western Blot procedureh The supernatant protein concentration was determined using a BCA assay (Albumin standard, ThermoScien- tific). The supernatants were diluted and boiled for 5 min at 95 °C in 5× Laemmli sample buffer. Cell lines (30 μg except for MATE 1 antibody in Bend.3 cells where 15 μg was utilized and PMAT antibody in hCMEC/D3 and bEnd.3 cells where 20 μg and 10 μg was utilized respec- tively), mouse samples (15 μg for MATE1, OCTN1 and 2) and (30 μg for MATE2, PMAT and OCT1), human sam- ples (10 μg for OCNT1 and 2) or 15–20 μg (for MATE1, MATE2, PMAT and OCT1) were loaded equally on 4–20% Mini-PROTEAN® TGX™ gels (Bio-Rad) along- side a molecular weight marker (Precision plus pro- tein, Bio-Rad). Samples underwent SDS-PAGE at 160 V for 1 h. Proteins were transferred onto 0.45 μm polyvi- nylidene fluoride membranes (GE Healthcare, UK) after methanol activation at 100 V for 1 h. Membranes were blocked to reduce nonspecific binding using 5% milk with PBS-TWEEN® tablets (PBS-T) (Calbiochem, USA) at room temperature (RT) for 1  h. Membranes were incubated overnight at 4 °C with primary antibodies in PBS-T (Table  1). Membranes were washed in PBS-T (3 × 10 min) and incubated with the secondary antibody in PBS-T at RT for 1 h. Further washing in PBS-T (3 × 10 hCMEC/D3 and b.End3 monolayers isolation Tissue was received on the basis that it will be han- dled, stored, used and disposed of within the terms of the Human Tissue Act 2004. Post-mortem brain cap- illaries from healthy individuals (Braak stage 0–II; 86.8 ± 1.5 years; 2 females, 3 males) and AD cases (Braak stage V–VI; 79.4 ± 3.7  years; 2 females, 3 males) were used to investigate the expression of transporters (Case details—Additional file 1: Table S2). Medication history of the cases was supplied by the Manchester Brain Bank (Additional file 1: Table S3). In this study we identified those drugs prescribed as sedatives, antidepressants and antipsychotics. In order to perform Western blots, cell lines were grown to confluence in T-75 flasks (Thermo Scientific, UK) and left for 3–4 days, as previously described. The flask was then transferred to ice and the medium removed, before the cells were washed twice using ice-cold PBS+. Then, 1 ml of ice-cold Radio-Immunoprecipitation Assay (RIPA) buffer (Sigma-Aldrich, Dorset, UK) with added protease inhibitors (10% v/v) (Thermo Scientific, Lough- borough, UK) was added to the flask to lyse the cells. A plastic cell scraper (Greiner Bio-One Ltd, Gloucester- shire, UK) was used to scrape the cells off the bottom of the flask and the cell lysate was transferred to a pre- cooled 1.5 ml Eppendorf tube which was left on ice for 20 min. The tubes were then centrifuged at 10,000 rpm for 10  min at 4  °C using a Thermo Electron Corpora- tion Heraeus Fresco17 bench-top micro-centrifuge. After centrifugation, the supernatant was transferred to another pre-cooled 1.5 ml Eppendorf and the pellet dis- carded. The resulting supernatant was taken for Western blot analysis. Mouse brain capillary isolation Brain capillaries from old-age (16 months) wild-type (3 females) and 3×TgAD (3 males, 1 female) mice were used to explore MATE1 expression. Brain capillaries were also isolated from elderly (24 month) age-matched wild-type C57BL6/129 mice (3 males, 2 females) and 3×TgAD mice (3 males, 2 females) for all other transporter stud- ies. The left ventricle of the heart was cannulated and perfused (5 ml/min) with an oxygenated artificial plasma (modified Krebs–Henseleit mammalian Ringer) for up to 2 min. The right atrium was sectioned before perfu- sion was started. The mice were then decapitated and the perfused brain removed. The brain was homogenized in physiological buffer (brain weight × 3) and 26% dextran (brain weight × 4). The homogenate was subjected to density gradient centrifugation (5400×g for 15  min at 4 °C) to give an endothelial cell-enriched pellet and the supernatant was discarded [42]. 300 µl of ice-cold RIPA: ThermoFisher Scientific cat#89900) buffer with added protease inhibitors was added to the pellet at 4 °C to lyse the tissue and then centrifuged at 8000×g for 15 min at 4  °C. The resulting supernatant was taken for Western blot analysis. Expression of results d Radioactive concentrations in the brain samples (dpm/g) were expressed as a percentage of that in the artificial plasma (dpm/ml) and termed ­RTISSUE (millilitres/100 g). All ­RTISSUE values for ­[3H]amisulpride were corrected for vascular/extracellular space by subtracting the ­[14C] sucrose ­RTISSUE value. Examination of the ­[14C]sucrose values (i.e. vascular space) and comparison to previ- ously published values determines if the result is an out- lier. This is acceptable for the WT animals, however, it is noted that ­[14C]sucrose (vascular space) may be affected in AD. No outliers were detected in this study. Sekhar et al. Fluids Barriers CNS (2019) 16:38 Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 7 of 19 OCT, OCTN, PMAT and MATE involvement Involvement of OCTs in ­[3H]amisulpride uptake was investigated by incubating hCMEC/D3 and bEnd.3 cells with the, OCT1 and 2 substrate, amantadine, the OCT1 and 3 substrate, prazosin, and the OCT3 substrate, corticosterone (Additional file  1: Figure S4). ­[3H]ami- sulpride accumulation did not change in the presence of amantadine in hCMEC/D3 cells, but significantly increased by 84% in bEnd.3 cells. In the presence of pra- zosin, there was a significantly reduced accumulation of ­[3H]amisulpride in hCMEC/D3 cells, but not in bEnd.3 Human tissue Human tissue was provided with informed consent via the brains for dementia research (BDR) and were anonymized. BDR has ethical approval granted by the national health service (NHS) health research author- ity (NRES Committee London-City & East, UK: REC reference: 08/H0704/128+5. IRAS project ID:120436). Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 8 of 19 min), membranes were then incubated with enhanced chemiluminescent reagent (ThermoScientific) for 30 s at RT. and a decrease of 50% in bEnd.3 cells observed after 2 h (Fig. 2). No significant differences were observed for ­[14C] sucrose between the treatments, except in the bEnd3 cells at 120 min where the presence of 20 μM amisulpride decreased the accumulation of ­[14C]sucrose (Additional file 1: Figure S1). However, all ­[14C]sucrose values were within the expected range for these in vitro models. Stud- ies also revealed that lower concentrations of unlabelled amisulpride (0.1 μM) did not affect accumulation of ­[3H] amisulpride in hCMEC/D3 (n = 5 passages) or bEnd.3 (n = 3 passages) cells at all time points (data not shown). Quantification of protein expression was determined by calculating the intensity ratio of the band of interest and the band of the loading control (tubulin or GAPDH). Band intensity ratio analysis was conducted using ImageJ software (NIH). Our group have previously published results from Pgp, BCRP, OCT1, OCT2 and OCT3 pro- tein expression studies of bEnd.3 and hCMEC/D3 cells [32, 34]. Data analysis Data are expressed as mean ± SEM. The data was ana- lysed by two-way ANOVA with Holm-Sidak post hoc test for the accumulation studies and in situ perfusion stud- ies, one-way ANOVA with Tukey’s post hoc test for MTT assay and Student’s t-test or two-way ANOVA for West- ern blot data using Sigmaplot version 13 (Systat, USA) or GraphPad Prism 7.03. p < 0.05 were considered as statis- tically significant. Exact p-values are provided in figure legends/“Results” section. ATP depletion did not affect the accumulation of ­[3H] amisulpride or ­[14C]sucrose in either cell line (Additional file  1: Figure S2). The P-gp substrate, dexamethasone, BCRP substrate, ko143, and inhibitor, pheophorbide A, and MRP family inhibitor, MK571, did not affect the accumulation of ­[3H]amisulpride or ­[14C]sucrose in either cell line (Additional file 1: Figure S3). Interaction with the positively charged anti‑psychotic drug, haloperidolhf The effect of the cationic drug, haloperidol (40 μM), on radiolabelled amisulpride accumulation was also inves- tigated. Incubation of unlabelled haloperidol with ­[3H] amisulpride did not yield any significant effects in either cell line (Additional file 1: Figure S9). No significant dif- ferences were found for ­[14C]sucrose between the treat- ments (Additional file 1: Figure S9). Incubation with the MATE1 inhibitor, famotidine (1 μM) resulted in no significant effect on the ­Vd of ­[3H] amisulpride in hCMEC/D3 cells (Fig. 4). The ­Vd of ­[14C] sucrose was not significantly different between control and test groups with (1 μM) famotidine during the stand- ard 2 h incubation period. An assessment of the effect of 2 μM famotidine on ­[14C]sucrose alone revealed a loss of hCMEC/D3 integrity at 2 h. Further assessment of famo- tidine (2  μM) did not affect either ­[3H]amisulpride or ­[14C]sucrose accumulation in hCMEC/D3 cells over a 1 h period (Fig. 4). No affect was observed with famotidine Amisulpride accumulation and saturable transport 3 All data have been corrected for ­[14C]sucrose and are expressed as mean ± S.E.M, n = 3 to 7 plates with 6 replicates (wells) per timepoint per plate (5 time-points) Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 9 of 19 (1  μM) in b.End3 with either ­[3H]amisulpride or ­[14C] sucrose (Additional file 1: Figure S7). cells. Corticosterone did not affect the accumulation of ­[3H]amisulpride in either cell line. No differences were found for ­[14C]sucrose between the treatments (Addi- tional file 1: Figure S4). i Incubation with the MATE2 inhibitor, nifekalant, resulted in no significant affect on the ­Vd of ­[3H]ami- sulpride in hCMEC/D3 cells (Additional file  1: Figure S8). The ­Vd of ­[14C]sucrose was not significantly differ- ent between control and test groups up to 120 min with nifekalant suggesting loss of membrane integrity at this time point. No affect was observed with this inhibitor in b.End3 with either ­[3H]amisulpride or ­[14C]sucrose (Additional file 1: Figure S7). i [3H]amisulpride accumulation in hCMEC/D3 and b.End3 was unaffected by the presence of ergothio- neine (OCTN1) and l-carnitine (OCTN2) respectively (Additional file 1: Figures S5 and S6). ­[14C]Sucrose ­Vd was not significantly different between the treatments, except significant differences were observed between ­[14C]sucrose and l-carnitine at 2 h suggestive that this time point for ­[3H]amisulpride should be ignored. Incubation with the PMAT inhibitor, lopinavir, resulted in a significant increase in the ­Vd of ­[3H]ami- sulpride by 63.9% at 20 min, 83.2% at 30 min, 85.1% at 60 min and by 68.6% at 120 min (Fig. 3). The ­Vd of ­[14C] sucrose was not significantly different between control and test groups (Fig. 3). No affect was observed with this inhibitor in the b.End3 cells (Additional file 1: Fig- ure S7). Amisulpride accumulation and saturable transport 3 p p [3H]Amisulpride was able to accumulate in both hCMEC/D3 and bEnd3 cell lines to a greater extent than the baseline marker, ­[14C]sucrose (Fig. 2 and Additional file 1: Figure S1). Incubation of hCMEC/D3 and bEnd3 cell lines with 20 µM amisulpride significantly decreased the accumulation of ­[3H]amisulpride (6.5 nM) at 5 min— with a significant decrease of 37% in hCMEC/D3 cells 5 20 30 60 120 5 20 30 60 120 0 5 10 15 20 hCMEC/D3 Incubation Time (minutes) Volume of distribution ( l/mg protein) *** *** *** **** **** [ 3 H]Amisulpride + 20 M Amisulpride 0 5 10 15 20 bEnd.3 Incubation Time (minutes) ** * ** **** µ µ Volume of distribution ( l/mg protein) µ Fig. 2  The effect of self-inhibition (20 μM) on the accumulation of ­[3H]amisulpride (6.5 nM) was determined in hCMEC/D3 (a) and bEnd.3 (b) cell lines. Significant differences compared to control were observed—*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. All data have been corrected for ­[14C]sucrose and are expressed as mean ± S.E.M, n = 3 to 7 plates with 6 replicates (wells) per timepoint per plate (5 time-points) 5 20 30 60 120 5 20 30 60 120 0 5 10 15 20 hCMEC/D3 Volume of distribution ( l/mg protein) *** *** *** **** **** [ 3 H]Amisulpride + 20 M Amisulpride 0 5 10 15 20 bEnd.3 ** * ** **** µ µ Volume of distribution ( l/mg protein) µ Incubation Time (minutes) Incubation Time (minutes) Fig. 2  The effect of self-inhibition (20 μM) on the accumulation of ­[3H]amisulpride (6.5 nM) was determined in hCMEC/D3 (a) and bEnd.3 (b) cell lines. Significant differences compared to control were observed—*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. All data have been corrected for ­[14C]sucrose and are expressed as mean ± S.E.M, n = 3 to 7 plates with 6 replicates (wells) per timepoint per plate (5 time-points) Fig. 2  The effect of self-inhibition (20 μM) on the accumulation of ­[3H]amisulpride (6.5 nM) was determined in hCMEC/D3 (a) and bEnd.3 (b) cell lines. Significant differences compared to control were observed—*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Characteristics of haloperidol accumulation in hCMEC/D3 and b.End3 cell lines hCMEC/D3 and b.End3 cells were incubated with unla- belled haloperidol (40  μM) along with ­[3H]haloperidol (10  nM). Incubation with unlabeled haloperidol signifi- cantly decreased the accumulation of radiolabelled halo- peridol by approximately 93% in hCMEC/D3 cell line and by 94% in bEnd.3 cell line at all times (***p < 0.001) (Addi- tional file 1: Figure S10). No significant differences were 5 20 30 60 120 0 20 40 60 80 hCMEC/D3 Incubation Time (minutes) [ 3H]Amisulpride + 2 M Lopinavir * * ** *** 0 20 40 60 80 hCMEC/D3 Incubation Time (minutes) [ 14 C]Sucrose + 2 M Lopinavir Volume of distribution ( l/mg protein) µ Volume of distribution ( l/mg protein) µ 5 20 30 60 120 µ µ Fig. 3  The effect of PMAT inhibition on the accumulation of ­[3H]amisulpride (3.7–7.7 nM) was determined in hCMEC/D3 cell lines. Significant increases were observed compared to control ****p ≤ 0.0001, ***p ≤ 0.001, **p = 0.01 and *p = 0.05. ­[3H]amisulpride data has been corrected for ­[14C]sucrose and are expressed as mean ± S.E.M, n = 5 passages (p30, 2 × p31, p32 and p34 for PMAT) with 6 replicates (wells) per timepoint per plate (5 time-points) 0 20 40 60 80 hCMEC/D3 [ 14 C]Sucrose + 2 M Lopinavir Volume of distribution ( l/mg protein) µ 5 20 30 60 120 µ Incubation Time (minutes) Incubation Time (minutes) Fig. 3  The effect of PMAT inhibition on the accumulation of ­[3H]amisulpride (3.7–7.7 nM) was determined in hCMEC/D3 cell lines. Significant increases were observed compared to control ****p ≤ 0.0001, ***p ≤ 0.001, **p = 0.01 and *p = 0.05. ­[3H]amisulpride data has been corrected for ­[14C]sucrose and are expressed as mean ± S.E.M, n = 5 passages (p30, 2 × p31, p32 and p34 for PMAT) with 6 replicates (wells) per timepoint per plate (5 time-points) Fig. 3  The effect of PMAT inhibition on the accumulation of ­[3H]amisulpride (3.7–7.7 nM) was determined in hCMEC/D3 cell lines. Significant increases were observed compared to control ****p ≤ 0.0001, ***p ≤ 0.001, **p = 0.01 and *p = 0.05. ­[3H]amisulpride data has been corrected for ­[14C]sucrose and are expressed as mean ± S.E.M, n = 5 passages (p30, 2 × p31, p32 and p34 for PMAT) with 6 replicates (wells) per timepoint per plate (5 time-points) Sekhar et al. Characteristics of haloperidol accumulation in hCMEC/D3 and b.End3 cell lines Fluids Barriers CNS (2019) 16:38 Page 10 of 19 f d f [14C] b t th t t t (Addi hCMEC/D3 ll d b 82% i bE d 3 ll d i th 0 20 40 60 80 hCMEC/D3 Incubation time (minutes) [ 3 H]Amisulpride + 1 M Famotidine 5 20 30 60 120 5 20 30 60 120 0 20 40 60 80 hCMEC/D3 Incubation time (minutes) [ 14 C]Sucrose + 1 M Famotidine 5 20 30 60 0 10 20 30 40 hCMEC/D3 Incubation Time (minutes) [ 3 H]Amisulpride + 2 M Famotidine 5 20 30 60 0 10 20 30 40 hCMEC/D3 Incubation time (minutes) [ 14 C]sucrose + 2 M Famotidine Volume of distribution ( l/mg protein) µ Volume of distribution ( l/mg protein) µ Volume of distribution ( l/mg protein) µ Volume of distribution ( l/mg protein) µ µ µ µ µ Fig. 4  The effect of MATE1 inhibition on the accumulation of ­[3H]amisulpride (3.7–7.7 nM) was determined in hCMEC/D3 cell lines. Significant increases were observed compared to control ****p ≤ 0.0001, ***p ≤ 0.001, **p = 0.01 and *p = 0.05. ­[3H]amisulpride data has been corrected for ­[14C]sucrose and are expressed as mean ± S.E.M, n = 4 passages (p30 × 2, p31 and p34 for MATE1 inhibitor falmotidine at 1 μM and n = 3 passages for MATE1 inhibitor falmotidine at 2 μM) with 6 replicates (wells) per timepoint per plate (5 time-points) 0 20 40 60 80 hCMEC/D3 Incubation time (minutes) [ 3 H]Amisulpride + 1 M Famotidine 5 20 30 60 120 5 20 30 60 120 0 20 40 60 80 hCMEC/D3 Incubation time (minutes) [ 14 C]Sucrose + 1 M Famotidine Volume of distribution ( l/mg protein) µ Volume of distribution ( l/mg protein) µ µ µ 5 20 30 60 0 10 20 30 40 hCMEC/D3 Incubation Time (minutes) [ 3 H]Amisulpride + 2 M Famotidine 5 20 30 60 0 10 20 30 40 hCMEC/D3 Incubation time (minutes) [ 14 C]sucrose + 2 M Famotidine Volume of distribution ( l/mg protein) µ Volume of distribution ( l/mg protein) µ µ µ Incubation time (minutes) Incubation Time (minutes) Fig. 4  The effect of MATE1 inhibition on the accumulation of ­[3H]amisulpride (3.7–7.7 nM) was determined in hCMEC/D3 cell lines. Significant increases were observed compared to control ****p ≤ 0.0001, ***p ≤ 0.001, **p = 0.01 and *p = 0.05. Cytotoxicity No cytotoxic effects of amisulpride (0.1–20  μM), 1  μM famotidine, 2  μM famotidine, 2  μM lopinavir, 20  μM ergothioneine, 5 μM l-carnitine, 3 μM nifekalant hydro- chloride and 1.5 μM ­[14C]sucrose (Additional file 1: Fig- ure S14A and B) and eflornithine (250–500  μM) were detected using the MTT assay (data not shown). A marker molecule ­([14C]sucrose) of extracellular/vascu- lar space was included in all ­[3H]amisulpride accumula- tion experiments and ensured that any measured effect Characteristics of haloperidol accumulation in hCMEC/D3 and b.End3 cell lines ­[3H]amisulpride data has been corrected for ­[14C]sucrose and are expressed as mean ± S.E.M, n = 4 passages (p30 × 2, p31 and p34 for MATE1 inhibitor falmotidine at 1 μM and n = 3 passages for MATE1 inhibitor falmotidine at 2 μM) with 6 replicates (wells) per timepoint per plate (5 time-points) found for ­[14C]sucrose between the treatments (Addi- tional file 1: Figure S10). hCMEC/D3 cells and by 82% in bEnd.3 cells and in the presence of prazosin—by 85% in hCMEC/D3 cells and by 82% in bEnd.3 cells (***p < 0.001) (Additional file 1: Fig- ure S12). No significant differences were found for ­[14C] sucrose between the treatments (Additional file 1: Figure S12). i ATP was depleted from both cell lines to determine the role of ABC transporters in the efflux of haloperi- dol. ATP depletion did not affect the accumulation of haloperidol in either cell line (Additional file  1: Figure S11). No significant differences were observed for ­[14C] sucrose between the treatments (Additional file 1: Figure S11). The hypothesis that haloperidol uptake is by OCT transporters was investigated by incubating the cells with OCT1 and 2 substrate amantadine (500 μM) and OCT1 and 3 substrate prazosin (100 μM). ­[3H]haloperidol accu- mulation significantly decreased in the presence of aman- tadine in both cell lines compared to control—by 89% in The effects of other cationic drugs—unlabeled pen- tamidine (100 μM), unlabeled efornithine (250 μM) and unlabelled amisulpride (20  μM) on radiolabelled halo- peridol accumulation in hCMEC/D3 was investigated. Unlabelled pentamidine significantly reduced the accu- mulation of radiolabeled haloperidol in the cell lines after 2 h—by 31% in hCMEC/D3 cells (***p < 0.001, **p < 0.01, and *p < 0.05). Unlabelled eflornithine significantly Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 11 of 19 on ­[3H]amisulpride values could be interpreted correctly and was not simply due to loss of membrane integrity caused by the cytotoxic nature of the drugs/inhibitors utilized. decreased the accumulation of radiolabelled haloperidol by 11% in hCMEC/D3 cells (***p < 0.001). Unlabelled ami- sulpride (20  μM) significantly decreased the accumula- tion of radiolabelled haloperidol in hCMEC/D3 cells—by 27% after 2 h (***p < 0.001) (Additional file 1: Figure S13). No significant differences were found for ­[14C]sucrose between the treatments (Additional file 1: Figure S13). Lipophilicityh The octanol-saline partition coefficient for ­[3H]amisul- pride was determined to be 0.0422 ± 0.0045 and for ­[3H] haloperidol was determined to be 0.6678 ± 0.1278. CNS amisulpride delivery in vivoh 3 The ­RTISSUE values for ­[3H]amisulpride did not differ from the ­RTISSUE values for ­[14C]sucrose in wildtype mice in all the age groups tested (Additional file 1: Table S5). There was also no effect of ageing on the brain distribution of ­[3H]amisulpride or ­[14C]sucrose. No differences were observed in the ­[14C]sucrose ­RTISSUE values in the frontal and occipital cortex between the wildtype and transgenic mice (Fig. 6). However, in transgenic mice the ­RTISSUE value for ­[3H]amisulpride was significantly higher than ­[14C]sucrose in the frontal cortex, but not the occipital cortex. Importantly, in transgenic mice the sucrose-cor- rected ­RTISSUE value for ­[3H]amisulpride was significantly higher than that of wildtype mice in the frontal cortex, but not the occipital cortex (Fig. 6). A similar molecular docking study suggested halop- eridol is a better substrate of both MATE1 and PMAT compared to amisulpride as it showed binding affin- ity of binding − 22.27  kcal/mol and − 21.72  kcal/mol for MATE1 and PMAT, respectively, which are notably higher than amisulpride (Fig. 5 and Additional file 1: Fig- ure S16). It showed good interaction with MATE1 with hydrogen bonds with Tyr45 and Ser74 and hydropho- bic interaction with Phe82 and Ala67 (Additional file 1: Fig. 6  The uptake of ­[3H]amisulpride was determined in wildtype and 3×transgenic AD mice. Significant differences were observed for ­[3H] amisulpride between wildtype (n = 5 frontal cortex and n = 6 occipital cortex) and transgenic mice (n = 4 each region)—**p < 0.005. ­[3H] Amisulpride data have been corrected for ­[14C]sucrose. ­[14C]Sucrose uptake is shown. No differences in paracellular permeability and membrane integrity were observed. All data are expressed as mean ± S.E.M, n = 4–6 mice, 2 years old. Perfusion time was 10 min. 6 C57BL6/129 mice (3 males and 3 females: weight 37.0 ± 1.8 g) and 4 transgenic (2 males, 2 females: weight 29.1 ± 1.0 g) were used. Also see Additional file 1: Table S5 Fig. 6  The uptake of ­[3H]amisulpride was determined in wildtype and 3×transgenic AD mice. Significant differences were observed for ­[3H] amisulpride between wildtype (n = 5 frontal cortex and n = 6 occipital cortex) and transgenic mice (n = 4 each region)—**p < 0.005. ­[3H] Amisulpride data have been corrected for ­[14C]sucrose. ­[14C]Sucrose uptake is shown. No differences in paracellular permeability and membrane integrity were observed. All data are expressed as mean ± S.E.M, n = 4–6 mice, 2 years old. Molecular docking studies with the ABC transporter—P‑gph Molecular docking studies with the ABC transporter—P‑gp The molecular docking study revealed that amisulpride was not a substrate for P-gp with a free energy binding of − 1.81 kcal/mol and the molecule was not able to inter- act favourably with the binding pocket of P-gp. P-gp sub- strates, dexamethasone and colchicine, showed notably superior interaction with P-gp with free energy of bind- ing values of − 31.83 kcal/mol and − 16.07 kcal/mol. Both dexamethasone and colchicine interacted with the bind- ing pocket employing hydrogen bonds and hydrophobic interactions (Additional file 1: Figure S17 and Table S4). Molecular docking studies with the ABC transporter—P‑gp The molecular docking study revealed that amisulpride was not a substrate for P-gp with a free energy binding of Amisulpride showed a similar level of interaction with MATE1 transporter with a free energy of bind- ing of − 14.32 kcal/mol. It fit snugly within the binding pocket (Fig. 5b) and formed hydrogen bonds with amino acids Tyr45, Ser294 and Gln301 as well as hydrophobic interactions with amino acid residues Met44, Phe78 and Tyr160. The hydrophobic interactions appeared to play an important role in its interaction with MATE1 com- pared to its interaction with OCT1. The interaction of amisulpride was relatively weaker with PMAT compared to both OCT1 and MATE1 with free energy of bind- ing − 11.4  kcal/mol. It formed a single hydrogen bond with Asn331 and interacted with hydrophobic interac- tions with amino acid residues Leu62, Leu236 and Ile239 through hydrophobic interactions (Fig. 5c). Molecular docking studies with the SLC transporters— OCT1, MATE1 and PMAT Amisulpride showed molecular interactions inside the binding site of OCT1 in the form of hydrogen bonds with amino acids Gln283, Asn288, Glu380 and Asn415 as well as hydrophobic interactions with amino acid residues Phe26 and Pro385 (Fig. 5a), while haloperidol showed hydrogen bonds with amino acids Gln 283, Gly384, Fig. 5  Molecular-level interactions of amisulpride within the binding site of OCT1 (a), MATE1 (b) and PMAT (c). Amisulpride is represented in stick-representation and amino acid residues in line-representations. Hydrogen bonds are represented in green dotted lines, and hydrophobic interactions are represented in pink dotted lines OCT1 (a), MATE1 (b) and PMAT (c). Amisulpride is represented in gen bonds are represented in green dotted lines, and hydrophobic Fig. 5  Molecular-level interactions of amisulpride within the binding site of OCT1 (a), MATE1 (b) and PMAT (c). Amisulpride is represented in stick-representation and amino acid residues in line-representations. Hydrogen bonds are represented in green dotted lines, and hydrophobic interactions are represented in pink dotted lines Fig. 5  Molecular-level interactions of amisulpride within the binding site of OCT1 (a), MATE1 (b) and PMAT (c). Amisulpride is represented in stick-representation and amino acid residues in line-representations. Hydrogen bonds are represented in green dotted lines, and hydrophobic interactions are represented in pink dotted lines Sekhar et al. Fluids Barriers CNS (2019) 16:38 Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 12 of 19 Trp388 and hydrophobic interactions with Gly384 and Pro385 while prazosin interacted with Asn288, Thr321, Ser324, Glu380, Asn411 through hydrogen bonds and hydrophobic interactions with Leu325, Val328, Gly384 and Pro385 (Additional file  1: Figure S15A). The best pose of amisulpride interacted with the binding pocket of OCT1 with a free energy of binding of − 14.28 kcal/mol while the free energy of binding for haloperidol and pra- zosin were − 29.97 kcal/mol and − 27.57 kcal/mol. Figure S16A). However, the interaction of haloperidol with PMAT was limited to single hydrogen bond with Asp34 and hydrophobic interaction with Leu62 (Addi- tional file 1: Figure S16B). Wildtype and transgenic AD miceh The total protein concentration measured in wildtype mice (188.2 ± 12.8  μg/100  μl) was not sig- nificantly different to that measured in 3×Tg AD mice (195.6 ± 13.3  μg/100  μl) brains, but this may be attrib- uted to the fact that it was not possible to assess regional differences in these small samples. Although slight vari- ability was observed, there was no significant differences in individual transporter (P-gp, OCT1, OCT2, OCT3, OCTN1, OCTN2, MATE1, MATE2 and PMAT) expres- sion between the wildtype and 3×TgAD mice (Additional file 1: Figures S20 and S21). Note mice were 24 months old except for MATE1 and PMAT studies where the mice were 16 and 12  months old. Data not shown for P-gp, OCT1, OCT2, OCT3 and PMAT. The expression of all these BBB transporters suggests that a capillary enriched sample had been assessed. Discussionh This study aimed to answer an important clinical ques- tion using an integrative approach to investigate the interaction between two drugs and BBB transporters and their potential pharmacodynamic relevance in AD. The in  vitro cell culture models and in silico computa- tional model allowed us to [1] identify the transporters, [2] assess the potential mechanisms of amisulpride and haloperidol transport [3] perform studies on human and mouse brain endothelium and [4] minimize animal stud- ies in line with the 3Rs (replacement, refinement and reduction principles). The in  situ brain perfusion tech- nique allowed us to [1] study the whole animal [2] and utilize a mouse model of AD. Importantly this is the only model to exhibit both amyloid-β40 and 42 and tau pathol- ogy, mimicking human AD [39, 43]. Both plaque and tan- gle pathology are mainly restricted to the hippocampus, amygdala and cerebral cortex. Transporter expression in capillaries isolated from AD and age-matched human cases and mouse brain were also assessed. Medication history of the cases was evaluated. Medication history of the casesi Additional file 1: Table S3 shows the medication history of the cases that have been used in this study. Sedatives, antidepressants and antipsychotic drugs (including halo- peridol) were identified and listed together. A separate column lists all other medications. Endothelial transporter expression Cell lines p < 0.05) and putamen (74.8%; p < 0.05) samples, respec- tively, compared to control (Fig. 7; Student’s t-test). No other significant differences were observed, however, fur- ther cases are required to explore this more fully. Please note it is also likely that transporter expression in caudate nucleus and putamen AD samples is even lower than the heathy controls shown here (Fig. 7; Additional file 1: Fig- ures S22–S28) as total protein expression is significantly reduced (Additional file 1: Table S6). The expression of all these BBB transporters would also indicate that a capil- lary enriched sample had been assessed. OCTN1, OCTN2, MATE1 and MATE2 expression was confirmed in hCMEC/D3 (passages 28 and 33) and bEnd.3 (passages 18, 19 and 23) cells (Additional file 1: Figures S18 and S19A). PMAT was expressed in hCMEC/ D3 cells (passages 28, 31 and 32) and bEnd.3 (passages 17, 18, 20 and 24) (Additional file 1: Figure S19B and C). OCTN1, OCTN2, MATE1 and MATE2 expression was confirmed in hCMEC/D3 (passages 28 and 33) and bEnd.3 (passages 18, 19 and 23) cells (Additional file 1: Figures S18 and S19A). PMAT was expressed in hCMEC/ D3 cells (passages 28, 31 and 32) and bEnd.3 (passages 17, 18, 20 and 24) (Additional file 1: Figure S19B and C). CNS amisulpride delivery in vivoh 3 Perfusion time was 10 min. 6 C57BL6/129 mice (3 males and 3 females: weight 37.0 ± 1.8 g) and 4 transgenic (2 males, 2 females: weight 29.1 ± 1.0 g) were used. Also see Additional file 1: Table S5 Fig. 6  The uptake of ­[3H]amisulpride was determined in wildtype and 3×transgenic AD mice. Significant differences were observed for ­[3H] amisulpride between wildtype (n = 5 frontal cortex and n = 6 occipital cortex) and transgenic mice (n = 4 each region)—**p < 0.005. ­[3H] Amisulpride data have been corrected for ­[14C]sucrose. ­[14C]Sucrose uptake is shown. No differences in paracellular permeability and membrane integrity were observed. All data are expressed as mean ± S.E.M, n = 4–6 mice, 2 years old. Perfusion time was 10 min. 6 C57BL6/129 mice (3 males and 3 females: weight 37.0 ± 1.8 g) and 4 transgenic (2 males, 2 females: weight 29.1 ± 1.0 g) were used. Also see Additional file 1: Table S5 Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 13 of 19 Page 13 of 19 Human brain b Human brain capillaries were isolated from the frontal cortex (for comparison with in  situ perfusion experi- ments), caudate nucleus and the putamen (forming the striatum where high D2 and D3 receptor occupancy is observed in individuals with AD with amisulpride usage) of healthy controls and age-matched AD affected indi- viduals (Additional file  1: Tables S2 and S3). The total protein concentration in the capillaries was found to be significantly lower in the caudate nucleus (by 37.4%) and putamen (by 32.5%), but not the frontal cortex samples, from individuals with AD compared to healthy controls (Additional file 1: Table S6). Cell culture studies revealed a slow accumulation of ­[3H]amisulpride indicating a low BBB permeability. This can be linked to its low lipophilicity, as measured by the octanol-saline partition coefficient, and its ina- bility to interact strongly with neutral and negatively charged lipid model systems thus its limited ability to passively diffuse across the lipid bilayer [22, 44]. Please note the plasma half-life of a single oral dose of ami- sulpride (50 or 200 mg) is ~12 h, which suggests that amisulpride will not have significantly degraded within the 2 h incubation period used in our study [45]. Fur- thermore the accumulation buffer does not contain plasma enzymes which would further increase the i Individual transporter expression in each brain region between AD and healthy cases is comparable as the same amount of protein has been loaded into each well. Note this amount was dependent on the antibody utilized so was variable. Transporter expression in the frontal cortex was less variable between healthy and AD cases than in the other regions studied with no significant differences in transporter expression being observed (Additional file 1: Figures S22–S28; Fig. 7). Expression of OCT1 did not change between control and individuals with AD in all the regions tested (Additional file 1: Figures S22, S23). PMAT and MATE1 expression was significantly lower in individuals with AD in the caudate nucleus (56.2%; Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 14 of 19 Page 14 of 19 Sekhar et al. Fluids Barriers CNS (2019) 16:38 half-life of amisulpride in our cell culture models. ( )i organelle membrane (e.g. lysozyme) [46]. Human brain b OCT1, OCT2 Control Cortex Intensity ratio MATE1 MATE2 PMAT PMAT PMAT PMAT PMAT PMAT MATE1 MATE2 MATE1 MATE2 MATE1 MATE2 MATE1 MATE2 MATE1 MATE2 0.0 0.5 1.0 1.5 2.0 BBN_20005 BBN_14792 BBN_6071 BBN_20006 BBN_22222 AD Cortex Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_24943 BBN_25109 BBN_24530 BBN_19609 BBN_25921 Control Caudate Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_20005 BBN_6071 BBN_20006 BBN_22222 BBN_14792 AD Caudate Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_25109 BBN_24530 BBN_19609 BBN_25921 BBN_24943 Control Putamen Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_20005 BBN_6071 BBN_20006 BBN_22222 BBN_14792 AD Putamen Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_24943 BBN_25109 BBN_24530 BBN_19609 BBN_25921 Fig. 7  Individual values have been plotted for the transporter expression in the capillaries of frontal cortex, caudate nucleus and caudate putamen samples from healthy and AD affected individuals. The numbers in the key indicate the MRC ID designated to each sample. Details of the samples can be found in Additional file 1: Tables S2 and S6 Control Cortex Intensity ratio MATE1 MATE2 PMAT PMAT MATE1 MATE2 0.0 0.5 1.0 1.5 2.0 BBN_20005 BBN_14792 BBN_6071 BBN_20006 BBN_22222 AD Cortex Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_24943 BBN_25109 BBN_24530 BBN_19609 BBN_25921 Control Cortex Intensity ratio MATE1 MATE2 PMAT 0.0 0.5 1.0 1.5 2.0 BBN_20005 BBN_14792 BBN_6071 BBN_20006 BBN_22222 PMAT MATE1 MATE2 5 2 6 2 AD Cortex Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_24943 BBN_25109 BBN_24530 BBN_19609 BBN_25921 PMAT PMAT MATE1 MATE2 MATE1 MATE2 Control Caudate Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_20005 BBN_6071 BBN_20006 BBN_22222 BBN_14792 AD Caudate Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_25109 BBN_24530 BBN_19609 BBN_25921 BBN_24943 PMAT MATE1 MATE2 Control Caudate Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_20005 BBN_6071 BBN_20006 BBN_22222 BBN_14792 PMAT MATE1 MATE2 AD Caudate Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_25109 BBN_24530 BBN_19609 BBN_25921 BBN_24943 Intensity ratio PMAT PMAT MATE1 MATE2 MATE1 MATE2 Control Putamen Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_20005 BBN_6071 BBN_20006 BBN_22222 BBN_14792 AD Putamen Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_24943 BBN_25109 BBN_24530 BBN_19609 BBN_25921 Fig. 7  Individual values have been plotted for the transporter expression in the capillaries of frontal cortex, caudate nucleus and caudate putamen samples from healthy and AD affected individuals. The numbers in the key indicate the MRC ID designated to each sample. Human brain b This is consistent with previous observations [22], which found higher amisulpride transport in the basolateral to apical direction (Pe 5.2 ± 3.6 × 10−6cm/s) compared to the api- cal to basolateral direction (Pe < 10−7cm/s) in porcine brain microvessel endothelium. The differences in the effect of inhibitors on the two cell lines may be explained by amisulpride being a substrate for multiple transport- ers and variations in the function/expression of OCT1, MATE and PMAT transporters possibly related to spe- cies differences [52, 53]. The absence of any effect of pra- zosin on ­[3H]amisulpride uptake in bEnd.3 cells can be explained by prazosin-associated toxicity in this cell line, which causes protein values to decrease over the course of the experiment, resulting in no net effect on ­VD val- ues [32]. It is unlikely that the efflux transporter identi- fied in our in vitro study is an ABC transporter, as neither ATP-depletion or substrates for P-gp, BCRP, or the MRP family had an effect on ­[3H]amisulpride accumulation in either cell line. Another study also revealed that ami- sulpride did not inhibit P-gp in an in  vitro efflux assay [23], although they also utilised P-gp knockout and WT mice and these in vivo distribution studies suggested it was a P-gp substrate. Interestingly they considered that this may have little therapeutic impact due to its unu- sual receptor profile. Our single transporter computa- tional studies revealed that amisulpride was not a P-gp substrate, but was a substrate for the SLC efflux trans- porters, PMAT and MATE1. Our in  vitro hCMEC/D3 inhibitor studies with lopinavir, also indicated that ami- sulpride could be effluxed by PMAT. PMAT is thought to use a proton gradient to drive organic cation efflux from cells [53] and we (and others) have found that PMAT protein is expressed on human and mouse brain capil- laries (Fig. 7, Additional file 1: Figures S19B, C and S28) Interestingly our in  vitro studies with the MATE1 inhibitor, famotidine, did not support the in silico data set which indicated that amisulpride was a MATE1 sub- strate. It is important to highlight that the lack of MATE1 inhibitor effect may not be conclusive proof of a lack of substrate interaction with the MATE1 transporter. It may be that the transporter was not sufficiently expressed [47, 52, 55, 56], although we could detect MATE1 protein in both our two cell lines, mouse and human brain capillary samples. Human brain b Details of the samples can be found in Additional file 1: Tables S2 and S6 PMAT MATE1 MATE2 Control Putamen Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_20005 BBN_6071 BBN_20006 BBN_22222 BBN_14792 PMAT MATE1 MATE2 AD Putamen Intensity ratio 0.0 0.5 1.0 1.5 2.0 BBN_24943 BBN_25109 BBN_24530 BBN_19609 BBN_25921 Fig. 7  Individual values have been plotted for the transporter expression in the capillaries of frontal cortex, caudate nucleus and caudate putamen samples from healthy and AD affected individuals. The numbers in the key indicate the MRC ID designated to each sample. Details of the samples can be found in Additional file 1: Tables S2 and S6 half-life of amisulpride in our cell culture models. Unlabelled amisulpride (20  µM) significantly reduced ­[3H]amisulpride accumulation in both cell lines, sug- gesting that there is a low affinity influx transporter at the plasma membrane or possibly at an intracellular organelle membrane (e.g. lysozyme) [46]. OCT1, OCT2 and 3 have been shown to be expressed in hCMEC/ D3 and b.End3 cells in an earlier study by our group [32] and OCTN1 and OCTN2 were shown to be expressed in both cell lines in this present study. How- ever, a study by Ohtsuki and colleagues found that the Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 15 of 19 protein expression of OCT1, OCT2, OCT3, OCTN1 and OCTN2 were below detectable limits in hCMEC/ D3 cells [47]. luminal and abluminal membrane of human, mouse and rat brain endothelial cells [53]. Other studies could not detect PMAT protein in isolated human capillaries and hCMEC/D3 cells [47, 56]. In line with the findings of [14] and our molecular docking studies, our accumulation studies suggested that OCT1 may be the influx transporter involved, as prazo- sin (substrate for OCT1 and 3) reduced ­[3H]amisulpride uptake in hCMEC/D3 cells after 2 h, whereas corticoster- one (OCT3 substrate) had no impact on ­[3H]amisulpride accumulation in either cell line. In addition, OCTN1 and 2 inhibitors did not affect accumulation of ­[3H]amisul- pride into hCMEC/D3 or bEnd.3 cells. However, in the presence of amantadine, a substrate for several trans- porters of organic cations (OCT1, OCT2 [48]), MATE 1, MATE 2 [49, 50] and PMAT [51]) there was no effect on hCMEC/D3 cells, and an increase in ­[3H]amisulpride accumulation in bEnd.3 cells after 2 h incubation, which suggests the involvement of an efflux transporter. Human brain b Further evidence for regional tissue changes with AD came from our total protein measures (Addi- tional file 1: Table S4). This may be linked to changes in the expression of other transporters such as P-gp [18] and GLUT1 [66] as well as the other SLC transporters measured in this present study. Interestingly regional differences are known to exist in the BBB transport and the intracellular distribution of antipsychotics [67]. The reduced MATE1 and PMAT expression observed in AD may therefore underpin the heightened sensitiv- ity to amisulpride observed in the clinical population especially as together our in silico and in vitro studies suggested that amisulpride was a substrate for MATE 1 and PMAT. Several medications listed in Additional file  1: Table  S3 (e.g. citalopram, metoclopramide and loratadine) have previously been identified as OCT1 inhibitors [25, 26]. It is not yet known if they also inter- act with MATE1 and PMAT. One of the medications (ranitidine) is an inhibitor of both OCT1 and MATE [64]. P-gp, although our inhibitor and in silico studies do not suggest amisulpride is a substrate for P-gp. Conversely when radiolabeled haloperidol (10  nM) was incubated with unlabeled amisulpride (20 μM) there was a signifi- cant decrease in accumulation. Overall these results may slight reflect differences in the interaction of amisulpride and haloperidol with different transporter binding sites. f g Further insight into the interaction of amisulpride and haloperidol with the transporters was obtained through the in silico computational studies, where amisulpride showed binding affinities towards the binding sites of the influx transporter, OCT1, as well as the efflux transport- ers, MATE1 and PMAT. The binding affinities of ami- sulpride towards the binding sites of these transporters showed comparable energies for OCT1 and MATE1 with molecular level interactions through hydrogen bonds and hydrophobic interactions with a number of amino acids within the binding pocket. The nature of the interaction of amisulpride with OCT1 is similar to that observed for haloperidol and prazosin which are known substrates for this transporter. The binding affinity and level of interac- tion were slightly weaker with PMAT compared to OCT1 and MATE1, but still considerable level of interactions were observed suggesting amisulpride is a weak sub- strate of PMAT. Haloperidol showed comparable affinity for both MATE1 and PMAT suggesting it is a substrate for both transporters. Human brain b MATE1 and PMAT are known to use a proton gradient to drive organic cation efflux from cells [53, 54, 62, 63]. MATE1, like P-gp, is thought to be expressed on the luminal membrane of the BBB, although this remains to be confirmed [64]. Human brain b Other considerations are that the inhibitor may need to reach a therapeutic concentration within the cell to elicit a response as has been previously observed with MATE1 inhibitors [50], the substrate and the inhibitor may bind to different binding sites, the non-specificity of the inhibitor, and that amisulpride interacts with both influx (OCT) and other efflux (PMAT) transporters mak- ing a response difficult to detect [26, 53, 57]. fi Haloperidol has been observed to have a high degree of dopamine receptor (D2) occupancy within the brain at very low doses suggesting that haloperidol is very effi- cient at crossing the BBB. This is in agreement with our in vitro BBB data. Please note that the half-life of halo- peridol has been reported to range 14.5–36.7  h (or up to 1.5 days) after a single oral dose so will not have been metabolized significantly over our 2 h incubation period [58]. Haloperidol may cross the BBB by passive diffusion, as supported by the relatively high octanol-saline parti- tion coefficient of haloperidol (0.6678 ± 0.1278) com- pared to amisulpride (0.0422 ± 0.0045), or may involve transporters. The use of transporters by haloperidol is confirmed by the self-inhibition studies in both hCMEC/ D3 and b.End3 cell lines. As haloperidol exists predomi- nately (94.8%) as a positively charged drug at physi- ological pH (pKa is 8.66) the transporter is likely to be OCT, which is expressed at the BBB. This was confirmed in  vitro using the OCT substrates, amantadine (OCT1 and 2) and prazosin (OCT1 and 3). We also investigated the involvement of ABC transporters in the transport of haloperidol. For this, ATP was depleted from the cells by incubating them with 10 mM 2-deoxy-d-glucose. No effects of ATP depletion were observed compared to con- trol in either cell line suggesting that haloperidol is not a substrate for the ABC transporters P-gp, BCRP, or the MRP family at the BBB as previously observed [59, 60]. Radiolabelled amisulpride (6.5 nM) was also incubated with haloperidol (OCT1 substrate and P-gp inhibitor) [25, 61]. Radiolabeled amisulpride accumulation was not affected by haloperidol (40 μM) in either cell line. This may be the result of interactions with both OCT1 and Sekhar et al. Fluids Barriers CNS (2019) 16:38 Sekhar et al. Fluids Barriers CNS (2019) 16:38 Page 16 of 19 Page 16 of 19 AD abnormality instead of from vascular comorbidi- ties [65]. Conclusionsh This study included a detailed evaluation of transporter expression and usage at the BBB using in silico computa- tional approaches, in vitro models and an in vivo animal model of AD as well as patient material. The datasets have provided evidence of an interaction of amisulpride and haloperidol with both influx (OCT1) and efflux (MATE1 and PMAT) transporters, which may be expressed at the luminal or abluminal membranes of the BBB and/or at an intracellular membrane. In vitro and in silico studies indicated that amisulpride was not a substrate for ABC transporters including P-gp. Furthermore, the study is of key importance as the results suggest that the heightened sensitivity to amisulpride observed in older people with AD is possibly due to previously unreported changes in SLC transporter expression, which increase amisulpride entry into, or possibly reduce clearance from the brain. This study is also the first step in the process of charac- terising age and AD-specific changes in SLC transport- ers of organic cations. Overall our study has implications beyond amisulpride prescribing, as it suggests that dose adjustments may be required for other drugs (e.g. halop- eridol) which are substrates for SLC transporters in par- ticular MATE1 and PMAT. i The in silico study supports the experimental obser- vations and provides further evidence that amisul- pride might be influxed through OCT1 and effluxed through PMAT and MATE1 but not P-gp. When ami- sulpride transport was investigated in vivo, a low BBB permeability to ­[3H]amisulpride was also observed in the wild-type mice and this did not change with age. Further studies in the 3×TG AD mice revealed an increased CNS uptake which was not accounted for by altered BBB integrity or changes in vascular space in the AD model mice, as there were no differences in ­[14C]sucrose uptake between the two groups; and nei- ther was it explained by non-expression of the trans- porters studied (P-gp, OCT1, OCT2, OCT3, OCTN1, OCTN2, MATE1, MATE2 and PMAT). However, in post-mortem human brains expression of the efflux transporter MATE1 was lower in individuals with AD compared to age-matched healthy controls in the puta- men; and PMAT showed a similar trend in the caudate nucleus, but there was no change in expression levels of these transporters in the frontal cortex. Importantly it has been reported that BBB impairments stem from Supplementary information Supplementary information accompanies this paper at https​://doi. org/10.1186/s1298​7-019-0158-1. Additional file 1. Acknowledgements We would like to thank Professor I. Romero, Professor B. Weksler, and Professor P. Couraud for the hCMEC/D3 cell line provided under MTA and Dr. Manasi Nandi for advice regarding the Western blots. Received: 10 October 2019 Accepted: 2 December 2019 Received: 10 October 2019 Accepted: 2 December 2019 Author details 1 Faculty of Life Sciences and Medicine, School of Cancer and Pharmaceuti- cal Sciences, King’s College London, Franklin‑Wilkins Building, 150 Stamford Street, Waterloo, London SE1 9NH, UK. 2 Faculty of Pharmacy, Misr Interna- tional University, Cairo 11431, Egypt. 3 Wolfson Centre for Age‑Related Dis- eases, King’s College London, Guy’s Campus, London SE1 1UL, UK. 4 Maurice Wohl Clinical Neuroscience Institute, King’s College London, 125 Coldharbour Lane, Camberwell, London SE5 9N, UK. 5 Division of Psychiatry, Faculty of Brain Sciences, University College London, 149 Tottenham Court Road, London W1T 7NF, UK. Authors’ contributions GNS co-designed the study with SAT. Performed the brain perfusions (with AF). Performed MTT assay for amisulpride and eflornithine, Western blot analysis on human tissue and the bEnd.3 and D3 cell culture studies with unlabelled amisulpride, ATP depletion, ATP-transporter and OCT inhibitors. Analysed the resulting data. Contributed to the writing of the manuscript. ALF performed the brain perfusions (with GNS). Performed Western blots for MATE1 in bEnd.3, hCMEC/D3, wild-type and 3×Tg AD mice. Analysed the resulting data. STB and RL performed the cell culture studies with MATE1, MATE2, PMAT, OCTN1 and OCTN2 inhibitors with hCMECD3 and bEnd.3 respectively. Analysed the resulting data. STB also performed MTT assay with ergothionine, l-carnitine, famotidine, lopinavir, nifekalant hydrochloride and ­[14C]sucrose. PMAT Western blot on hCMEC/D3. Analysed the resulting data. HR performed the Western blots for OCTN1, OCTN2 and MATE2 on bEnd.3, hCMEC/D3, wild-type and 3×Tg AD mice. Also Western blot on OCTN1 and OCTN2 on human control and AD cases. Analysed the resulting data. HW provided cell culture guidance to STB and RL. DBF and KMR performed the in silico simulations, analysed the data and contributed to the writing of the manuscript. MB provided the WT and 3×AD mice. SR provided clinical insight into the project, separated the medications into the two groups and contributed to the writing of the manuscript. SAT co-designed, directed and co-ordinated the complete study, developed the concept and wrote the majority of the manuscript. Applied for human tissue through the brains for dementia research fund. Contributed to analysing the data. All authors read and approved the final manuscript. GNS co-designed the study with SAT. Performed the brain perfusions (with AF). Performed MTT assay for amisulpride and eflornithine, Western blot analysis on human tissue and the bEnd.3 and D3 cell culture studies with unlabelled amisulpride, ATP depletion, ATP-transporter and OCT inhibitors. Analysed the resulting data. Contributed to the writing of the manuscript. ALF performed the brain perfusions (with GNS). Performed Western blots for MATE1 in bEnd.3, hCMEC/D3, wild-type and 3×Tg AD mice. Analysed the resulting data. STB and RL performed the cell culture studies with MATE1, Consent for publication Not applicable. Consent for publication Not applicable. OCT: organic cation transporters; OCTN: organic cation transporters novel; hCMEC/D3: immortalized human cerebral microvessel endothelial cell line; MATEs: multi-drug and toxic compound extrusion proteins; PMAT: plasma membrane monoamine transporter; 3×TgAD: triple transgenic AD model; FBS: foetal bovine serum; BBB: blood–brain barrier; AD: Alzheimer’s disease; P-gp: P-glycoprotein; BCRP: breast cancer resistance protein; MRP: multi-drug resistance protein; RIPA: radio-immunoprecipitation assay; SLC: solute carrier; bEnd.3: mouse brain endothelial cell lines; VLOSLP: very late-onset (> 60 years) schizophrenia-like psychosis; NHS: national health service; MTT: 3-(4,5-dimeth- ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; APP: amyloid precursor protein; PBS-T: PBS-TWEEN® tablets; RT: room temperature; BDR: brains for dementia research; Vd: volume of distribution; WB: Western blot. References 1. Ballard C, Howard R. Neuroleptic drugs in dementia: benefits and harm. Nat Rev Neurosci. 2006;7(6):492–500. https​://doi.org/10.1038/nrn19​26. 2. Schneider LS, Tariot PN, Dagerman KS, Davis SM, Hsiao JK, Ismail MS, et al. Effectiveness of atypical antipsychotic drugs in patients with Alzheimer’s disease. N Engl J Med. 2006;355(15):1525–38. https​://doi.org/10.1056/ NEJMo​a0612​40. 3. Reeves S, McLachlan E, Bertrand J, Antonio FD, Brownings S, Nair A, et al. Therapeutic window of dopamine D2/3 receptor occupancy to treat psychosis in Alzheimer’s disease. Brain. 2017;140(4):1117–27. https​://doi. org/10.1093/brain​/aww35​9. 3. Reeves S, McLachlan E, Bertrand J, Antonio FD, Brownings S, Nair A, et al. Therapeutic window of dopamine D2/3 receptor occupancy to treat psychosis in Alzheimer’s disease. Brain. 2017;140(4):1117–27. https​://doi. org/10.1093/brain​/aww35​9. 4. Caravaggio F, Graff-Guerrero A. Is antipsychotic sensitivity in Alzheimer’s disease secondary to abnormal blood-brain barrier integrity? Brain. 2017;140(4):865–7. 5. 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This research was supported by the Medical Research Council (Ph.D. student- ship for Ms. Sekhar Ph.D. studies MR/K500811/1), the King’s College London Alzheimer’s Research UK (ARUK) network centre seed fund (to Dr. Suzanne Reeves, Dr. Sarah Thomas and Ms. Gayathri Sekhar), The Edmond and Lily Safra Research Foundation (Dr. Broadstock) and a multi-user equipment grant from The Wellcome Trust (Dr. Thomas (PI), Professors Francis, McMahon, Malcangio, and Rattray 080268). Ms Fleckney is a BBSRC-CASE funded PhD student (BB/ L01534X/1). Ms. Sevda Boyanova is a Guy’s and St Thomas’ Charity funded MRes-Ph.D. studentship. Mr Huzefa Rupawala is on a MRC DTP Ph.D. student- ship (MR/N013700/1). Dr. Suzanne Reeves is funded by the UCLH NIHR BRC. y 9. Schoemaker H, Claustre Y, Fage D, Rouquier L, Chergui K, Curet O, et al. Neurochemical characteristics of amisulpride, an atypical dopamine D2/ D3 receptor antagonist with both presynaptic and limbic selectivity. 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A Comparative Study of Relational Learning Capacity in Honeybees (Apis mellifera) and Stingless Bees (Melipona rufiventris)
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Abstract 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 exists. * E-mail: j.reinhard@uq.edu.au A Comparative Study of Relational Learning Capacity in Honeybees (Apis mellifera) and Stingless Bees (Melipona rufiventris) Antonio Mauricio Moreno1,2,3, Deisy das Grac¸as de Souza1,2, Judith Reinhard3* Antonio Mauricio Moreno1,2,3, Deisy das Grac¸as de Souza1,2, Judith Reinhard3* 1 Departamento de Psicologia, Universidade Federal de Sa˜o Carlos, Sa˜o Carlos, Sa˜o Paulo, Brazil, 2 National Institute of Science and Technology on Behavior, Cognition, and Teaching, Universidade Federal de Sa˜o Carlos, Sa˜o Carlos, Sa˜o Paulo, Brazil, 3 Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia 1 Departamento de Psicologia, Universidade Federal de Sa˜o Carlos, Sa˜o Carlos, Sa˜o Paulo, Brazil, 2 National Institute of Science and Technology on Behavior, Cognition, and Teaching, Universidade Federal de Sa˜o Carlos, Sa˜o Carlos, Sa˜o Paulo, Brazil, 3 Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia Abstract Background: Learning of arbitrary relations is the capacity to acquire knowledge about associations between events or stimuli that do not share any similarities, and use this knowledge to make behavioural choices. This capacity is well documented in humans and vertebrates, and there is some evidence it exists in the honeybee (Apis mellifera). However, little is known about whether the ability for relational learning extends to other invertebrates, although many insects have been shown to possess excellent learning capacities in spite of their small brains. Methodology/Principal Findings: Using a symbolic matching-to-sample procedure, we show that the honeybee Apis mellifera rapidly learns arbitrary relations between colours and patterns, reaching 68.2% correct choice for pattern-colour relations and 73.3% for colour-pattern relations. However, Apis mellifera does not transfer this knowledge to the symmetrical relations when the stimulus order is reversed. A second bee species, the stingless bee Melipona rufiventris from Brazil, seems unable to learn the same arbitrary relations between colours and patterns, although it exhibits excellent discrimination learning. Conclusions/Significance: Our results confirm that the capacity for learning arbitrary relations is not limited to vertebrates, but even insects with small brains can perform this learning task. Interestingly, it seems to be a species-specific ability. The disparity in relational learning performance between the two bee species we tested may be linked to their specific foraging and recruitment strategies, which evolved in adaptation to different environments. Citation: Moreno AM, de Souza DdG, Reinhard J (2012) A Comparative Study of Relational Learning Capacity in Honeybees (Apis mellifera) and Stingless Bees (Melipona rufiventris). PLoS ONE 7(12): e51467. doi:10.1371/journal.pone.0051467 Editor: Nigel E. Raine, Royal Holloway University of London, United Kingdom Citation: Moreno AM, de Souza DdG, Reinhard J (2012) A Comparative Study of Relational Learning Capacity in Honeybees (Apis mellifera) and Stingless Bees (Melipona rufiventris). PLoS ONE 7(12): e51467. doi:10.1371/journal.pone.0051467 Editor: Nigel E. Raine, Royal Holloway University of London, United Kingdom Received March 30, 2012; Accepted November 7, 2012; Published December 1 Copyright:  2012 Moreno 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. Funding: AMM was supported by the Foundation for Research Support in the State of Sao Paulo (FAPESP grants no. 08/50576-8 and no. 08/57705-8) and by the National Council for Scientific and Technological Development of Brazil (CNPq grant no. 573972/2008-7). December 2012 | Volume 7 | Issue 12 | e51467 PLOS ONE | www.plosone.org Introduction common honeybee Apis mellifera, but also in bumblebees [7–12], and even new-world stingless bees, such as Melipona rufiventris and M. quadrifasciata [13–17]. Using Y-maze set-ups, where bees are presented with a sample stimulus followed by two choice stimuli, Giurfa et al. [18] demonstrated that Apis mellifera can learn matching-to-sample and non-matching-to-sample relations be- tween visual stimuli such as colours. They further showed that Apis mellifera is also able to transfer the concepts of ‘‘sameness’’ and ‘‘difference’’ from colours to novel stimuli such as patterns and vice versa, and even cross-modally from odours to colours [18]. Prior to this study, such a level of learning performance was demonstrated only in vertebrates. Relational learning defines the capacity to acquire knowledge about the relations, associations, and interactions between different stimuli, objects or events. Matching-to-sample procedures [1] are typically used for investigating relational learning. When presented with a sample stimulus, a human or non-human subject can be taught to select, from an array of comparison stimuli, a second stimulus identical to this sample (identity matching-to-sample), or to select the stimulus that differs from the sample (oddity matching- to-sample). The subject can also learn to associate stimuli that do not share physical similarities (arbitrary or symbolic matching-to-sample), for example selecting a yellow stimulus when presented with a sample stimulus consisting of black and white vertical patterns and selecting a blue stimulus when presented with a sample stimulus consisting of black and white horizontal patterns. Honeybees can also learn arbitrary relations between stimuli: symbolic matching-to-sample procedures have been used to train Apis mellifera to associate patterns with colours, and vice versa [19,20]. In the study by Zhang et al. [19], bees learnt the relations between three visual stimuli (grating orientation, colour, and pattern) that were presented in a specific order. When the stimulus sequence was changed, i.e. the stimuli were presented in a different It has long been known that bees have an impressive capacity to learn and discriminate colours, shapes, patterns and odours [2–6]. These learning abilities have been investigated mostly in the December 2012 | Volume 7 | Issue 12 | e51467 1 December 2012 | Volume 7 | Issue 12 | e51467 PLOS ONE | www.plosone.org Relational Learning in Bees Figure 1. Schematic of the Y-maze apparatus used for in- vestigating learning of arbitrary relations in honeybees. Introduction Bees entered a tunnel, flew through the maze entrance (marked with the sample stimulus) and then flew through one of two exits (each marked with a comparison stimulus). A correct choice (S+) led to a feeder containing sugar solution. An incorrect choice (S–) led to an empty feeder. doi:10.1371/journal.pone.0051467.g001 order, the bees still showed a significant percentage of correct choices, which could be interpreted as demonstration that bees had learnt emergent symmetries between the stimuli. However, in the study by Zhang et al. [19] correct responses were always rewarded even when stimulus sequences were reversed. Therefore, the bees might have merely learnt new relations between the stimuli in a different order. To investigate whether honeybees truly have the capacity for learning emergent symmetries within stimulus relations, the stimuli need to be presented without positive reinforcement in a reversal test. While Apis mellifera’s capacity for learning symmetrical relations remains to be investigated, it is known that its relational learning ability extends beyond patterns and colours. Honeybees can learn cross-modal arbitrary relations, i.e. associating stimuli from different sensory modalities with each other, such as lemon scent with the colour blue, and mango scent with the colour yellow [21]. This capacity for cross-modal associations was further confirmed by field experiments, in which honeybees learnt to associate scents with different feeder colours, and returned to the coloured feeders whenever the corresponding scent was blown into the hive, irrespective of the feeder location [22]. Stingless Melipona bees are known to have considerable associative learning capacities both in the visual and the olfactory domain [14–17], but their capacity for relational or other complex forms of learning has not been well investigated. One early study explored relational learning of light intensities and colours in Melipona rufiventris [23]. Here, bees had to learn the relation between the light condition of a chamber (light on or off), and the colour around the feeder. However, due to the design of the experiment it could not be shown conclusively whether Melipona learnt the relations between the stimuli or merely stimulus configurations. In the present study, we therefore investigated the capacity for learning arbitrary relations between colours and patterns in Melipona rufiventris, in direct comparison to the relational and symmetrical learning abilities of Apis mellifera, demonstrating existence of this capacity in one species, but not the other. Figure 1. Introduction Schematic of the Y-maze apparatus used for in- vestigating learning of arbitrary relations in honeybees. Bees entered a tunnel, flew through the maze entrance (marked with the sample stimulus) and then flew through one of two exits (each marked with a comparison stimulus). A correct choice (S+) led to a feeder containing sugar solution. An incorrect choice (S–) led to an empty feeder. doi:10.1371/journal.pone.0051467.g001 order to the training, and the bees’ choices were unrewarded. That is, the stimuli changed their function with sample stimuli becoming comparison stimuli and vice versa. The bees that were trained to relate patterns to colours were tested with the corresponding colour-pattern combinations, and bees trained to relate colours to patterns were tested with the corresponding pattern-colour combinations (Fig. 2). In spite of successfully learning arbitrary relations, Apis mellifera bees were not able to transfer this knowledge to the symmetry test. The mean percentage of correct responses with symmetrical relations was 51.7% (Fig. 2A) and 47.5% (Fig. 2B), respectively, both results not differing statistically from chance (one-sample t-test, P = 0.307 and P = 0.548). Results Learning of Arbitrary Relations in Apis mellifera We trained 20 individually marked Apis mellifera workers to navigate a Y-maze (Fig. 1), where they first encountered a sample stimulus (black and white pattern, either horizontal or vertical) and then two comparison stimuli (the colours blue and yellow). The bees had to learn the arbitrary relation between horizontal pattern and the colour blue, and vertical pattern and the colour yellow. They received a sugar reward when choosing the correct comparison stimulus. A second group of 20 individual A. mellifera bees were trained in the reverse sequence, namely relating a blue sample stimulus to a horizontal pattern, and a yellow sample stimulus to a vertical pattern. The bees were trained in this way in six sessions, each consisting of seven 30-minute training blocks, over six consecutive days (for details of the procedure see Materials and Methods). The bees’ choices were pooled per block for each session. A. mellifera honeybees easily learnt the arbitrary pattern- colour (Fig. 2A) and colour-pattern (Fig. 2B) relations. Learning performance, measured as percentage of correct choices per session, significantly increased over the sessions from 56.2% to 68.2% for pattern-colour relations (F5,30 = 7.91, P,0.001), and from 51.5% to 73.3% for colour-pattern relations (F5,30 = 13.71, P,0.001). December 2012 | Volume 7 | Issue 12 | e51467 PLOS ONE | www.plosone.org Learning of Arbitrary Relations in Melipona rufiventris Bars: mean percentage (6 s.d.) of correct choices in six rewarded training sessions of seven training blocks each, during which one group of 8 bees learnt the relations patterns-colours (A), and a second group of 8 bees learnt the relations colours-patterns (B). Numbers inside bars indicate the total number of choices per session. doi:10.1371/journal.pone.0051467.g003 Figure 2. Learning of arbitrary relations in Apis mellifera honeybees. Black bars: mean percentage (6 s.d.) of correct choices in six rewarded training sessions of seven training blocks each, during which one group of 20 bees learnt the relations patterns-colours (A), and a second group of 20 bees learnt the relations colours-patterns (B). Grey bar: mean percentage (6 s.d.) of correct choices in an unrewarded symmetry test session (ST) during which the bees were presented with the stimuli in reverse order to the training sessions. Numbers inside bars indicate the total number of choices per session. Asterisks above columns indicate sessions in which the mean number of correct choices was significantly different to chance (50%) at P,0.001. doi:10.1371/journal.pone.0051467.g002 interpreted as a demonstration of emergent symmetry. However, in this study all sessions were conducted under reinforcement conditions; that is responses to the correct stimuli were always rewarded even during sequence reversal [19]. It is therefore conceivable that the bees in the Zhang et al [19] study did not demonstrate emergent symmetry, but merely learnt the new relations between the stimuli expedited by the fact that they were already familiar with one relationship between the stimuli. In contrast, our study presented the stimuli without positive re- inforcement during the stimulus reversal, and is thus a true test for symmetrical relations. investigated whether their poor learning performance could be due to an inability to discriminate the pattern and colour stimuli. Therefore, the 16 M. rufiventris bees received simple discrimination training with horizontal vs vertical patterns and blue vs yellow colours, with one group of 8 bees trained with vertical and yellow as rewarded stimuli (Fig. 4A), and the second group of 8 bees trained with horizontal and blue as rewarded stimuli (Fig. 4B). Each bee received eight consecutive 15-minute training blocks until 100% correct responses in at least two consecutive blocks was reached. All bees learnt to discriminate patterns as well as colours rapidly, achieving the learning criterion of 100% correct choice after 4, 6 or 7 blocks, respectively (Fig. 4). Learning of Arbitrary Relations in Melipona rufiventris Numbers inside bars indicate the total number of choices per session. Asterisks above columns indicate sessions in which the mean number of correct choices was significantly different to chance (50%) at P,0.001. doi:10.1371/journal.pone.0051467.g002 Figure 3. Learning of arbitrary relations in Melipona rufiventris stingless bees. Bars: mean percentage (6 s.d.) of correct choices in six rewarded training sessions of seven training blocks each, during which one group of 8 bees learnt the relations patterns-colours (A), and a second group of 8 bees learnt the relations colours-patterns (B). Numbers inside bars indicate the total number of choices per session. doi:10.1371/journal.pone.0051467.g003 Relational Learning in Bees Figure 2. Learning of arbitrary relations in Apis mellifera honeybees. Black bars: mean percentage (6 s.d.) of correct choices in six rewarded training sessions of seven training blocks each, during which one group of 20 bees learnt the relations patterns-colours (A), and a second group of 20 bees learnt the relations colours-patterns (B). Grey bar: mean percentage (6 s.d.) of correct choices in an unrewarded symmetry test session (ST) during which the bees were presented with the stimuli in reverse order to the training sessions. Numbers inside bars indicate the total number of choices per session. Asterisks above columns indicate sessions in which the mean number of correct choices was significantly different to chance (50%) at P,0.001. doi:10.1371/journal.pone.0051467.g002 Figure 3. Learning of arbitrary relations in Melipona rufiventris stingless bees. Bars: mean percentage (6 s.d.) of correct choices in six rewarded training sessions of seven training blocks each, during which one group of 8 bees learnt the relations patterns-colours (A), and a second group of 8 bees learnt the relations colours-patterns (B). Numbers inside bars indicate the total number of choices per session. doi:10.1371/journal.pone.0051467.g003 Figure 3. Learning of arbitrary relations in Melipona rufiventris stingless bees. Bars: mean percentage (6 s.d.) of correct choices in six rewarded training sessions of seven training blocks each, during which one group of 8 bees learnt the relations patterns-colours (A), and a second group of 8 bees learnt the relations colours-patterns (B). Numbers inside bars indicate the total number of choices per session. doi:10.1371/journal.pone.0051467.g003 Figure 3. Learning of arbitrary relations in Melipona rufiventris stingless bees. Learning of Arbitrary Relations in Melipona rufiventris Our results seem to suggest that Apis mellifera do not learn stimulus relations in a symmetrical fashion, in line with their inability to establish transitive inferences [26]. When presented with multiple stimulus relations, bees guide their choices by recency effects combined with associative strength of the stimuli [26]. In our study the comparison stimuli were directly associated to the reward, while the sample stimulus may function as a navigational ‘landmark’ for the bees and therefore have lower associative strength. When the stimuli were reversed in the symmetry test, the bees might only pay attention to the sample stimulus, which previously was directly associated with the sugar reward, and consider the comparison stimuli as not immediately relevant for obtaining the food. This would result in random choice, as observed in our test. Learning of Arbitrary Relations in Melipona rufiventris Learning of Arbitrary Relations in Melipona rufiventris We repeated the experiment on learning of arbitrary relations with Melipona rufiventris, a stingless honeybee from Brazil. Due to the difficulties in training this species compared to Apis (see Materials and Methods), only eight individual M. rufiventris workers were used per group. As for Apis, one group was trained to relate patterns to colours, and the second group trained to relate colours to patterns, in six sessions over six consecutive days with each session consisting of seven 30-minute training blocks. Although fewer individuals were used with Melipona, the overall number of choices per session was in a similar range to Apis, because the experimental apparatus was installed right next to the Melipona colony allowing more frequent return visits per session. In spite of the extensive training, M. ruvifentris workers did not learn the arbitrary relations between patterns and colours (Fig. 3A), or colours and patterns (Fig. 3B). Learning performance did not increase over the six sessions for pattern-colour relations (F5,30 = 0.745, P = 0.596), nor for colour-pattern relations (F5,30 = 0.71, P = 0.62), remaining at chance levels. After having learnt these arbitrary relations the bees were then tested for emergent symmetrical relations between the training stimuli, where the respective stimuli were presented in reverse Due to their inability to learn arbitrary relations, M. rufiventris bees were not tested for the symmetrical relations. Instead, we December 2012 | Volume 7 | Issue 12 | e51467 PLOS ONE | www.plosone.org 2 Relational Learning in Bees interpreted as a demonstration of emergent symmetry. However, in this study all sessions were conducted under reinforcement conditions; that is responses to the correct stimuli were always rewarded even during sequence reversal [19]. It is therefore conceivable that the bees in the Zhang et al [19] study did not demonstrate emergent symmetry, but merely learnt the new Figure 2. Learning of arbitrary relations in Apis mellifera honeybees. Black bars: mean percentage (6 s.d.) of correct choices in six rewarded training sessions of seven training blocks each, during which one group of 20 bees learnt the relations patterns-colours (A), and a second group of 20 bees learnt the relations colours-patterns (B). Grey bar: mean percentage (6 s.d.) of correct choices in an unrewarded symmetry test session (ST) during which the bees were presented with the stimuli in reverse order to the training sessions. December 2012 | Volume 7 | Issue 12 | e51467 Discussion One group of 8 bees was trained with vertical and yellow as rewarded stimuli (A), and a second group of 8 bees was trained with horizontal and blue as rewarded stimuli (B). The learning criterion was 100% correct choices in at least two consecutive blocks. Numbers inside bars indicate the total number of choices per session. doi:10.1371/journal.pone.0051467.g004 Figure 4. Discrimination learning in Melipona rufiventris stingless bees. Bars: percentage of correct choices in eight consecutive training blocks, during which bees learnt to discriminate a vertical pattern from a horizontal pattern, and yellow from blue. One group of 8 bees was trained with vertical and yellow as rewarded stimuli (A), and a second group of 8 bees was trained with horizontal and blue as rewarded stimuli (B). The learning criterion was 100% correct choices in at least two consecutive blocks. Numbers inside bars indicate the total number of choices per session. doi:10.1371/journal.pone.0051467.g004 argument is hypothetical at this stage and requires the support of detailed comparative studies of the food sources and visual stimuli that each species uses during foraging – from flower cues to navigational landmarks. Such studies would have to take into account the specific foraging environment a particular colony and individual foragers experience. For example, an A. mellifera colony might exploit a larger variety of food sources than an M. rufiventris colony in general; but depending on the particular colony site, individual A. mellifera bees may be exposed to either rapidly changing food sources (and hence an individual would have to learn a variety of different flowers in its life time), or bees might have access to food sources that flower for extended periods (and hence individual bees would only need to learn one or two flower types in their life time). from our laboratory using a modified matching-to-sample pro- cedure in a T-maze indicate similar results to the findings presented here: both M. rufiventris and M. quadrifasciata seem to acquire identity relations very quickly, but fail to learn arbitrary relations involving visual stimuli as well as olfactory stimuli (Moreno and de Souza, pers. comm.). There is one study suggesting M. rufiventris can learn relations between colours and light intensities [23]. However, because both stimuli were presented simultaneously in that study, and not successively (as is the case in matching-to-sample procedures), it is possible that the bees had learnt stimuli configurations instead of relations [28]. Discussion Nevertheless, it is clear that Melipona species have good learning abilities. Why then are they not able to learn arbitrary relations? An animal’s learning abilities presumably have evolved to suit the lifestyle within its ecological niche, and species-specific variations in learning performance are thought to be an adaptation to the particular environment in which the animal operates [29]. Melipona’s inability to learn arbitrary relations may be intrinsically linked to its foraging biology. Melipona bees including M. rufiventris have much narrower food niches than Apis mellifera; the spectrum of plants visited by Melipona bees is rather restricted, and they are less generalistic than Apis in terms of flower choice [30–33]. Melipona focuses on mass-flowering trees that produce a burst of dense inflorescence clusters for a short period of time. Due to their small colonies of less than 1000 individuals Melipona species do not require numerous different food sources that are available at the same time to sustain themselves [34]. Their narrower foraging niche may reduce the need to simulta- neously learn multiple visual cues from a broad spectrum of flower types that are distributed over a large foraging area, as Apis mellifera does. It is conceivable that under the latter conditions flexibility in learning food-related cues is more important, and may have driven the evolution of relational learning in A. mellifera. Of course, this Another aspect that might contribute to the difference in relational learning capacity between Apis and Melipona, is their differences in recruitment strategies. Melipona species use compar- atively simple communication strategies to recruit nest-mates to food sources. Although there are a diversity of species-specific recruitment mechanisms in meliponine bees, such as jostling, spinning, body contacts and sound production inside the nest, visual guidance flights, odour trails and foot-print marks on food sources [35–37], none of these mechanisms reach the sophistica- tion and complexity of the honeybee dance language produced exclusively by bees of the genus Apis [3,38]. The simpler recruitment and foraging mechanisms in Melipona, and the lack of a complex dance language might have prevented the evolution of more ‘‘abstract’’ learning abilities in stingless bees such as required for learning of arbitrary relations. Species-specific differences in learning performance have been shown in many insect groups including bees, parasitoid wasps, and butterflies [29,39–42]. However, natural within-species variations can affect between-species comparisons. December 2012 | Volume 7 | Issue 12 | e51467 Discussion Our study showed that Apis mellifera is capable of learning arbitrary relations between stimuli, confirming previous findings revealing that this small insect has impressive cognitive capacities [18–22,24,25]. However, Apis mellifera was apparently unable to use this relational information when tested with symmetrical relations, where the stimulus order was reversed. These findings contrast with earlier results, where Apis mellifera workers learnt the relations between three visual stimuli (gratings A vs A’, colours B vs B’, patterns C vs C’) [19]. In this earlier study, bees learnt the sequences ABC and A’B’C’, with learning performance equivalent to our study. When presented with different stimulus sequences, e.g. BAC vs B’A’C’ or CBA vs C’B’A’, the bees still showed a significant percentage of correct choices, which could be In contrast to honeybees, Melipona rufiventris seems incapable of learning the arbitrary relations of patterns and colours tested here, and hence were not tested for symmetrical relations. M. rufiventris’ apparent inability for relational learning is not due to sensory limitations as they are clearly able to discriminate between the stimuli used. Nor is it due to a general inability or inadequacy to learn: associative learning in Melipona bees has been shown both for the visual and olfactory domain [13–17], and Melipona rufiventris is even capable of operant learning [27]. Recent preliminary work December 2012 | Volume 7 | Issue 12 | e51467 3 December 2012 | Volume 7 | Issue 12 | e51467 3 PLOS ONE | www.plosone.org Relational Learning in Bees Figure 4. Discrimination learning in Melipona rufiventris stingless bees. Bars: percentage of correct choices in eight consecutive training blocks, during which bees learnt to discriminate a vertical pattern from a horizontal pattern, and yellow from blue. One group of 8 bees was trained with vertical and yellow as rewarded stimuli (A), and a second group of 8 bees was trained with horizontal and blue as rewarded stimuli (B). The learning criterion was 100% correct choices in at least two consecutive blocks. Numbers inside bars indicate the total number of choices per session. doi:10.1371/journal.pone.0051467.g004 g Figure 4. Discrimination learning in Melipona rufiventris stingless bees. Bars: percentage of correct choices in eight consecutive training blocks, during which bees learnt to discriminate a vertical pattern from a horizontal pattern, and yellow from blue. Relational Learning in Bees w/w sucrose solution), the incorrect comparison stimulus (S–) led to an empty feeder. A paper barrier placed 2 cm behind the holes prevented the bees from seeing the feeders while making their decision. After bees had made their decision, they were released by lifting the lid from the cylinders and returned to the hive. The stimuli were black and white gratings with 3 cm wide bars, printed with a high-resolution laser printer, and colours were pieces of either blue (39N) and yellow (3N) artist-quality cardboard (K+E Stuttgart, Germany). Each stimulus was 14614 cm in size, with a 4 cm hole in the centre that fitted over the holes in the cylinders. differences in learning performance depending on the population or colony have been shown for both honeybees and bumblebees [39,43–45]. It has to be taken into account that our study was carried out with only one colony per species and a rather limited number of Melipona individuals. To provide conclusive evidence that the observed differences in relational learning performance between A. mellifera and M. rufiventris are indeed species-specific, the results would need to be confirmed with more colonies. Furthermore, the study was carried out in two different countries due to the fact that Melipona rufiventris is native to Brazil and cannot be introduced into Australia for ecological reasons. Although the experimental apparatus and protocol were the same, and we tried to keep the settings as similar as possible, different weather conditions, differences in food availability and colony health could potentially have contributed to the species differences in learning performance. There is also the possibility that our experimental set-up may not have been suitable for demonstrating arbitrary learning in Melipona rufiventris. Indeed, the fact that we kept experimental conditions identical could have been a disad- vantage. Finding differences in learning between two species that were tested under the same experimental conditions might reflect true species differences or, alternatively, may reflect differences in how each species responds to the experimental apparatus/ protocol. Stingless bees are notoriously difficult to work with in a laboratory setting compared to Apis mellifera. It is well known that behavioural protocols for bee learning need to be adapted to the specific needs of a species, in order to obtain robust results [11,12,16,46]. Relational Learning in Bees It is therefore not impossible that a different set-up, for example under natural conditions in the field using more natural stimuli, might prove successful in demonstrating some degree of arbitrary learning in Melipona. Individually marked Apis mellifera foragers, all experimentally naı¨ve, were initially trained to navigate the Y-maze without stimuli, until they had familiarized themselves with the set-up. During this pre-training phase, the feeder containing sucrose solution was placed first inside the tunnel entrance, then moved step by step through the tunnel, into the first cylinder, and then into the second cylinders alternating position between left and right cylinder (Fig. 1). This procedure, which lasted approximately 2 hours, allowed bees to learn that they had to fly through the holes and into either of the second cylinders to obtain the sugar reward. Bees were considered to be familiar with the set-up, once each individual bee flew straight through the Y-maze and found the feeder at the end without slowing down or turning around. Then, the visual stimuli were placed on the cylinders to investigate learning of arbitrary relations. One group of bees was trained to relate patterns to colours: when the sample showed the horizontal pattern, the cylinder containing the sucrose was marked with blue; when the sample was the vertical pattern, the cylinder containing the sucrose was marked with yellow. Bees were trained to these stimuli relations in six sessions on six consecutive days. Each session lasted approximately 4–6 hours and consisted of seven training blocks of 30 minutes each. During a training block the sample pattern stimulus and their corresponding colour stimuli were presented alternately, that is bees were first shown one sample stimulus (e.g. horizontal pattern) for 15 minutes, and then the second sample stimulus (vertical pattern) for 15 minutes. During each 15-minute period, the position of the colour comparison stimuli was swapped every 7.5 minutes to prevent positional learning. Typically, a bee returned to the experimental apparatus every 5–8 minutes. This ensured that the bee experienced all stimulus positions at least once during a training block. Bees were individually trained in this way and the experiment was repeated until 20 bees had been trained altogether. The present study is the first to investigate the acquisition of arbitrary relations in Melipona rufiventris, using a symbolic match- ing-to-sample procedure. Relational Learning in Bees Stingless bees have not been as in- tensively studied as honeybees, and thus our understanding of their biology and cognitive capacity is at an early stage relative to what is known about Apis mellifera. The differences in learning performance between the two bee species could be intrinsic and associated with specific foraging and recruitment strategies that have evolved in adaptation to differing foraging environments. However, to provide a conclusive answer, whether and how the cognitive capacities of these bees are driven by their foraging ecology, and whether indeed some bee species are ‘‘smarter’’ than others, detailed studies on the cognitive ecology of a variety of stingless bee species using many different colonies are needed. At the end of the six training sessions, after the bees had learnt the arbitrary relations, they were given a test for symmetrical relations. In this test, the stimuli changed their functions: that is, the stimuli previously used as sample were used as comparison stimuli, whereas the stimuli previously used as comparison stimuli were used as sample stimuli. Therefore, the bees were tested for the relations blue-horizontal and yellow-vertical (symmetrical to the relations horizontal-blue and vertical-yellow). A test session consisted of four unrewarded symmetry tests of 15 minutes each; that is, each test sample stimulus was presented twice. Tests were interspersed with 30-minute training blocks with the stimuli in their original function and responses to S+ rewarded to maintain bee motivation. Only the data from the unrewarded tests were used for analysis of the test session. December 2012 | Volume 7 | Issue 12 | e51467 Discussion Significant intraspecific December 2012 | Volume 7 | Issue 12 | e51467 December 2012 | Volume 7 | Issue 12 | e51467 PLOS ONE | www.plosone.org 4 Relational Learning in Bees Materials and Methods Apis mellifera Experiments Acknowledgments We thank Michael Hrncir and Stefan Jarau for helpful discussions, and four anonymous reviewers for constructive comments on earlier versions of the manuscript. All experiments were conducted in strict accordance with national guidelines and regulations for animal research in Australia and Brazil. Melipona rufiventris Experiments The experiments with Melipona rufiventris bees were carried out at the Universidade Federal de Sa˜o Carlos, Brazil. A M. rufiventris hive was mounted inside the laboratory, which was illuminated by natural daylight, and the experimental apparatus was set up next to the colony to ensure easy access and frequent visits by the bees. The apparatus including UV-transparent Perspex lids and the stimuli were the same as those used for Apis mellifera (Fig. 1). The experimental procedure for investigating relational learning in M. rufiventris was identical to the one described above for A. mellifera. One group of M. rufiventris workers, all experimentally naı¨ve was trained to relate patterns to colours, and a second group was trained to relate colours to patterns. As for Apis, training was conducted in six sessions of seven 30-minute training blocks each, over 6 consecutive days. Training Melipona in complex learning tasks such as matching-to-sample is difficult and time-consuming: it can take many days to ‘entice’ a Melipona worker to enter and navigate its way through an experimental apparatus such as the Y- maze used here, and to continually participate in an experiment. Only data from Melipona individuals that participated in each consecutive training session were used for analysis. This is in contrast to Apis workers, which are easily trained within a couple of hours to participate in practically any behavioural experiment. Therefore, due to the difficulties in training Melipona honeybees, only eight Melipona ruvifentris workers per group were used. Although fewer bees were used than for Apis, the total number of choices per session was in the same range for both species: Melipona bees visited the apparatus more frequently as it was set up Author Contributions Conceived and designed the experiments: AMM DGS JR. Performed the experiments: AMM. Analyzed the data: AMM JR. Wrote the paper: AMM DGS JR. Conceived and designed the experiments: AMM DGS JR. Performed the experiments: AMM. Analyzed the data: AMM JR. Wrote the paper: AMM DGS JR. Relational Learning in Bees they were tested for the symmetrical relations horizontal-blue and vertical-yellow. Again the experiment was repeated until a total of 20 bees were trained and tested. right next to the hive allowing rapid return visits. For analysis, all responses to the comparison stimuli were recorded. The choices from all bees per group were pooled for each block per training session, and the mean percentage of correct choices per session calculated. Learning performance for arbitrary relations was analyzed using repeated-measures ANOVA. For analysis, all responses to the comparison stimuli were recorded. The choices from all bees per group were pooled for each block per training session as well as for the four symmetry tests of the test session, and the mean percentage of correct choices per session calculated. Learning performance during training was analyzed using repeated-measures ANOVA with session as within- subject-factor, and performance during the symmetry test was analyzed using a two-tailed one-sample t-test against a reference value of 50%. y g p Due to the outcome of the training sessions investigating relational learning, M. rufiventris bees were not tested for symmetrical relations. Instead, they received simple discrimination training with the stimuli used above, to investigate whether they could discriminate the two colours and the two patterns. Discrimination training was conducted straight after the last session of the relational learning experiment. The eight bees from group 1 were trained individually with vertical (S+) versus horizontal (S–) and yellow (S+) versus blue (S–), whereas the other eight bees from group 2 were trained individually with horizontal (S+) versus vertical (S–) and blue (S+) versus yellow (S–). Each bee received eight 15-minute blocks of discrimination training until 100% correct responses in at least two consecutive blocks was reached. The position (left-right) of the stimuli followed a pseudo-random sequence, with the rewarded stimulus being presented on either side for 7.5 minutes during a 15-minute block. Half of the bees in each group were first trained to pattern discrimination and then to colour discrimination; for the other half of the group the sequence was reversed. We did not find any effect of sequence, and thus the choices from all bees per group were pooled for each block irrespective of training sequence, and the percentage of correct choices per block calculated. Apis mellifera Experiments The experiments with Apis mellifera ligustica were carried out at the Queensland Brain Institute, Australia, in an indoor honeybee flight facility illuminated by natural daylight. Free-flying honey- bees from one experimental hive entered the facility through a small window, which was connected to the experimental apparatus. The apparatus consisted of a wooden tunnel (H6W6L = 256256130 cm), and three vertical plastic cylinders, (25 cm internal diameter (ID) 625 cm high: Fig. 1). The cylinders were interconnected via small holes (4 cm ID) to form a Y-shaped maze. The tunnel and the cylinders were covered with UV- transparent Perspex lids. Bees entering the tunnel would fly toward the first cylinder that served to present the sample stimulus. Inside the first cylinder, the bees were then presented with two comparison stimuli (S+ or S–). The correct comparison stimulus (S+) led into another cylinder with a food reward (feeder with 50% The entire experiment, i.e. six training sessions over six days for arbitrary relations followed by a test session for symmetric relations, was repeated with a new set of bees, which was trained and tested as described above, but with colours as sample stimuli and patterns as comparison stimuli. The bees had to learn the arbitrary relations blue-horizontal and yellow-vertical, and then December 2012 | Volume 7 | Issue 12 | e51467 5 PLOS ONE | www.plosone.org Relational Learning in Bees References 1. Cumming WW, Berryman R (1965) The complex discriminated operant: Studies of matching-to-sample. In: Mostofsky DI, editor. Stimulus generaliza- tion. Stanford: Stanford University Press. 284–330. 13. Pessotti I (1967) Inverso˜es de discriminac¸a˜o com dois ‘‘manipulanda’’ em Melipona (M) rufiventris. Rev Psicol Normal Patolo´g 3–4: 171–182. ( ) f g 14. 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(2011) Natural variation in learning rate and memory dynamics in parasitoid wasps: opportunities for converging ecology and neuroscience. Proc R Soc B 278: 889– 897. 40. Dukas R, Real LA (1991) Learning foraging tasks by bees: a comparison between social and solitary species. Anim Behav 42: 269–276. 40. Dukas R, Real LA (1991) Learning foraging tasks by bee between social and solitary species. Anim Behav 42: 269–276. 41. Raine NE, Ings TC, Dornhaus A, Saleh N, Chittka L (2006) Adaptation, genetic drift, pleiotropy, and history in the evolution of bee foraging behavior. Adv Study Behav 36: 305–354. 30. Ramalho M, Kleinert-Giovannini A, Imperatriz-Fonseca VL (1990) Important bee plants for stingless bees (Melipona and Trigonini) and Africanized honeybees (Apis mellifera) in neotropical habitats: a review. Apidologie 21: 469–488. 42. References Naturwiss 91: 224–227. 20. Cooke MH, Couvillon PA, Bitterman ME (2007) Delayed symbolic matching in honeybees (Apis mellifera). J Comp Psych 121: 106–108. 21. Srinivasan MV, Zhang SW, Zhu H (1998) Honeybees link sights to smells. Nature 396: 637–638. 10. Dyer AG, Spaethe J, Prack S (2008) Comparative psychophysics of bumblebee and honeybee colour discrimination and object detection. J Comp Physiol A 194: 617–627. 22. Reinhard J, Srinivasan MV, Guez D, Zhang SW (2004) Floral scents induce recall of navigational and visual memories in honeybees. J Exp Biol 207: 4371– 4381. 11. Riveros A., Gronenberg W (2009) Olfactory learning and memory in the bumblebee Bombus occidentalis. Naturwiss 96: 851–856. 23. Pessotti I (1981) Aprendizagem em abelhas. VI – Discriminac¸a˜o condicional em Melipona rufiventris. Rev Brasil Psicol 41: 681–693. 12. Toda NRT, Song J, Nieh JC (2009) Bumblebees exhibit the memory spacing effect. Naturwiss 96: 1185–1191. 6 PLOS ONE | www.plosone.org December 2012 | Volume 7 | Issue 12 | e51467 December 2012 | Volume 7 | Issue 12 | e51467 Relational Learning in Bees Kandori I, Yamaki T, Okuyama S, Sakamoto N, Yokoi T (2009) Interspecific and intersexual learning rate differences in four butterfly species. J Exp Biol 212: 3810–3816. 31. Wilms W, Imperatriz-Fonseca VL, Engels W (1996) Resource partitioning between highly eusocial bees and possible impact of the introduced Africanized honey bee on native stingless bees in the Brazilian Atlantic rainforest. Stud Neotrop Fauna & Environm 31: 137–151. 43. Chittka L, Ings TC, Raine NE (2004) Chance and adaptation in the evolution of island bumblebee behaviour. Pop Ecol 46: 243–251. p 32. Wilms W, Wiechers B (1997) Floral resource partitioning between native Melipona bees and the introduced Africanized honey bee in the Brazilian Atlantic rain forest. Apidologie 28: 339–355. 44. Raine NE, Chittka L (2008) The correlation of learning speed and natural foraging success in bumble-bees. Proc R Soc B 275: 803–808. foraging success in bumble-bees. Proc R Soc B 275: 803–80 45. Ings TC, Raine NE, Chittka L (2009) A population comparison of the strength and persistence of innate colour preference and learning speed in the bumblebee Bombus terrestris. Behav Ecol Sociobiol 63: 1207–1218. p g 33. Biesmeijer JC, Slaa EJ (2006) The structure of eusocial bee assemblages in Brazil. Apidologie 37: 240–258. 34. Ramalho M, Kleinert-Giovannini A, Imperatriz-Fonseca VL (1989) Utilization of floral resources by species of Melipona (Apidae, Meliponinae): floral preferences. Apidologie 20: 185–195. 46. Vorel CA, Pitts-Singer TL (2010) The proboscis extension reflex not elicited in Megachilid bees. J Kansas Entomol Soc 83: 80–83. PLOS ONE | www.plosone.org December 2012 | Volume 7 | Issue 12 | e51467 PLOS ONE | www.plosone.org 7 7
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Inhibition of Angiotensin Converting Enzyme Impairs Anti-staphylococcal Immune Function in a Preclinical Model of Implant Infection
Frontiers in immunology
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ORIGINAL RESEARCH published: 11 September 2020 doi: 10.3389/fimmu.2020.01919 Inhibition of Angiotensin Converting Enzyme Impairs Anti-staphylococcal Immune Function in a Preclinical Model of Implant Infection Rishi Trikha 1, Danielle Greig 1, Benjamin V. Kelley 1, Zeinab Mamouei 1, Troy Sekimura 1, Nicolas Cevallos 1, Thomas Olson 1, Ameen Chaudry 1, Clara Magyar 1, Daniel Leisman 2, Alexandra Stavrakis 1, Michael R. Yeaman 3,4 and Nicholas M. Bernthal 1* Rishi Trikha 1, Danielle Greig 1, Benjamin V. Kelley 1, Zeinab Mamouei 1, Troy Sekimura 1, Nicolas Cevallos 1, Thomas Olson 1, Ameen Chaudry 1, Clara Magyar 1, Daniel Leisman 2, Alexandra Stavrakis 1, Michael R. Yeaman 3,4 and Nicholas M. Bernthal 1* 1 Department of Orthopaedic Surgery, University of California, Los Angeles, CA, United States, 2 Icahn School of Medicine at Mount Sinai, New York, NY, United States, 3 Divisions of Molecular Medicine and Infectious Diseases, Department of Medicine, Harbor-UCLA Medical Center, Torrance, CA, United States, 4 The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA, United States Edited by: Semih Esin, University of Pisa, Italy Edited by: Semih Esin, University of Pisa, Italy Reviewed by: Nihal Engin Vrana, Sparta Medical, France Hridayesh Prakash, Amity University, India *Correspondence: Nicholas M. Bernthal nbernthal@mednet.ucla.edu Reviewed by: Nihal Engin Vrana, Sparta Medical, France Hridayesh Prakash, Amity University, India Methods: In vitro antimicrobial effects of ACEi and ARBs were first assessed. C57BL/6J mice subsequently received either an ACEi (lisinopril; 16 mg/kg/day), an ARB (losartan; 30 mg/kg/day), or no treatment. Conditioned mice blood was then utilized to quantify respiratory burst function as well as Staphylococcus aureus Xen36 burden ex vivo in each treatment group. S. aureus infectious burden for each treatment group was then assessed in vivo using a validated mouse model of implant infection. Real-time quantitation of infectious burden via bioluminescent imaging over the course of 28 days post-procedure was assessed. Host response via monocyte and neutrophil infiltration within paraspinal and spleen tissue was quantified by immunohistochemistry for F4/80 and myeloperoxidase, respectively. *Correspondence: Nicholas M. Bernthal nbernthal@mednet.ucla.edu *Correspondence: Nicholas M. Bernthal nbernthal@mednet.ucla.edu Specialty section: This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology Received: 07 May 2020 Accepted: 16 July 2020 Published: 11 September 2020 Received: 07 May 2020 Accepted: 16 July 2020 Results: Blood from mice treated with an ACEi demonstrated a decreased ability to eradicate bacteria when mixed with Xen36 as significantly higher levels of colony forming units (CFU) and biofilm formation was appreciated ex vivo (p < 0.05). Mice treated with an ACEi showed a higher infection burden in vivo at all times (p < 0.05) and significantly higher CFUs of bacteria on both implant and paraspinal tissue at the time of sacrifice (p < 0.05 for each comparison). There was also significantly decreased infiltration and respiratory burst function of immune effector cells in the ACEi group (p < 0.05). Background: Evidence suggests the renin-angiotensin system (RAS) plays key immunomodulatory roles. In particular, angiotensin-converting enzyme (ACE) has been shown to play a role in antimicrobial host defense. ACE inhibitors (ACEi) and angiotensin receptor blockers (ARB) are some of the most commonly prescribed medications, especially in patients undergoing invasive surgery. Thus, the current study assessed the immunomodulatory effect of RAS-modulation in a preclinical model of implant infection. Edited by: Semih Esin, University of Pisa, Italy INTRODUCTION neutrophils led to a 6-fold reduction in the clearance of a subcutaneous infection with methicillin-resistant Staphylococcus aureus (MRSA) (26). The purported mechanisms underlying any possible immunosuppressive effect of ACE inhibition include dysregulation of TNF-α, IL-6 and/or TGF-β response (47, 48), IL- 12 suppression (49), decreased neutrophil superoxide production (26), dysfunctional macrophage activity (34, 35, 40), impaired chemotactic function (34), and decreased pro-inflammatory cytokine production (39, 40). Such mechanisms may impact innate and/or adaptive roles of antimicrobial host defense. Implant-associated surgical site infections (SSI) represent significant morbidity and mortality for the patient as well as massive economic strain to the current healthcare system (1–7). Despite increasing efforts to prevent SSI through perioperative antibiotic management and the optimization of aseptic surgical technique, infection rates still range from 1.2% in primary joint replacements to 8.6% in ventral hernia mesh repair to as high as 12.9% in ventriculoperitoneal shunts (8–16). Although infection rates and treatment approaches vary by implant type, the overwhelming majority of patients who develop SSI ultimately require surgical implant removal, as bacteria form protective glycocalyx layers on avascular surfaces knows as biofilm (8, 17, 18). In high risk surgeries such as cardiac device implantation and spinal instrumentation, this can lead to catastrophic outcomes such as cardiovascular compromise, spinal column collapse, or death (19, 20). Even in hip and knee replacement surgery, an implant infection carries a worse 5-year mortality rate than breast cancer, renal cell cancer, or HIV/AIDS (21–25). Given the absence of effective treatment for implant infections, prevention is thus paramount. To that end, the identification and optimization of safe and short-term host- targetable risk factors represent crucial, innovative opportunities to prevent SSI. p It is crucial to ensure immunocompetency at the time of surgery as the immunoprofile of patients prior to implantation are inextricably linked with the development of a SSI (50–52). It is undoubtedly true that certain patients will not be able to achieve lifelong immunocompetency, however the optimization of the immune system at the time of surgery remains vitally important to minimize SSI, as the majority of implant associated infections occur at the time of surgery (51, 52). Ethics Statement All animal studies were performed in accordance with protocols reviewed and approved by the Chancellor’s Animal Research Committee (ARC) at University of California, Los Angeles (ARC #2012-104-21J). These practices are adherent to National Institute of Health and Public Health Service policies. While the role of the renin-angiotensin system for blood pressure regulation is well-known, emerging evidence suggests this system also has an immunologic function. Of the components involved in this system, ACE appears to have a particularly important role in antimicrobial host defense. Multiple human and animal studies have demonstrated that ACE overexpression increases immune cell response and facilitates host defense against bacterial infections (26, 33–46). In one murine study, selectively reducing ACE expression in Citation: Trikha R, Greig D, Kelley BV, Mamouei Z, Sekimura T, Cevallos N, Olson T, Chaudry A, Magyar C, Leisman D, Stavrakis A, Yeaman MR and Bernthal NM (2020) Inhibition of Angiotensin Converting Enzyme Impairs Anti-staphylococcal Immune Function in a Preclinical Model of Implant Infection. Front. Immunol. 11:1919. doi: 10.3389/fimmu.2020.01919 Trikha R, Greig D, Kelley BV, Mamouei Z, Sekimura T, Cevallos N, Olson T, Chaudry A, Magyar C, Leisman D, Stavrakis A, Yeaman MR and Bernthal NM (2020) Inhibition of Angiotensin Converting Enzyme Impairs Anti-staphylococcal Immune Function in a Preclinical Model of Implant Infection. Front. Immunol. 11:1919. doi: 10.3389/fimmu.2020.01919 Conclusion: ACEi, but not ARB, treatment resulted in increased S. aureus burden and impaired immune response in a preclinical model of implant infection. These results September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org Trikha et al. ACE Inhibition Increases Staphylococcal Burden suggest that perioperative ACEi use may represent a previously unappreciated risk factor for surgical site infection. Given the relative interchangeability of ACEi and ARB from a cardiovascular standpoint, this risk factor may be modifiable. suggest that perioperative ACEi use may represent a previously unappreciated risk factor for surgical site infection. Given the relative interchangeability of ACEi and ARB from a cardiovascular standpoint, this risk factor may be modifiable. Keywords: implant, infection, angiotensin-converting enzyme inhibitor, angiotensin II receptor blocker, bioluminescence Frontiers in Immunology | www.frontiersin.org INTRODUCTION Although the immunological impact of ACEi has been studied, to some extent, in vitro and in short-term models in vivo (33–35, 38, 40– 42, 46), there is a lack of longitudinal in vivo data to quantify this effect or explore its potential mechanistic basis, and no study to our knowledge has investigated this phenomenon in a surgical model. Thus, the purpose of this study was to assess whether perioperative ACEi treatment impacts the host immune response and determine whether any purported impact would be sufficient to affect infection rates and severity in a well- validated in vivo mouse model of implant infection (53–56). This study also aimed to assess whether a reasonable alternative drug with a similar cardiovascular profile could avoid such host immunomodulation, thus optimizing host immunity to minimize perioperative infectious risk. p Angiotensin-converting enzyme inhibitors (ACEi), which block the conversion of angiotensin I to angiotensin II, and angiotensin II receptor blockers (ARB) are two of the most commonly used drugs for the treatment of hypertension (26– 29). In 2017, 73 million Americans were prescribed at least one cardiovascular medication, of which, 28 million Americans were prescribed an ACEi and another 15 million were prescribed an ARB (30). Furthermore, according to the CDC, an estimated 11.4% of Americans between 40 and 59 years old, and 21.3% from the age of 60–79 have taken an ACEi in the last 30 days (31). The prevalence of these medications is perhaps even greater in the surgical population. In one multi-institutional study performed across 12 surgery centers, 4,802 out of 14,687 (32.7%) patients who underwent inpatient non-cardiac surgery were taking an ACEi or ARB perioperatively (32). Mice and Medication Administration Mice and Medication Administration Eight to twelve-week-old, 20–25 g C57BL/6 wildtype mice (Jackson Laboratories, Bar Harbor, ME) were housed (four mice per standard cage) and stored with a 12 h light and dark cycle with food and water ad libitum. Veterinary staffassessed all mice on a daily basis to ensure well-being throughout the entirety of the experiment. Mice were randomized to receive treatment with either: an ACEi (lisinopril; 16 mg/kg/day PO; LKT Laboratories, St. Paul, MN) (ACEi group), an ARB (losartan; 30 mg/kg/day PO, LKT Laboratories, St. Paul, MN) (ARB group), or no treatment (control group) with dosing as per prior independent protocols that demonstrated a percent reduction in blood pressure akin to that of humans (40, 61). Medications were suspended in 250 mL containers of drinking water. Ten milliliters of sucralose was added to the drinking water of all mice and intake was recorded daily to ensure each mouse drank 3–5 mL/day. For all ex vivo experiments, mice received medication treatment for 4 weeks prior to cardiac puncture and sacrifice. For in vivo Selection and Preparation of Bioluminescent Xen36 Staphylococcus aureus Staphylococcus aureus strain Xen36 (PerkinElmer, Waltham, MA), a bioluminescent strain derived from ATCC-29525 (Wright), was used as the study organism. This strain September 2020 | Volume 11 | Article 1919 2 ACE Inhibition Increases Staphylococcal Burden Trikha et al. expresses a genomically integrated luxABCDE operon (53, 57, 58). Consequently, Xen36 generates a bioluminescent blue- green signal with a maximal emission wavelength of 490 nm from viable, metabolically active organisms. Previous studies demonstrated this strain to be ideal for research targeting the longitudinal monitoring of S. aureus infections due to its strength and consistency of signal (57–59). ARB, vancomycin, each at a concentration of 0.5 mg/mL, or normal saline was aliquoted onto separate 6 mm filter paper disks and left to dry for 30 min. One disk from each group was then placed into a separate quadrant of a TSA plate that had been flooded and spread with 200 µL of 1 × 106 S. aureus Xen36. This was performed for four total plates. This procedure was replicated on four additional plates using a concentration of 1 mg/mL of study therapeutics. Plates were left to incubate at 37◦C for 16 h and zone of inhibition(s) were then analyzed. Bacterial inocula were prepared following previously published protocols (53–57). In brief, Xen36 was isolated on kanamycin to select for purity and affirm possession of the kanamycin-resistance marker integral to the lux operon. The authenticated Xen36 strain was then quadrant-streaked onto tryptic soy agar (TSA; Beckton-Dickinson) and incubated for 24 h at 37◦C. Single colonies were then isolated and cultured in tryptic soy broth (TSB) for 16 h at 37◦C in a shaking incubator (196 rpm) (MaxQ 4,450, Thermo). A subsequent 2 h subculture of a 1:50 dilution of this culture was used to obtain a mid- logarithmic phase bacteria. Lastly, after centrifugation, cells were pelleted, resuspended, and washed in PBS. Bacterial inocula were quantitated and standardized by spectrophotometry (OD, 600 nm; BioMate 3; ThermoFisher Scientific). A schematic overview of our ex vivo and in vivo experiments is provided (Figure 1). Ex vivo Quantification of the Biofilm Biomass experiments, treatment began three weeks preoperatively and continued postoperatively for 4 weeks until sacrifice. These time points were selected based on previous studies demonstrating sufficiently altered immune profiles of mice blood after 7–10 days (26, 35). Five mice in each group underwent whole blood collection as above. One-hundred microliters of whole blood from each mouse were mixed with 100 µL of 1 × 107 S. aureus Xen36 CFU/mL for a final inoculum of 106 CFU in 200 µL. This solution was added to each well within a 96-well-flat bottom plate. Six additional control wells containing 200 µL of saline were also included for standardization. After 24 h of incubation at 37◦C, each well was washed with PBS three times to remove residual blood cells and non-adherent bacteria. A well-validated crystal violet assay (Abcam, Cambridge, United Kingdom) (62–64) was performed to quantify the biomass of the residual biofilm formation by OD at 595 nm. Ex vivo Quantification of Respiratory Burst Following 4 weeks of medication treatment, blood was collected from six mice in each group via cardiac puncture under 2% isoflurane inhalation anesthesia, followed by immediate euthanasia. Ethylenediaminetetraacetic acid (EDTA) was added to blood samples in a 1:10 ratio to prevent coagulation. One- hundred microliters were added from each mouse to each well within a 96-well flat bottom plate (Corning Costar, Corning, New York). Reactive oxygen species (ROS) activity of whole blood were assessed using a dihydrorhodamine (DHR) 123 assay. Briefly, 10 µL of DHR 123 Assay Reagent followed by 25 µL of Phorbol myristate acetate followed by 2 mL of Red Blood Cell Lysis Buffer was added to each plate. Mean fluorescent intensity was read with an excitation filter of 485 nm and an emission filter of 520 nm using a fluorescent plate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany). In vitro Determination of Direct Staphylococcal Growth Effect of ACEi or ARB In order to confirm that ACEi or ARB do not have any direct antimicrobial effects against S. aureus, a Kirby Bauer diffusion susceptibility test was performed (60). Briefly, 20 µL of ACEi, FIGURE 1 | Schematic diagram of ex vivo and in vivo experiments. FIGURE 1 | Schematic diagram of ex vivo and in vivo experiments. FIGURE 1 | Schematic diagram of ex vivo and in vivo experiments FIGURE 1 | Schematic diagram of ex vivo and in vivo experiments. FIGURE 1 | Schematic diagram of ex vivo and in vivo experiments. September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org 3 ACE Inhibition Increases Staphylococcal Burden Trikha et al. Ex vivo CFU Quantification of S. aureus Mixed With Whole Blood Six mice in each group underwent whole blood collection as above. Ten microliters of blood from each mouse were then gently mixed with 10 µL of 1 × 103 S. aureus Xen36 and incubated at 37◦C for 1 h. After 1 h, the entire 20 µL of solution was quantitatively cultured on TSA and incubated at 37◦C for 16 h. Resulting CFUs were then counted for a minimum of n = 6 replicates in each group. FIGURE 2 | Mouse spinal implant surgery procedures. (A) Mice were prepped with six alternating scrubs of povidone-iodine and alcohol and subsequently draped in a sterile fashion. (B) A 2 cm midline dorsal incision was made. Dissection was carried through the fascia and muscle and directed laterally along the L4 spinous process. (C) The L4 spinous process was reamed with a 25-gauge needle. (D) The short arm of the implant was press-fit into the spinous process and the long arm was laid longitudinally parallel along the spine directed cranially. (E) The wound was then prepared for closure using polyglycolic acid 5-0 sutures. Prior to these sutures being tied, 1 × 102 CFUs of Xen36 in a volume of 2 µL was inoculated directly onto the long arm of the implant. (F) Deep sutures were then tied and a running 5-0 vicryl suture was used to close the skin. (G) Proper placement of the implant was confirmed with high resolution X-rays on post-operative day 0 using the IVIS Lumina X5 (PerkinElmer, Waltham, MA). FIGURE 2 | Mouse spinal implant surgery procedures. (A) Mice were prepped with six alternating scrubs of povidone-iodine and alcohol and subsequently draped in a sterile fashion. (B) A 2 cm midline dorsal incision was made. Dissection was carried through the fascia and muscle and directed laterally along the L4 spinous process. (C) The L4 spinous process was reamed with a 25-gauge needle. (D) The short arm of the implant was press-fit into the spinous process and the long arm was laid longitudinally parallel along the spine directed cranially. (E) The wound was then prepared for closure using polyglycolic acid 5-0 sutures. Prior to these sutures being tied, 1 × 102 CFUs of Xen36 in a volume of 2 µL was inoculated directly onto the long arm of the implant. (F) Deep sutures were then tied and a running 5-0 vicryl suture was used to close the skin. In vivo and Longitudinal Monitoring of Bacterial Burden and Implant and Paraspinal Tissue CFU Quantification p Twenty-six total mice were randomized into the following groups: 2 in the sterile control group, 8 in the infected control group, 8 in the ACEi group and 8 in the ARB group. Mouse spinal implant surgery and inoculation with 1 × 102 S. aureus Xen36 was performed as described in prior protocols (53–56). Briefly, a midline dorsal incision was made and dissection was performed through the fascia and muscle directed laterally along the L4 spinous process. The L4 process was manually reamed with a 25-gauge needle. An “L-shaped” surgical grade 0.1 mm diameter titanium implant (Custom Wire Technologies, Port Washington, WI) was then press-fit into the L4 process. The long arm of the implant measured 6.5 mm in length and the short arm measured 3.5 mm in length (Figure 2). The IVIS Lumina X5 (PerkinElmer, Waltham, MA) was used to capture bioluminescent images representative of S. aureus Xen36 burden Statistical Analysis y Probability (p) values were calculated using a Student’s t- test (one or two-tailed where indicated), while data analysis among three or more groups were compared using a one- way ANOVA. Longitudinal bioluminescent data were analyzed using a linear mixed effects regression model. Data were expressed as mean ± standard error of the mean (SEM). Stata-14 software (Statacorp, College Station, TX) was used for all statistical analyses and statistical significance was set at p < 0.05. Histologic Analysis and Quantification of Monocyte and Neutrophil Infiltration y p An additional 12 mice were randomized into the following groups: four in the infected control group, four in the ARB group and four in the ACEi group. Mice underwent spinal implant surgery and infection as described above. Two mice in each group were sacrificed on POD1 and two mice in each group were sacrificed at POD4. At the time of sacrifice, paraspinal and splenic tissue samples were harvested and stained with hematoxylin and eosin (H&E), F4/80 antibody (representing monocyte infiltration), and myeloperoxidase (MPO) (representing neutrophil infiltration). Histologic images were de-identified and qualitatively reviewed by a board-certified pathologist to assess for F4/80 and MPO signals. Brightfield slides were digitized on a ScanScope AT (Leica Biosystems, Inc., Vista, CA) and morphometric analysis performed with Definiens Tissue Studio (Definiens Inc., Parsippany, Ex vivo CFU Quantification of S. aureus Mixed With Whole Blood (G) Proper placement of the implant was confirmed with high resolution X-rays on post-operative day 0 using the IVIS Lumina X5 (PerkinElmer, Waltham, MA). September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org ACE Inhibition Increases Staphylococcal Burden Trikha et al. on postoperative day (POD) 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, and 28. Bioluminescent quantification of bacterial burden was confirmed directly by CFU quantification of the implant and surrounding tissue. On POD 28, each mouse was sacrificed and CFU of bacteria adherent to the implant as well as in the paraspinal tissue quantified. To do so, the implant was sonicated in 500 µL 0.3% Tween-80 (ThermoFisher Scientific) in TSB and the paraspinal tissue was homogenized (Pro200H Series homogenizer; Pro Scientific). Samples from implants and paraspinal tissue were then plated onto a TSA plate and incubated overnight. Resulting CFUs per plate were counted and total CFUs harvested from the paraspinal tissue and implant were expressed as CFUs/mL. NJ) to quantify monocyte and neutrophil counts. Briefly, a stain specific algorithm was created using the pre-defined cellular detection module and classification tool, through which positive and negative stained cells within a tissue core were identified. The data were exported to Excel for further statistical analysis. Ex vivo Quantification of Respiratory Burst, CFU, and Biofilm Biomass 0 and 3 (p < 0.05). The only significant difference between the ARB and infected control group occurred at POD 14, when the ARB group was significantly higher than the infected control (Figures 7A,B). Respiratory burst, represented by mean fluorescent intensity from a DHR 123 assay, was significantly higher in the blood of mice from the ARB group (5.5 × 104 ± 2.2 × 103) and the infected control group (5.7 × 104 ± 1.8 × 103) when compared to the ACEi group (4.7 × 104 ± 1.9 × 103, p < 0.05; Figure 4). Viable S. aureus CFUs were identified in 0 of 2 (0%) implants from the sterile control group, 2 of 8 (25%) implants from the infected control group, 2 of 8 (25%) implants from the ARB group, and 3 of 8 (37.5%) implants from the ACEi group. The mean CFU/mL cultured from the harvested implant in the ACEi group (1.1 × 104 ± 8.5 × 103) was significantly higher than either the ARB (9.8 × 102 ± 7.0 × 102) or infected control groups (3.3 × 102 ± 2.9 × 102, p < 0.05; Figure 8A). The mean CFU/mL of S. aureus Xen36 in whole blood was significantly higher for the ACEi group than the ARB group (1.3 × 104 ± 9.5 × 102 vs. 9.1 × 103 ± 2.5 × 102, p < 0.05), which was significantly higher than the infected control group (4.6 × 103 ± 2.2 × 102, p < 0.05; Figure 5). Biofilm biomass, represented by absorbance units, was significantly higher in the ACEi group (1.9 ± 0.3) as compared to both the ARB (1.3 ± 0.3) and control (1.1 ± 0.1) groups (p < 0.05; Figure 6). The mean CFU/mL cultured from the excised paraspinal tissue in the ACEi group (1.6 × 106 ± 5.1 × 105) was significantly higher than the ARB (6.1 × 105 ± 4.2 × 105) and infected control groups (6.4 × 105 ± 3.7 × 105, p < 0.05; Figure 8B). In vitro Determination of Direct Staphylococcal Growth Effect of ACEi or ARB FIGURE 5 | CFU counts demonstrating significantly increased S. aureus burden ex vivo in the blood of mice treated with an ACEi as compared to those treated with an ARB and the untreated control group. * denotes p < 0.05, ** denotes p < 0.01. In vitro Determination of Direct Staphylococcal Growth Effect of ACEi or ARB No zones of inhibition were appreciated in any plates for the normal saline, ACEi or ARB disks at any concentrations. A zone of inhibition of 18.5 ± 0.3 mm was measured around disks with 0.5 mg/mL of vancomycin. A zone of inhibition of 20.5 ± 0.3 mm was measured around disks with 1.0 mg/mL of vancomycin (Figures 3A,B). FIGURE 3 | Kirby–Bauer disk diffusion susceptibility test showing no direct effect of ACEi or ARB on S. aureus growth when study therapeutics are dosed at 0.5 mg/mL (A) and 1.0 mg/mL (B). Thus, any purported effect on bacterial burden in ACEi and ARB-treated mice were not due to any anti-staphylococcal properties of the therapeutics themselves. FIGURE 3 | Kirby–Bauer disk diffusion susceptibility test showing no direct effect of ACEi or ARB on S. aureus growth when study therapeutics are dosed at 0.5 mg/mL (A) and 1.0 mg/mL (B). Thus, any purported effect on bacterial burden in ACEi and ARB-treated mice were not due to any anti-staphylococcal properties of the therapeutics themselves. September 2020 | Volume 11 | Article 1919 5 Frontiers in Immunology | www.frontiersin.org ACE Inhibition Increases Staphylococcal Burden Trikha et al. FIGURE 5 | CFU counts demonstrating significantly increased S. aureus burden ex vivo in the blood of mice treated with an ACEi as compared to those treated with an ARB and the untreated control group. * denotes p < 0.05, ** denotes p < 0.01. FIGURE 5 | CFU counts demonstrating significantly increased S. aureus burden ex vivo in the blood of mice treated with an ACEi as compared to those treated with an ARB and the untreated control group. * denotes p < 0.05, ** denotes p < 0.01. FIGURE 4 | Measurement of respiratory burst ex vivo showing significantly decreased respiratory burst in the blood of mice treated with an ACEi as compared to those treated with an ARB and the untreated control group. *p < 0.05. FIGURE 4 | Measurement of respiratory burst ex vivo showing significantly decreased respiratory burst in the blood of mice treated with an ACEi as compared to those treated with an ARB and the untreated control group. *p < 0.05. FIGURE 4 | Measurement of respiratory burst ex vivo showing significantly decreased respiratory burst in the blood of mice treated with an ACEi as compared to those treated with an ARB and the untreated control group. *p < 0.05. Histologic Analysis and Quantification of Monocyte and Neutrophil Infiltration Following a review by a board-certified pathologist, there was no qualitative difference in monocyte or neutrophil infiltration to the spleen at POD1 or POD4 between the ACEi, ARB, or control treatment groups based on MPO and F4/80 stains. Bioluminescent signal was higher in the ACEi group than both the ARB and infected control groups at all time points. This difference reached significance at all time points other than POD September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org 6 ACE Inhibition Increases Staphylococcal Burden Trikha et al. FIGURE 6 | Quantification of biomass of residual biofilm demonstrating a significantly increased S. aureus burden ex vivo in the blood of mice treated with an ACEi as compared to those treated with an ARB and the untreated group (A). *p < 0.05. Ninety-six well-plate after 24 h of incubation with whole blood and Xen36 for 24 h and stained with crystal violet (B). FIGURE 6 | Quantification of biomass of residual biofilm demonstrating a significantly increased S. aureus burden ex vivo in the blood of mice treated with an ACEi as compared to those treated with an ARB and the untreated group (A). *p < 0.05. Ninety-six well-plate after 24 h of incubation with whole blood and Xen36 for 24 h and stained with crystal violet (B). FIGURE 7 | S. aureus burden in vivo was higher at all time points in mice treated with an ACEi as compared to those treated with an ARB and the untreated group. Statistical significance (p < 0.05) was reached at all time points other than POD 0 and 3 (A). Representative Xen36 S. aureus bioluminescent images at three selected postoperative time points overlaid on top of grayscale images of mice (B). FIGURE 7 | S. aureus burden in vivo was higher at all time points in mice treated with an ACEi as compared to those treated with an ARB and the untreated group. FIGURE 7 | S. aureus burden in vivo was higher at all time points in mice treated with an ACEi as compared to those treated with an ARB and the untreated group. Statistical significance (p < 0.05) was reached at all time points other than POD 0 and 3 (A). Representative Xen36 S. aureus bioluminescent images at three selected postoperative time points overlaid on top of grayscale images of mice (B). Histologic Analysis and Quantification of Monocyte and Neutrophil Infiltration September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org 7 ACE Inhibition Increases Staphylococcal Burden Trikha et al. FIGURE 8 | Bacteria harvested from both the implant (A) and the paraspinal soft tissue (B) demonstrate a significantly increased S. aureus burden in mice treated with an ACEi than those with an ARB and untreated mice. *p < 0.05. FIGURE 8 | Bacteria harvested from both the implant (A) and the paraspinal soft tissue (B) demonstrate a significantly increased S. aureus burden in mice treated with an ACEi than those with an ARB and untreated mice. *p < 0.05. rom both the implant (A) and the paraspinal soft tissue (B) demonstrate a significantly increased S. aureus burden in mice treated ARB and untreated mice. *p < 0.05. population (31, 68). Given the prevalence of both ACEi and ARB, the potential impact of these therapies could be enormous if they modify immune response or efficacy in ways that subvert host defense. However, there was a qualitative difference in monocyte and neutrophil infiltration to the paraspinal tissue between the groups at POD 4 (Figures 9A,B, 10A,B). The number of nuclei stained at POD 4 per tissue area sum in samples stained with F4/80 was significantly lower in the ACEi group (1.9 × 10−4 ± 5.6 × 10−5) compared with both the ARB group (6.0 × 10−4 ± 2.7 × 10−4) and the infected control group (5.4 × 10−4 ± 3.9 × 10−4; p < 0.05; Figure 9C). The number of cells stained at POD 4 per tissue area sum in samples stained with MPO was significantly lower in the ACEi group vs. the ARB group (1.9 × 10−4 ± 4.2 × 10−5 vs. 3.8 × 10−4 ± 1.1 × 10−4, p < 0.05; Figure 10C). The current findings first showed that neither ACEi nor ARB had any direct anti-staphylococcal activity in vitro. The rationale behind this in vitro experiment was to show that any purported impact of ACEi or ARB impact on bacterial burden ex vivo or in vivo would not have been due to any anti-staphylococcal effects of the actual therapeutics themselves. This study also shows that blood from mice treated with ACEi demonstrated significantly decreased respiratory burst capacity as well as a significantly decreased ability to suppress S. aureus infection ex vivo as compared to ARB-treated or control mice blood. Histologic Analysis and Quantification of Monocyte and Neutrophil Infiltration Mice treated with an ACEi also had higher S. aureus burden in vivo as measured by bioluminescent signal throughout the entirety of the experiment compared to ARB-treated or infected control groups. September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org DISCUSSION Implant-associated SSI is a catastrophic complication. Moreover, staphylococcal species represent roughly two thirds of implant- associated SSI and often compound the issue due to multi-drug resistant phenotypes and a high propensity to form biofilms (65–67). Regardless of implant type, conservative, non-invasive treatment measures frequently fail to eradicate an infection and high-risk surgical intervention is often required (17, 19, 20). Thus, there is a significant and unmet need to identify modifiable risk factors that may optimize host immune protection against such infections. ACEi and ARB are amongst the most widely prescribed medications, particularly in the aging surgical Congruent with these findings, CFU burden measured on implants as well as paraspinal tissue in the ACEi group was significantly higher than either the ARB or infected control groups. Furthermore, viable S. aureus CFUs were identified in 37.5% of the implants in the ACEi group as compared to 25% in both the ARB and infected control group. Paralleling these microbiologic findings, mice treated with ACEi had significantly decreased monocyte and neutrophil infiltration to the paraspinal tissue on POD 4 compared with infected controls or ARB-treated September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org 8 ACE Inhibition Increases Staphylococcal Burden Trikha et al. FIGURE 9 | F4/80 stain representing monocyte infiltration to the paraspinal tissue in mice treated with an ARB (A) and an ACEi (B). (C) Mice treated with both an ARB and ACEi had significantly lower levels of monocyte infiltration than the control group at POD 1. Mice treated with an ACEi had significantly lower monocyte infiltration than mice treated with an ARB and the control group at POD 4 (C). *p < 0.05. FIGURE 9 | F4/80 stain representing monocyte infiltration to the paraspinal tissue in mice treated with an ARB (A) and an ACEi (B). (C) Mice treated with both an ARB and ACEi had significantly lower levels of monocyte infiltration than the control group at POD 1. Mice treated with an ACEi had significantly lower monocyte infiltration FIGURE 9 | F4/80 stain representing monocyte infiltration to the paraspinal tissue in mice treated with an ARB (A) and an ACEi (B). (C) Mice treated with both an ARB and ACEi had significantly lower levels of monocyte infiltration than the control group at POD 1. Frontiers in Immunology | www.frontiersin.org DISCUSSION Mice treated with an ACEi had significantly lower monocyte infiltration than mice treated with an ARB and the control group at POD 4 (C). *p < 0.05. The increased infectious burden that developed with ACEi treatment is consistent with emerging literature that the renin- angiotensin system, and in particular ACE-1, may play an important role in innate pathogen defense (34). Khan et al. (26) recently found that selective neutrophil underexpression of ACE- 1 markedly increased the susceptibility of mice to cutaneous methicillin resistant S. aureus (MRSA) infection, whereas mice. These data suggest that monocyte and neutrophil infiltration and/or functional ROS generation might be impaired by ACEi-related mechanism(s) and may be responsible for the increased susceptibility to S. aureus. Taken together, these findings suggest that perioperative ACEi treatment may represent a previously unappreciated risk factor to be considered prior to high-risk surgery such as implant instrumentation. September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org 9 ACE Inhibition Increases Staphylococcal Burden Trikha et al. FIGURE 10 | Myeloperoxidase stain representing neutrophil infiltration to the paraspinal tissue in mice treated with an ARB (A) and an ACEi (B). (C) Mice treated with both an ARB and ACEi had significantly lower levels of neutrophil infiltration than the control group at POD 1. Mice treated with an ACEi had significantly lower neutrophil infiltration than mice treated with an ARB and the control group at POD 4 (C). *p < 0.05. neutrophil ACE-1 overexpression reduced susceptibility. Similar However, other studies implicate the angiotensin type-1 FIGURE 10 | Myeloperoxidase stain representing neutrophil infiltration to the paraspinal tissue in mice treated with an ARB (A) and an ACEi (B). (C) Mice treated with both an ARB and ACEi had significantly lower levels of neutrophil infiltration than the control group at POD 1. Mice treated with an ACEi had significantly lower neutrophil infiltration than mice treated with an ARB and the control group at POD 4 (C). *p < 0.05. FIGURE 10 | Myeloperoxidase stain representing neutrophil infiltration to the paraspinal tissue in mice treated with an ARB (A) and an ACEi (B). (C) Mice treated with both an ARB and ACEi had significantly lower levels of neutrophil infiltration than the control group at POD 1. Mice treated with an ACEi had significantly lower neutrophil infiltration than mice treated with an ARB and the control group at POD 4 (C). *p < 0.05. DISCUSSION FIGURE 10 | Myeloperoxidase stain representing neutrophil infiltration to the paraspinal tissue in mice treated with an ARB (A) and an ACEi (B). (C) Mice treated with both an ARB and ACEi had significantly lower levels of neutrophil infiltration than the control group at POD 1. Mice treated with an ACEi had significantly lower neutrophil infiltration than mice treated with an ARB and the control group at POD 4 (C). *p < 0.05. However, other studies implicate the angiotensin type-1 receptor in diverse leukocyte functions including neutrophil chemotaxis (69) as well as natural killer cell proliferation and chemotaxis (70). Activated neutrophils are also a source of angiotensin-II generation, which are produced both ACE- dependently and independently (71). In our study, ARB-treated mice treated with ACEi showed significantly higher ex vivo neutrophil ACE-1 overexpression reduced susceptibility. Similar results have been reported with selective ACE-1 expression modulation in macrophages when challenged with both MRSA and Listeria monocytogenes (40). In both of these studies, the effect of ACE-1 to enhance pathogen clearance appeared angiotensin type-1 receptor independent, consistent with the majority of the results of the present study. September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org 10 ACE Inhibition Increases Staphylococcal Burden Trikha et al. CFU counts and significantly lower neutrophil and monocyte infiltration on POD 1 as compared to the infected control group. Therefore, while ARBs may influence pathogen defense-related pathways, these effects appear far less pronounced than ACE1 inhibition. Whether this distinction has clinical relevance warrants future investigation. Moreover, unlike well-established modifiable host risk factors such as obesity and diabetes, switching a patient from ACEi to ARB treatment may be relatively easy, safe, and inexpensive. To this end, it may be possible that certain patients undergoing elective surgery could safely be switched from an ACEi to an ARB during the perioperative period to minimize any purported infectious risk associated with the immunomodulatory effects of ACEi treatment. g There are limitations to this study. It is important to consider the clinical, translational limitations of this implant infection model as it is a simplification of the complex steps involved in human spinal implant surgery. Limitations to this model include being unilateral, involving only the posterior elements of the spine, and use of a single stainless-steel metal implant (53). Furthermore, only S. aureus Xen36 was used in this study. DISCUSSION Although this has been shown to be a well- validated, representative strain from a clinical isolate (57–59) the authors cannot extrapolate the findings reported to different staphylococcal strains or other microbial organisms. Given that this model allows for a safe, feasible, well-powered, and reproducible way to longitudinally quantify infection in vivo, these advantages are widely viewed to outweigh the accepted limitations. Another limitation to this study is the documented differences between murine and human physiology (38, 72, 73). Although doses of study therapeutics have been well-established and verified (40, 61), dose equivalent adjustments to humans as well as murine-specific pharmacological properties of these therapeutics are further limitations. However, mice treated with ACEi have been shown to respond similarly to humans in that they develop hypotension, increased levels of angiotensin I and decreased levels of ACE expression in myeloid cells (34, 38). Lastly, although this study showed that neither ACEi nor ARB exerted any direct antimicrobial effects on the growth potential of S. aureus in vitro, the potential direct effects of these study therapeutics on S. aureus metabolism, gene expression and/or virulence in vivo could also contribute to differences in outcomes observed. Preoperative host optimization is a key component to mitigating the risk of SSI and its devastating sequelae. The results of this study add to the growing body of literature suggesting that ACEi treatment may represent an under-appreciated, modifiable infectious risk factor. Future clinical studies investigating the relation between SSI and choice of antihypertensives are warranted to help develop guidelines regarding the perioperative use of ACEi. ETHICS STATEMENT The animal study was reviewed and approved by ARC (Animal Research Committee) at the University of California, Los Angeles (UCLA). The animal study was reviewed and approved by ARC (Animal Research Committee) at the University of California, Los Angeles (UCLA). FUNDING This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health, Award Number 5K08AR069112-01 and the H&H Lee Research Program. The research presented is the sole responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. AUTHOR CONTRIBUTIONS RT, DG, BK, ZM, TS, NC, MY, and NB contributed to conception and design of the study. All experiments were performed by RT, DG, BK, ZM, TS, TO, AC, CM, DL, NC, AS, and NB. Mouse surgical procedures were performed by RT with assistance from DG, BK, ZM, TS, TO, AC, NC, and NB. NC performed the statistical analysis. RT, DG, TS, NC, and DL completed all reference formatting. Figure generation was done by RT, DG, BK, ZM, CM, and NB. RT, DG, BK, ZM, TS, NC, TO, AC, CM, DL, AS, MY, and NB all assisted in writing the first and all subsequent drafts of the manuscript. All authors read and approved the submitted manuscript The current findings provide ex vivo and in vivo evidence that perioperative ACEi treatment as compared to ARB treatment increases S. aureus burden in a manner that corresponds to a reduction in immune effector responses in a longitudinal murine implant infection model. These results in conjunction with the overall body of literature on ACEi immunomodulation suggest that perioperative ACEi treatment could pose additional infectious risks to patients. It is, however, important to consider the balance between any purported immunomodulatory effects of ACEi and its protective cardiovascular effects. ACE inhibition has been shown to improve arterial compliance (74, 75) and, by inhibiting angiotensin II formation, decrease left ventricular hypertrophy, generalized coagulability and possibly systemic sympathetic activity in diabetic and hypertensive patients (76– 78). Therefore, the discontinuation of ACEi perioperatively is not without cardiologic risk. In patients lacking specific indications for particular antihypertensives, ACEi and ARB are often both considered first line therapy (79). Fortunately, ARB have been shown to exert protective cardiovascular effects to a similar, and perhaps greater, extent than ACEi (76, 78, 80, 81). Thus, the cardiovascular sequelae of switching a patient from an ACEi to an ARB perioperatively may not be substantially different. Frontiers in Immunology | www.frontiersin.org REFERENCES 18. 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Mechanisms by which angiotensin- converting enzyme inhibitors prevent diabetes and cardiovascular September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org 13 Trikha et al. ACE Inhibition Increases Staphylococcal Burden disease. Am J Cardiol. (2003) 91:30H−7H. doi: 10.1016/S0002-9149(03) 00432-6 80. Ferrari R, Boersma E. The impact of ACE inhibition on all-cause and cardiovascular mortality in contemporary hypertension trials: a review. Expert Rev Cardiovasc Ther. (2013) 11:705–17. doi: 10.1586/erc.13.42 75. Winer N, Weber MA, Sowers JR. The effect of antihypertensive drugs on vascular compliance. Curr Hypertens Rep. (2001) 3:297–304. doi: 10.1007/s11906-001-0092-9 81. Misra S, Stevermer JJ. ACE inhibitors and ARBs: one or the other–not both–for high-risk patients. J Fam Pract. (2009) 58:24–7. 81. Misra S, Stevermer JJ. ACE inhibitors and ARBs: one or the other–not both–for high-risk patients. J Fam Pract. (2009) 58:24–7. 76. Klein IH, Ligtenberg G, Oey PL, Koomans HA, Blankestijn PJ. Enalapril and losartan reduce sympathetic hyperactivity in patients with chronic renal failure. J Am Soc Nephrol. (2003) 14:425–30. doi: 10.1097/01.ASN.0000045049. 72965.B7 Conflict of Interest: NB and MY have or may receive grant support or related benefits from the National Institutes of Health. MY receives grant support from the U.S. Department of Defense, and is founder and shareholder of NovaDigm Therapeutics, Inc., which develops novel vaccines and immunotherapeutics targeting infection. 77. Schmieder RE, Hilgers KF, Schlaich MP, Schmidt BM. Renin- angiotensin system and cardiovascular risk. Lancet. (2007) 369:1208–19. doi: 10.1016/S0140-6736(07)60242-6 The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 78. Tsuda K. Renin-Angiotensin system and sympathetic neurotransmitter release in the central nervous system of hypertension. Int J Hypertens. (2012) 2012:474870. doi: 10.1155/2012/474870 79. Whelton PK, Carey RM, Aronow WS, Casey DE Jr, Collins KJ, Dennison Himmelfarb C, et al. 2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA guideline for the prevention, detection, evaluation, and management of high blood pressure in adults: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. Hypertension. (2018) 71:1269–324. doi: 10.1161/HYP.0000000000 000075 79. Frontiers in Immunology | www.frontiersin.org September 2020 | Volume 11 | Article 1919 REFERENCES Whelton PK, Carey RM, Aronow WS, Casey DE Jr, Collins KJ, Dennison Himmelfarb C, et al. 2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA guideline for the prevention, detection, evaluation, and management of high blood pressure in adults: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. Hypertension. (2018) 71:1269–324. doi: 10.1161/HYP.0000000000 000075 79. Whelton PK, Carey RM, Aronow WS, Casey DE Jr, Collins KJ, Dennison Himmelfarb C, et al. 2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Copyright © 2020 Trikha, Greig, Kelley, Mamouei, Sekimura, Cevallos, Olson, Chaudry, Magyar, Leisman, Stavrakis, Yeaman and Bernthal. 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. September 2020 | Volume 11 | Article 1919 Frontiers in Immunology | www.frontiersin.org 14
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Renal function after liver transplantation: Real-world experience with basiliximab induction and delayed reduced-dose tacrolimus
Digestive and Liver Disease/Digestive and liver disease
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Renal function after liver transplantation: Real-world experience with basiliximab induction and delayed reduced-dose tacrolimus Cederborg, Anna; Noren, Asa; Barten, T.R.M.; Lindkvist, Bjorn; Bennet, William; Herlenius, Gustaf; Marschall, Hanns-Ulrich; Aberg, Fredrik 2022, Article / Letter to editor (Digestive and Liver Disease, 54, 8, (2022), pp. 1076-1083) Doi link to publisher: https://doi.org/10.1016/j.dld.2021.12.006 Renal function after liver transplantation: Real-world experience with basiliximab induction and delayed reduced-dose tacrolimus Cederborg, Anna; Noren, Asa; Barten, T.R.M.; Lindkvist, Bjorn; Bennet, William; Herlenius, Gustaf; Marschall, Hanns-Ulrich; Aberg, Fredrik 2022, Article / Letter to editor (Digestive and Liver Disease, 54, 8, (2022), pp. 1076-1083) Doi link to publisher: https://doi.org/10.1016/j.dld.2021.12.006 ∗Corresponding author at: Mag- och tarmmottagningen, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden. E-mail address: anna.cederborg@gu.se (A. Cederborg). a r t i c l e i n f o Conclusion: Basiliximab induction with delayed reduced-dose tacrolimus is associated with less kidney injury when compared to standard-dose tacrolimus, without increased risk of rejection, graft loss or death. Results: Mean mGFR was similar between groups at wait-listing (85.3 vs 84.1 ml/min/1.73m ², p = 0.60), but higher in the revised treatment group at 3 (56.8 vs 63.4 ml/min/1.73m ², p = 0.004) and 12 months post-transplant (60.9 vs 69.7 ml/min/1.73m ², p < 0.001); this difference remained after correcting for mul- tiple confounders and was independent of pre-transplant mGFR. In the revised treatment group, biopsy proven acute rejection rate was lower (38% vs. 21%, p < 0.001), and graft-survival better ( p = 0.01). Conclusion: Basiliximab induction with delayed reduced-dose tacrolimus is associated with less kidney injury when compared to standard-dose tacrolimus, without increased risk of rejection, graft loss or death. © 2021 The Authors. Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana Thi i ti l d th CC BY li (htt // ti /li /b / © 2021 The Authors. Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) icity is controversial, but is thought to result from a combination of CNI-associated hemodynamic changes and a direct toxic effect [6] . Renal function after liver transplantation: Real-world experience with basiliximab induction and delayed reduced-dose tacrolimus Renal function after liver transplantation: Real-world experience with basiliximab induction and delayed reduced-dose tacrolimus Anna Cederborg a , ∗, ˚Asa Norén b , Thijs Barten c , Björn Lindkvist a , William Bennet b , Gustaf Herlenius b , Maria Castedal b , Hanns-Ulrich Marschall a , Fredrik ˚Aberg b , d a Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, and Department of Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden b Transplant Institute, Sahlgrenska University Hospital and Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden c Department of Gastroenterology and Hepatology, Radboud University Medical Center, Nijmegen, the Netherlands d Transplantation and Liver Surgery Clinic, Helsinki University Hospital and University of Helsinki, Finland a r t i c l e i n f o Article history: Received 10 September 2021 Accepted 9 December 2021 Available online 26 December 2021 Keywords: Clinical research Hepatology Immunosuppression Kidney injury Article history: Received 10 September 2021 Accepted 9 December 2021 Available online 26 December 2021 Keywords: Clinical research Hepatology Immunosuppression Kidney injury Article history: Received 10 September 2021 Accepted 9 December 2021 Available online 26 December 2021 Keywords: Clinical research Hepatology Immunosuppression Kidney injury Background: Routine use of delayed reduced-dose calcineurin-inhibitor treatment with induction im- munosuppression in liver transplantation to minimize post-operative kidney injury is still scarce. Background: Routine use of delayed reduced-dose calcineurin-inhibitor treatment with induction im- munosuppression in liver transplantation to minimize post-operative kidney injury is still scarce. Aim: To evaluate real-world experience of basiliximab induction with delayed reduced-dose tacrolimus. Methods: In a retrospective cohort study, kidney function was evaluated pre- and postoperatively by mea- sured glomerular filtration rate (mGFR). Adult patients undergoing liver transplantation between 20 0 0 and 2017 were divided into a conventional treatment group (immediate-introduction of tacrolimus, tar- get trough levels 10–15 ng/mL, and corticosteroids, n = 203) and a revised treatment group (basiliximab induction, reduced-dose tacrolimus, target through levels 5–8 ng/mL, delayed until day three, and my- cophenolate mofetil 20 0 0 mg/day, n = 343). Aim: To evaluate real world experience of basiliximab induction with delayed reduced dose tacrolimus. Methods: In a retrospective cohort study, kidney function was evaluated pre- and postoperatively by mea- sured glomerular filtration rate (mGFR). Adult patients undergoing liver transplantation between 20 0 0 and 2017 were divided into a conventional treatment group (immediate-introduction of tacrolimus, tar- get trough levels 10–15 ng/mL, and corticosteroids, n = 203) and a revised treatment group (basiliximab induction, reduced-dose tacrolimus, target through levels 5–8 ng/mL, delayed until day three, and my- cophenolate mofetil 20 0 0 mg/day, n = 343). Results: Mean mGFR was similar between groups at wait-listing (85.3 vs 84.1 ml/min/1.73m ², p = 0.60), but higher in the revised treatment group at 3 (56.8 vs 63.4 ml/min/1.73m ², p = 0.004) and 12 months post-transplant (60.9 vs 69.7 ml/min/1.73m ², p < 0.001); this difference remained after correcting for mul- tiple confounders and was independent of pre-transplant mGFR. In the revised treatment group, biopsy proven acute rejection rate was lower (38% vs. 21%, p < 0.001), and graft-survival better ( p = 0.01). Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Digestive and Liver Disease 54 (2022) 1076–1083 Contents lists available at ScienceDirect https://doi.org/10.1016/j.dld.2021.12.006 1590-8658/© 2021 The Authors. Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) ed by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. This is an open access article under the CC BY license 4 0/) 1. Introduction Chronic renal failure is an important factor affecting outcome after liver transplantation (LT), present in 15–30% of patients af- ter 5 years [ 1 , 2 ]. Post-transplant kidney function evaluated at 1 year, or as early as at 3 months, is associated with long-term renal outcome [3–5] . Several pre-, intra-, and post-LT factors are known to affect kidney function, and immunosuppressive treatment with calcineurin inhibitors (CNI) has been associated with both acute and chronic kidney injury [6] . A dose-dependent afferent arteriolar vasoconstriction is the main driver of acute CNI nephrotoxicity and can be ameliorated by CNI dose reduction. Chronic CNI nephrotox- Induction immunosuppression has been shown to reduce acute cellular rejection, i. e. T-cell-mediated rejection, in kidney trans- plantation [7] but in LT, with a lower incidence of acute cellular rejection, the benefit was initially not as clear [8] . In 2010, Cai et al. showed that induction therapy in LT was related to improved graft and patient survival up to 5 years post-LT [9] . The only non- depleting induction therapy (NDI) currently on the market is basil- iximab, a chimeric anti-CD25 (IL-2 receptor) monoclonal antibody that selectively targets activated T-cells with a sustained effect for 1–2 months after administration [10] . In the early 20 0 0s, several single-center clinical trials suggested that NDI therapy and delayed introduction of CNI, or immediate low-dose CNI, in patients with pre-operative kidney dysfunction, A. Cederborg, ˚A. Norén, T. Barten et al. Digestive and Liver Disease 54 (2022) 1076–1083 The conventional treatment group included patients undergoing LT between 20 0 0 and 20 07 receiving TAC (immediate release for- mula) and prednisolone from POD 0 with TAC trough levels of 10– 15 ng/mL in the first 2 months and thereafter 5–10 ng/mL. Pred- nisolone was initiated at 200 mg/day, and then tapered stepwise to 20 mg/day on POD 4 and to 5 mg/day at 3 months. Mycophenolate mofetil (MMF) was only added in case of impaired renal function or in case of graft rejection. In the transitional period of 2008– 2009, the patients undergoing LT received an immunosuppressive protocol according to the clinician ´s judgement and could therefore receive the conventional protocol or induction therapy with NDI. The protocol for CNI use under NDI was not standardized during this time period. Therefore, we excluded these patients from the analysis. 2.6. Statistical analysis Differences in baseline characteristics between patients in the study groups were analyzed with independent or paired samples t -test for continuous variables, if normally distributed, or Mann- Whitney U test if non-normally distributed, and with the Chi- squared test for categorical variables. The association between treatment protocol and renal outcomes was assessed by univari- ate and multivariable linear regression analyses. Factors associated with mGFR at 12 months post-LT with a P-value < 0.1 in univari- ate linear regression (Supplementary Table 1) were adjusted for as confounders in multivariable linear regression analysis (model 1 1. Introduction The revised treatment group included patients undergoing LT in 2010–2017 receiving NDI (basiliximab) administered 20 mg intravenously on POD 0 and 4. Standard protocol consisted of TAC (immediate release formula), initiated on POD 3, with trough levels of 5–8 ng/mL for 3 months, and 3–5 ng/mL thereafter, and MMF 10 0 0 mg twice daily, with dose-reduction in case of side-effects. All patients also received 10 0 0 mg of methylprednisolone intraop- eratively. In primary sclerosing cholangitis and autoimmune hep- atitis, the maintenance protocol also included oral steroids, initi- ated at 20 mg of prednisolone on POD 1 and tapered stepwise to 5 mg after 3 months. All other patients were steroid free post-LT. improves post-operative renal function with a similar or lower in- cidence of acute cellular rejection [ 11 , 12 ]. Randomized clinical tri- als followed, using NDIs (daclizumab or basiliximab) with delayed reduced-dose CNI after LT, only including patients without signif- icant kidney dysfunction pre-operatively. Early kidney injury was reduced [13] and renal function remained the same [14] or im- proved at 1 year compared to standard-dose tacrolimus and cor- ticosteroids administered immediately post-LT. There were no ad- verse effects on rejection rates or patient survival [14–17] . Never- theless, reduced-dose CNI without delayed introduction failed to significantly reduce nephrotoxicity [15] . A later study, using NDI and delayed introduction of CNI in patients with early post-LT re- nal insufficiency could not show any association between the level of achieved post-LT renal recovery prior to delayed CNI initiation and renal outcomes [18] . In 2019 [19] , a study on patients with renal insufficiency pre-transplant or acute kidney injury on post- operative day one (POD 1) the renal sparing protocol using NDI and delayed introduction of CNI could not show a reduced cu- mulative probability of advanced (stage 4–5) chronic kidney dis- ease. These trials evaluated kidney function using serum creatinine or estimated glomerular filtration rate (eGFR), both of which have several known limitations in this context [20–23] . Also, the renal benefit observed seemed to be partly dependent on which specific equation was being used to estimate GFR [13–15] . Importantly, re- ports of post-trial real-world experience with these protocols are scarce, and the use of NDI with delayed-onset CNI is still uncom- mon [16] . 2.4. Evaluation of kidney function Pre- and post-operative direct measurements of GFR was per- formed by chrome-EDTA or iohexol-clearance at baseline (wait- listing), and 3- and 12-months post-LT. For patients on dialysis at 3- or 12-months post-LT, we set the mGFR to 5 ml/min/1,73m 2 at that time-point for the purpose of statistical analyses. 2.5. Study outcome The primary study outcome was mGFR at 12 months post-LT. Secondary outcomes were mGFR at 3 months after LT, the change in mGFR from baseline (wait-listing) to 3- and 12-months, biopsy- proven acute rejection (BPAR), as well as graft and patient survival. Both absolute and percentage changes in mGFR were analyzed. All acute rejection episodes were biopsy confirmed. Inclusion criteria were age 18 years and older, LT between 20 0 0 and 2017 at Sahlgrenska University Hospital, Gothenburg, Swe- den. Exclusion criteria were missing data on wait-listing (base- line) mGFR or at both 3 and 12 months post-LT ( n = 207; pa- tients were included if only either 3 or 12 months mGFR was miss- ing), split or reduced graft or auxiliary LT ( n = 73), ABO incom- patibility ( n = 35), multi-visceral transplantation ( n = 14), domino LT ( n = 14), combined liver and kidney/heart/lung transplantation ( n = 14) or prior non-liver transplantation ( n = 6). 2.1. Study population This was a retrospective single-center cohort study of patients who underwent LT in Gothenburg, Sweden, in 20 0 0–2017, when all organ donations were after brain death. Patients were identi- fied using the Nordic Liver Transplant Registry (NLTR), which keeps record of all LT patients in the Nordic countries. The study was ap- proved by the regional ethical review board in Gothenburg (diary number 048–13). 2.3. Data collection From the NLTR and patient records, we collected data on serum creatinine and bilirubin, international normalized ratio (INR), mGFR, model for end-stage liver disease (MELD) score, body mass index (BMI), all from the time of wait-listing (baseline) and the day of transplantation, and cold ischemia time (CIT) and intra- operative blood loss. In Gothenburg, the immunosuppression protocol was changed in 2010 for all LT patients to a regimen with routine basiliximab induction and delayed-onset of reduced-dose tacrolimus (TAC). We have a long tradition of performing routine direct measurements of GFR by chrome-EDTA or iohexol-clearance [ 24 , 25 ] in all trans- planted patients both before and after LT. In this study we report the effects on kidney function, rejec- tion episodes, and survival rates of our real-world experience with basiliximab induction and delayed-onset reduced-dose TAC given routinely to all patients undergoing LT regardless of pre-transplant kidney function. We compare this protocol to the previous one with immediate standard-dose TAC and adjust for multiple con- founders. 2. Methods We also calculated the estimated GFR (eGFR) at baseline by Cockcroft-Gault equation [26] and the Modification of Diet in Renal Disease equation [27] (excluding race). 2.2. Treatment regimens The patients were grouped according to the treatment proto- col used at the time of transplantation into either a conventional group or revised group. 1077 A. Cederborg, ˚A. Norén, T. Barten et al. Digestive and Liver Disease 54 (2022) 1076–1083 Fig. 1. Flowchart of patients included from the Nordic Liver Transplant Registry (NLTR) allocated in groups according to the immunosuppression protocol implemented at the time of liver transplantation. Conventional treatment group (20 0 0–20 07), tacrolimus and prednisolone from POD 0, revised treatment group (2010–2017) receiving induction therapy with basiliximab in combination with reduced and delayed dose tacrolimus and MMF and the transitional period (20 08–20 09) in which patients received either the conventional or the revised treatment according to the handling physician’s discretion. mGFR = measured Glomerular Filtration rate, POD = post-operative day, MMF = Mycophenolate mofetil. A. Cederborg, ˚A. Norén, T. Barten et al. Digestive and Liver Disease 54 (2022) 1076–1083 Fig. 1. Flowchart of patients included from the Nordic Liver Transplant Registry (NLTR) allocated in groups according to the immunosuppression protocol implemented at the time of liver transplantation. Conventional treatment group (20 0 0–20 07), tacrolimus and prednisolone from POD 0, revised treatment group (2010–2017) receiving induction therapy with basiliximab in combination with reduced and delayed dose tacrolimus and MMF and the transitional period (20 08–20 09) in which patients received either the conventional or the revised treatment according to the handling physician’s discretion. mGFR = measured Glomerular Filtration rate, POD = post-operative day, MMF = Mycophenolate mofetil. group) and 2010–2013 and 2014–2017 (revised group) to examine whether there was a progressive change in kidney outcomes over time within each study group. adjustment). To limit collinearity, among variables with a Pearson correlation coefficient of > 0.7, we chose the variable judged to be clinically more important. Another multivariable analysis was per- formed with adjustment for clinical confounders, i.e. factors that have been associated with kidney function after LT in previous studies [28–33] (model 2 adjustment); these comprised recipient age and sex, donor age, donor BMI, alcohol-related liver disease or hepatitis C (HCV) as primary indication for LT, mGFR at baseline, intraoperative blood loss, CIT, difference ( ࢞) between serum crea- tinine at baseline and on the day of LT, serum bilirubin and INR on the day of LT, time on waiting list and hemodialysis prior to LT [31–33] . A p-value < 0.05 was considered statistically significant. 2.2. Treatment regimens Data were analyzed with IBM SPSS version 26. 3. Results Overall, 990 patients underwent LT during 20 0 0–2017. After applying the exclusion criteria, 627 patients were included, 203 and 343 patients in the conventional treatment and revised treat- ment groups, respectively. Furthermore, the patients transplanted in the transitional period were excluded (81 patients) due to non- standardized immunosuppression protocol. The flow of patients is outlined in Fig. 1 . Subgroup analyses were performed by baseline mGFR ( < vs. ≥60 ml/min/1.73m ² [34] ), MELD score ( < vs. ≥15 and < vs. ≥25 at LT day), HCV (yes vs. no), and intraoperative bleeding ( < vs. ≥40 0 0 mL). Patient and graft survival rates were evaluated with Kaplan-Meier analysis and log rank tests. As outcome differences between the groups might be confounded by a time-effect, we an- alyzed the possible time-effect on kidney outcomes. We divided calendar-time into years 20 0 0–20 03 and 20 04–20 07 (conventional Baseline recipient and donor demographics and characteristics are outlined in Table 1 . No difference was found in mean base- line mGFR between the conventional and revised treatment groups (85.3 and 84.1 ml/min/1.73 m ², respectively, P = 0.60). In the conventional treatment group, compared to the revised treatment 1078 Digestive and Liver Disease 54 (2022) 1076–1083 A. Cederborg, ˚A. Norén, T. Barten et al. Table 1 Baseline recipient and donor characteristics. Results are presented as mean ( ±SD) or N (%) unless otherwise specified. 3. Results Conventional treatment ( N = 203) Revised treatment ( N = 343) P-value Age, years 51 (11) 54 (12) 0.006 Male 139 (68.5%) 232 (67.6%) 0.84 BMI, kg/m 2 25.2 (4.5) 26.5 (4.8) 0.004 Donor age, years 49 (17) 57 (16) < 0.0001 Donor type N (% living) 5 (2.5%) 1 (0.3%) 0.02 Donor BMI, kg/m 2 24.4 (3.4) 26.1 (4.5) < 0.0001 mGFR at baseline, ml/min/1.73m 2 85.3 (28.9) 84.1 (26.1) 0.60 ≥90 95 (46.8%) 152 (44.3%) 0.57 89–60 69 (34.0%) 128 (37.3%) 0.43 59–30 32 (15.8%) 57 (16.6%) 0.79 < 30 7 (3.4%) 6 (1.7%) 0.21 eGFR at baseline, ml/min (CG) 95.8 (39.9), N = 161 108.2 (46.2), N = 343 0.004 eGFR at baseline, ml/min/1.73m 2 (MDRD) 80.8 (35.4), N = 200 87.6 (34.1), N = 343 0.03 Intraoperative blood loss, liters a 5.0 (2.5, 8.4) 2.4 (1.1, 4.2) < 0.0001 Cold ischemia time, hours a 9.0 (7.3, 11.5) 7.2 (6.1, 9.2) < 0.0001 Creatinine LT-day , μmol/L 95.1 (49.8) 87.3 (49.2) 0.08 ࢞Creatinine baseline Creatinine LT-day , μmol/L b 1.6 (42.2) 6.5 (41.1) 0.19 Bilirubin LT-day , μmol/L b 90.4 (120) 89.8 (142) > 0.99 INR LT-day b 1.4 (0.4) 1.6 (0.8) 0.001 Hemodialysis LT-day 3 (1.5%) 5 (1.5%) 0.93 Waiting list time, days a 29.0 (13, 67) 57 (23.0, 121) < 0.0001 MELD score baseline a 13.5 (10.6, 17.9) 13.2 (9.4, 17.8) 0.38 MELD score LT-day a 14.7 (10.8, 19.5) 13.5 (10.1, 18.5) 0.31 Primary indication for LT: Acute liver disease 1 (1%) 8 (2%) 0.10 Metabolic liver disease 5 (3%) 12 (4%) 0.62 Cryptogenic cirrhosis 16 (8%) 27 (8%) > 0.99 Autoimmune liver disease 10 (5%) 17 (5%) > 0.99 HCV 37 (18%) 87 (25%) 0.055 Cholestatic liver disease 64 (32%) 85 (25%) 0.09 Alcohol-related liver disease 41 (20%) 82 (24%) 0.32 HCC c 31 (15%) 122 (36%) < 0.0001 Conventional treatment group (20 0 0–20 07), tacrolimus and prednisolone from POD 0, revised treatment group (2010–2017) receiving induction therapy with basiliximab in combination with reduced and delayed dose tacrolimus and MMF. 3. Results 57 y, p < 0.001), and donor BMI (24.4 vs. 26.1 kg/m 2 , p < 0.001) were lower, while intraoperative blood loss (median 5.0 vs. 2.3 L, p < 0.001) was higher and CIT longer (median 9.0 vs. 7.2 h, P < 0.001). Frequencies of hepatocellular carcinoma (HCC) (15% vs. 36%, p < 0.001) and HCV (18% vs. 25%, p = 0.04) were lower, although 30% vs. 83% ( p < 0.001) of patients with HCV also had HCC as primary indication for LT. Numbers of missing data are reported in Supplementary Table 2. By univariate linear regression analysis, factors associated with 12-months mGFR at the pre-selected p-value of < 0.1 were recipi- ent age, sex, alcohol-related liver disease, HCC, mGFR at baseline, intraoperative blood loss, CIT, creatinine on the day of LT, time on the waiting list, and hemodialysis prior to LT (Supplementary Ta- ble 1); these factors were adjusted for in model 1. In multivariable linear regression analysis adjusted for either model 1 or model 2 factors, baseline mGFR did not differ between groups, but accord- ing to both adjustment models mGFR were higher in the revised treatment group at 3- and 12-months post-LT ( Table 2 ). 3. Results p ( Q ) b ࢞Creatinine baseline - Creatinine LT-day ; median (IQR) μmol/L: 1( −4.5, 9) vs. 3( −5, 11.3), p = 0.403, Bilirubin LT; median (IQR) μmol/L: 49 (21, 110) vs. 33 (18, 91), p = 0.049, INR LT-day ; median (IQR): 1.3 (1.1, 1.5) vs. 1.4 (1.2, 1.7), p = 0.09. c patients with HCC may also have another etiology of liver disease. both significantly lower in the revised treatment group ( Table 2 ). There was no difference in the need for hemodialysis between the conventional and revised treatment group at 3- (4 [2%] vs 3 [0.9%], p = 0.19) or 12-months (2 [1%] vs 1 [0.3%], p = 0.27) post-LT. How- ever, the frequency of mGFR < 30 ml/min/1.73m 2 was higher in the conventional treatment group at both 3- (19 [11.7%] vs 16 [5.1%], p = 0.009) and 12-months (15 [8.6%] vs 9 [2.9%], p = 0.006) post- LT. group, mean recipient age (51 vs. 54 y, p = 0.006), recipient BMI (25.2 vs. 26.5 kg/m 2 , p = 0.004), donor age (49 vs. 57 y, p < 0.001), and donor BMI (24.4 vs. 26.1 kg/m 2 , p < 0.001) were lower, while intraoperative blood loss (median 5.0 vs. 2.3 L, p < 0.001) was higher and CIT longer (median 9.0 vs. 7.2 h, P < 0.001). Frequencies of hepatocellular carcinoma (HCC) (15% vs. 36%, p < 0.001) and HCV (18% vs. 25%, p = 0.04) were lower, although 30% vs. 83% ( p < 0.001) of patients with HCV also had HCC as primary indication for LT. Numbers of missing data are reported in Supplementary Table 2. both significantly lower in the revised treatment group ( Table 2 ). There was no difference in the need for hemodialysis between the conventional and revised treatment group at 3- (4 [2%] vs 3 [0.9%], p = 0.19) or 12-months (2 [1%] vs 1 [0.3%], p = 0.27) post-LT. How- ever, the frequency of mGFR < 30 ml/min/1.73m 2 was higher in the conventional treatment group at both 3- (19 [11.7%] vs 16 [5.1%], p = 0.009) and 12-months (15 [8.6%] vs 9 [2.9%], p = 0.006) post- LT. group, mean recipient age (51 vs. 54 y, p = 0.006), recipient BMI (25.2 vs. 26.5 kg/m 2 , p = 0.004), donor age (49 vs. 3.1. Renal outcomes Mean mGFR at baseline was lower in both the conven- tional and the revised treatment groups compared to eGFR, using the Cockcroft-Gault equation (83.4 vs. 95, 83.7 vs. 108.2 ml/min/1.73m 2 , both p < 0.001). When using the Modifica- tion of Diet in Renal Disease equation, the mean mGFR was also lower than the mean eGFR in the revised treatment group (84.1 vs. 87.6 ml/min/1.73m 2 , p = 0.015), but no difference was found in the conventional group (84.9 vs. 80.8 ml/min/1.73m 2 , p = 0.075). 3. Results IQR = interquartile range, SD = standard deviation, baseline = during evaluation for liver transplantation, BMI = body mass index, mGFR = measured glomerular filtration rate, eGFR = estimated glomerular filtration rate, CG = Cockcroft-Gault equation, MDRD = Modification of Diet in Renal Disease-equation, LT-day = day of liver transplantation, MELD = model for end stage liver disease, Acute = viral and toxic acute liver failure, Metabolic = NAFLD, Wilson, Cryptogenic = cryptogenic cirrhosis, Autoimmune = autoimmune hepatitis, Cholestatic = primary biliary cirrhosis, primary sclerosing cholangitis, choledochal cyst, secondary biliary cirrhosis, HCV = , hepatitis C, HCC = Hepatocellular carcinoma. a presented as median (IQR) liters. b ࢞Creatinine baseline - Creatinine LT-day ; median (IQR) μmol/L: 1( −4.5, 9) vs. 3( −5, 11.3), p = 0.403, Bilirubin LT; median (IQR) μmol/L: 49 (21, 110) vs. 33 (18, 91), p = 0.049, INR LT-day ; median (IQR): 1.3 (1.1, 1.5) vs. 1.4 (1.2, 1.7), p = 0.09. c patients with HCC may also have another etiology of liver disease. Table 1 Baseline recipient and donor characteristics. Results are presented as mean ( ±SD) or N (%) unless otherwise specified. Conventional treatment group (20 0 0–20 07), tacrolimus and prednisolone from POD 0, revised treatment group (2010–2017) receiving induction therapy with basiliximab in combination with reduced and delayed dose tacrolimus and MMF. IQR = interquartile range, SD = standard deviation, baseline = during evaluation for liver transplantation, BMI = body mass index, mGFR = measured glomerular filtration rate, eGFR = estimated glomerular filtration rate, CG = Cockcroft-Gault equation, MDRD = Modification of Diet in Renal Disease-equation, LT-day = day of liver transplantation, MELD = model for end stage liver disease, Acute = viral and toxic acute liver failure, Metabolic = NAFLD, Wilson, Cryptogenic = cryptogenic cirrhosis, Autoimmune = autoimmune hepatitis, Cholestatic = primary biliary cirrhosis, primary sclerosing cholangitis, choledochal cyst, secondary biliary cirrhosis, HCV = , hepatitis C, HCC = Hepatocellular carcinoma. a presented as median (IQR) liters. presented as median (IQR) liters. b ࢞Creatinine baseline - Creatinine LT-day ; median (IQR) μmol/L: 1( −4.5, 9) vs. 3( −5, 11.3), p = 0.403, Bilirubin LT; median (IQR) μmol/L: 49 (21, 110) vs. 33 (18, 91), p = 0.049, INR LT-day ; median (IQR): 1.3 (1.1, 1.5) vs. 1.4 (1.2, 1.7), p = 0.09. c patients with HCC may also have another etiology of liver disease. 3.2. Subgroup analyses 3.2. Subgroup analyses Mean mGFR at both 3- (56.8 vs 63.4 ml/min/1.73m ²; p = 0.004) and 12-months post-LT (60.9 vs 69.7 ml/min/1.73m ²; p < 0.001) were significantly higher in the revised treatment group ( Fig. 2 ). The absolute decline in mean mGFR from baseline to 3 months ( −27.4 vs −20.9 ml/min/1.73m ²; p = 0.005), and from baseline to 12 months ( −25.1 vs −14.8 ml/min/1.73m ², p < 0.001) post-LT were In the subgroup of patients with baseline mGFR ≥60 ml/min/1.73m ², mean mGFR at both 3- (61.3 vs. 67.3 ml/min/1.73m 2 , p = 0.017) and 12-months (64.8 vs. 73.5 ml/min/1.73m 2 , p = 0.0 0 02) post-LT were higher in the revised treatment group. Similarly, in the subgroup of patients 1079 1079 A. Cederborg, ˚A. Norén, T. Barten et al. Digestive and Liver Disease 54 (2022) 1076–1083 Fig. 2. Comparison of unadjusted mean mGFR values between conventional and revised treatments groups at different time points. Time point zero is transplantation day. mGFR = measured glomerular filtration rate, LT = liver transplantation. Digestive and Liver Disease 54 (2022) 1076–1083 A. Cederborg, ˚A. Norén, T. Barten et al. Fig. 2. Comparison of unadjusted mean mGFR values between conventional and revised treatments groups at different time points. Time point zero is transplantation day. mGFR = measured glomerular filtration rate, LT = liver transplantation. 3.3. Effect of transplantation periods with mGFR < 60 ml/min/1.73m ², a higher mean mGFR was found in the revised treatment group (44.4 vs. 53.8 ml/min/1.73m 2 , p = 0.03) at 12 months post-LT ( Table 3 and Supplementary figure 1). When evaluated as percentage change in mean mGFR in the subgroup with baseline mGFR ≥60 ml/min/1.73m ², the decline was less pronounced from baseline to 3- and 12-months post-LT in the revised treatment group ( −34.9% vs −26.3%, and −30.8% vs 19.4%, respectively, both p < 0.001). However, a similar effect was not seen in the subgroup with baseline mGFR < 60 ml/min/1.73m ², neither at 3- nor at 12-months post-LT (8.9% vs −0.7%, p = 0.45; 5.8% vs. 17.5%. p = 0.36, respectively; Fig. 3 ). No significant difference in mGFR development from base- line was found within the conventional treatment group, when comparing the time periods 20 0 0–20 03 and 20 04–20 07, at 3- ( −27.4 vs. −27.3, p > 0.99) or 12-months post-LT ( −25.1 vs. −25.1, p > 0.99). The same was true within the revised treatment group when comparing the time periods 2010–2013 and 2014–2017 at 3- ( −19.7 vs. −22.0, p = 0.39) or 12-months post-LT ( −13.3 vs. −15.9, p = 0.32) (Supplementary figure 7). Therefore, no time-effect bias was seen within the study groups. In further subgroup analyses, the decrease in mGFR from base- line to 12 months post-LT was worse in the conventional treat- ment group, both when stratified by a MELD score of 15 (MELD < 15; −25.6% vs. −15.4%, p = 0.006, MELD ≥15; −25.2% vs. −9.3%, p = 0.03), MELD score below 25 ( −25.3% vs. −14.2%, p = 0.003), blood loss of less than 4 liters ( −25,8% vs. −11.6%, p = 0.006), and HCV (HCV, −28.2% vs. −14.8%, p = 0.02; no HCV, −23.2% vs. −12%, p = 0.004) as shown in Supplementary figures 2–5. 3.4. Survival Patient survival rates in the conventional versus the revised treatment group were 97% vs. 99% at 1 year and 84% vs. 87% at 5 years post-LT; Kaplan-Meier analysis showed no difference in pa- tient survival between groups ( p = 0.16) (Supplementary figure 8). The major causes of death within 10 years post-LT in the conven- tional and the revised treatment groups were cancer 49% vs. 61%, cardiovascular disease 11% vs. 12% and infection 8% vs. 6%. Graft survival rates were higher in the revised treatment group; 91% vs. 97% at 1 year and 75% vs. 84% at 5 years post-LT, p = 0.01 (Sup- plementary figure 9). After availability of direct-acting antiviral (DAA) treatment of HCV in 2013, 31 HCV patients were HCV-PCR-negative at LT and 56 were HCV-PCR-positive, of whom 13 received DAA therapy post-LT and 8 became negative by 12 months, while 48 were still HCV-PCR-positive. The decline in mGFR from baseline to 12 months post-LT was similar in HCV-negative patients and in pa- tients who became HCV-PCR-negative by 12 months ( −20.0 vs. −29.0 ml/min/1.73m ², p = 0.28), and in those who were still HCV-PCR-positive at 12 months ( −20.0 vs. −12.8 ml/min/1.73m ², p = 0.16), Supplementary Table 3. Successful DAA treatment be- fore LT had no effect on graft survival ( p = 0.22, Supplementary figure 6). 3.5. Biopsy-proven acute rejection Biopsy-proven acute rejection (BPAR) was diagnosed in 170 patients (31% of all patients) during follow-up, and 148 of these (87%) occurred within 12 months. BPAR was more frequent in the conventional treatment group at both 3- (37% vs. 14%, p < 0.001), 6 (38% vs. 18%, p < 0.001) and 12-months (38% vs. 21%, p < 0.001) post-LT. 1080 1080 Digestive and Liver Disease 54 (2022) 1076–1083 A. Cederborg, ˚A. Norén, T. Barten et al. Table 2 Comparison of mean mGFR (ml/min/1,73m 2 ) values between conventional and revised treatment groups at different time points. 3.5. Biopsy-proven acute rejection Unadjusted Conventional treatment mean ( ±SD) Revised treatment mean ( ±SD) difference (95%CI) p-value mGFR baseline 85.3 (28.9) 84.1 (26.1) 1.2 ( −3.5 to 6.0) 0.60 mGFR 3 months post-LT 56.8 (24.9) 63.4 (23.0) −6.6 ( −11.1 to −2.1) 0.004 mGFR 12 months post-LT 60.9 (23.3) 69.7 (22.5) −8.9 (13.1 to −4.6) < 0.0001 ࢞mGFR baseline to 3 months post-LT −27.4 (24.2) −20.9 (22.7) −6.4 ( −10.8 to-1.9) 0.005 ࢞mGFR baseline to 12 months post-LT −25.1 (24.7) −14.8 (22.9) −10.3 ( −14.7 to −5.9) < 0.0001 ࢞mGFR 3 months to 12 months post-LT 3.1 (16.5) 5.7 (15.1) −2.6 ( −5.8 to 0.6) 0.11 Model 1 a Conventional treatment mean (95% CI) Revised treatment mean (95% CI) mean difference (95%CI) p-value mGFR baseline 84.9 (81.1–88.7) 83.2 (80.6–85.8) 1.7 ( −3.1 to 6.5) 0.50 mGFR 3 months post-LT 55.4 (51.8–59.1) 63.7 (61.4–66.1) −8.3 ( −12.9 to-3.7) 0.0004 mGFR 12 months post-LT 59.6 (56.3–63.0) 69.7 (67.5–72.0) −10.1 ( −14.3 to −5.8) < 0.0001 ࢞GFR baseline to 3 months post-LT −28.1 ( −31.8 to −24.5) −19.8 ( −22.2 to −17.5) −8.3 ( −12.9 to −3.7) 0.0004 ࢞GFR baseline to 12 months post-LT −24.6 ( −28.0 to −21.3) −14.6 ( −16.8 to −12.3) −10.1 ( −14.3 to −5.8) < 0.0001 ࢞GFR 3 months to 12 months post-LT 4.9 (1.6–8.2) 4.9 (2.8–6.9) 0.06 ( −4.0 to 4.1) 0.98 Model 2 b mGFR baseline 81.8 (75.8–87.7) 83.4 (80.5–86.3) −1.6 ( −8.6 to 5.3) 0.64 mGFR 3 months post-LT 54.5 (49.5–59.4) 63.7 (61.4–65.9) −9.2 ( −14.8 to −3.7) 0.001 mGFR 12 months post-LT 55.8 (51.1–60.6) 69.4 (67.1–71.7) −13.6 ( −19.0 to −8.1) < 0.0001 ࢞GFR baseline to 3 months post-LT −28.9 ( −33.8 to −24.0) −19.7 ( −21.9 to −17.4) −9.2 ( −14.8 to −3.7) 0.001 ࢞GFR baseline to 12 months post-LT −27.1 ( −31.9 to −22.4) −13.6 ( −15.9 to −11.3) −13.5 ( −19.0 to −8.1) < 0.0001 ࢞GFR 3 months to 12 months post-LT 4.5 (0.1–8.9) 5.2 (3.3–7.1) −0.7 ( −5.6 to 4.2) 0.77 mGFR = measured glomerular filtration rate, baseline = during evaluation for liver transplantation, LT = liver transplantation, HCC = hepatocellular carcinoma, BMI = body mass index, ࢞= difference between two time points, INR = international normalized ratio. 3.5. Biopsy-proven acute rejection a Adjusted for statistical confounders ( P < 0.1 in univariate linear regression analysis): age, sex, alcoholic liver disease or HCC as primary indication for liver transplan- tation, mGFR at baseline, intraoperative blood loss, cold ischemia time, s-creatinine on the day of transplantation, time on waiting list and hemodialysis pre-LT. b Adjusted for clinical confounders identified from the literature: recipient age, sex, BMI, donor age, donor BMI, alcoholic liver disease or Hepatitis C as primary indication for liver transplantation, mGFR at baseline, intraoperative blood loss, cold ischemia time, ࢞s-creatinine (baseline-LT-day), s-bilirubin and INR on the day of liver transplantation, time on waiting list and hemodialysis pre-LT. min/1,73m 2 ) values between conventional and revised treatment groups at different time points. = measured glomerular filtration rate, baseline = during evaluation for liver transplantation, LT = liver transplantation, HCC = hepatoce ndex, ࢞= difference between two time points, INR = international normalized ratio. a Adjusted for statistical confounders ( P < 0.1 in univariate linear regression analysis): age, sex, alcoholic liver disease or HCC as primary indication for liver transplan- tation, mGFR at baseline, intraoperative blood loss, cold ischemia time, s-creatinine on the day of transplantation, time on waiting list and hemodialysis pre-LT. b Adjusted for clinical confounders identified from the literature: recipient age, sex, BMI, donor age, donor BMI, alcoholic liver disease or Hepatitis C as primary indication for liver transplantation, mGFR at baseline, intraoperative blood loss, cold ischemia time, ࢞s-creatinine (baseline-LT-day), s-bilirubin and INR on the day of liver transplantation, time on waiting list and hemodialysis pre-LT. a Adjusted for statistical confounders ( P < 0.1 in univariate linear regression analysis): age, sex, alcoholic liver disease or HCC as primary indication for liver transplan- tation, mGFR at baseline, intraoperative blood loss, cold ischemia time, s-creatinine on the day of transplantation, time on waiting list and hemodialysis pre-LT. b Adjusted for clinical confounders identified from the literature: recipient age, sex, BMI, donor age, donor BMI, alcoholic liver disease or Hepatitis C as primary indication for liver transplantation, mGFR at baseline, intraoperative blood loss, cold ischemia time, ࢞s-creatinine (baseline-LT-day), s-bilirubin and INR on the day of liver transplantation, time on waiting list and hemodialysis pre-LT. Fig. 3. Mean change in mGFR (percent) from baseline to 3 months post-LT and to 12 months post-LT in the conventional and revised treatment groups including subgroups of mGFR at baseline < 60 or ≥60 ml/min/1.73m2. 3.5. Biopsy-proven acute rejection mGFR = measured glomerular filtration rate, baseline = during evaluation for liver transplantation/wait-listing, LT = liver transplantation, CI = confidence interval. Fig. 3. Mean change in mGFR (percent) from baseline to 3 months post-LT and to 12 months post-LT in the conventional and revised treatment groups including subgroups of mGFR at baseline < 60 or ≥60 ml/min/1.73m2. mGFR = measured glomerular filtration rate, baseline = during evaluation for liver transplantation/wait-listing, LT = liver transplantation, CI = confidence interval. 4. Discussion Conse- quently, we consider this finding as strong evidence for the im- provement in kidney outcomes not being a mere time-effect, but rather due to the change in immunosuppression protocol. This is further supported by multivariable confounder adjustments. Although the advent of DAA for HCV has resulted in a decline in HCV as a primary indication for LT during more recent years, the proportion of patients with HCV increased in the revised treatment group. This was largely driven by HCV-related HCC. We confirmed the kidney benefit associated with the revised protocol in subgroup analysis by HCV status irrespective of DAA treatment or treatment response. In conclusion, this is to our knowledge the first large real-world experience evaluating routine NDI therapy with basiliximab and delayed-onset, reduced-dose TAC using measured glomerular filtra- tion rate to evaluate kidney function, both pre- and post-LT for all study patients. Our study shows that this immunosuppression pro- tocol is associated with less kidney impairment post-LT, both in patients with mGFR pre-LT < 60 and ≥60 ml/min/1.73m 2 , with a lower rate of BPAR and improved overall graft-survival. The strengths of our study are the real-world setting, relatively large sample size and use of the gold standard measurement of GFR to evaluate kidney function in all patients, both pre- and post- LT. Limitations of the study include the retrospective design and the lack of data on some risk factors, such as hypertension and di- abetes, for kidney impairment in the different treatment groups. In the revised treatment group, patients were significantly older and with a higher BMI at LT which, as risk factors for perioper- ative kidney injury, could bias findings towards the null hypoth- esis; nonetheless, a significantly higher mGFR post-LT was found and remained when adjusting for these confounders. A potential study limitation is the inability to evaluate the independent ef- fect of MMF on kidney function post-LT, the rates of BPAR or graft survival in this retrospective study. MMF was used in all patients in the revised treatment group but only in selected patients (usu- ally those with renal dysfunction) in the conventional group. An- other potential study limitation is the lack of data on the measured trough level of TAC post-LT. The trough levels aimed for at different time points are known, but the registries used do not contain data on the actual level reached for each individual patient. 4. Discussion tion, the risk of rejection, graft loss or death were not increased, and we did not find confounders based on MELD score or HCV status. The level of kidney function post-LT is associated with long- term survival [ 35 , 36 ], thus preventing loss of renal function is clinically important. This real-world experience using mGFR demonstrates that NDI therapy with basiliximab and delayed- onset reduced-dose TAC routinely in all liver transplanted pa- tients irrespective of pre-transplant kidney function, is associ- ated with a significantly reduced deterioration in kidney function when compared to standard-dose TAC, at both 3- and 12-months post-LT, even after correcting for multiple confounders. In addi- Furthermore, in the subgroup of patients with pre-LT mGFR ≥60 ml/min/1.73m 2 , the loss of renal function was smaller in the revised treatment group at both 3- and 12-months post-LT, regardless of whether change in mGFR was evaluated as abso- lute or percentage change. Among patients with pre-LT mGFR < 60 ml/min/1.73m 2 , kidney function improved from the pre-LT level in the revised treatment group compared to the conventional treatment group at 12 months post-LT. 1081 Digestive and Liver Disease 54 (2022) 1076–1083 A. Cederborg, ˚A. Norén, T. Barten et al. Table 3 Subgroup analysis of baseline mGFR below or above 60ml/min/1.73m 2 . Table 3 Subgroup analysis of baseline mGFR below or above 60ml/min/1.73m 2 . baseline mGFR < 60 Conventional treatment, n = 41 Mean ( ±SD) baseline mGFR < 60 Revised treatment, n = 67 Mean ( ±SD) mean diffence (95%CI) p-value mGFR baseline 44.8 (14.4) 47.2 (11.4) −2.4 ( −7.4 to 2.5) 0.34 mGFR 3 months post-LT 39.8 (13.7) 46 . 5 (19.3) −6.7 ( −14.2 to 0.8) 0.08 mGFR 12 months post-LT 44.4 (19.1) 53.8 (21.0) −9.5 ( −18.2 to 0.8) 0.03 baseline mGFR ≥60 Conventional treatment n = 162 Mean ( ±SD) baseline mGFR ≥60 Revised treatment n = 276 Mean ( ±SD) mean diffence (95%CI) p-value mGFR baseline 95.6 (21.8) 93.0 (20.1) 2.6 ( −1.5 to 6.6) 0.21 mGFR 3 months post-LT 61 . 3 (25.3) 67 . 3 (22.0) −6.0 ( −10.9 to −1.1) 0.017 mGFR 12 months post-LT 64 . 8 (22.5) 73 . 5 (21.2) −8.7 ( −13.2 to −4.2) 0.0002 mGFR = measured glomerular filtration rate, baseline = during evaluation for liver transplantation/wait-listing, LT = liver transplantation, SD = standard deviation, CI = con- fidence interval. 4. Discussion The results from the present study are in accordance with pre- vious randomized studies [ 15 , 17 ] reporting that NDI with delayed [17] and delayed reduced-dose TAC [ 13 , 15 ] reduces kidney im- pairment post-LT, as evaluated by estimated GFR (eGFR). How- ever, eGFR has numerous acknowledged limitations leading to both over- and underestimation of GFR. In the Respect trial [15] , the study findings depended in part on which equation was used to es- timate GFR. Accuracy of eGFR in patients with end-stage liver dis- ease depends on factors such as decreased skeletal muscle mass, decreased hepatic creatine synthesis and increased tubular cre- atinine secretion [ 21 , 22 , 37 , 38 ]. Moreover, the immunosuppressive medication used post-LT also affects serum creatinine and urea ni- trogen levels independent of GFR, thus reducing the accuracy of eGFR in the post-LT setting [ 23 , 38 , 39 ]. In accordance with previ- ous studies [13–15] , we also evaluated eGFR at baseline by both the Cockcroft-Gault and the Modification of Diet in Renal Disease equations. There was a significant difference in mean GFR at base- line, using both equations, which further supports the importance of correct measurement of kidney function. Furthermore, the administration of TAC is delayed until POD 3 in the revised treatment group allowing for no/lower blood concen- tration of TAC in the perioperative period, which is shown to be an important factor to reduce kidney injury [15] . Recent Italian recommendations propose the use of induction therapy in LT for critically ill patients, including patients with re- nal dysfunction, followed by MMF treatment and a reduction of early post-transplant CNI through levels to 3–5 ng/mL and, from 3 months onward, to 2–3 ng/mL [40] . Nonetheless, there is paucity of studies evaluating this recommendation, as previous studies mostly included patients without renal impairment. In our study, the difference between study groups could poten- tially be explained by a time-effect, as factors such as improve- ments in surgical techniques, increased focus on minimizing peri– operative bleeding, patient selection and so forth, likely evolved over time. However, we found no evidence of a time-effect within neither the conventional group nor within the revised group when stratifying these groups into smaller calendar-time periods. Funding Grants from the Swedish state under the agreement between the Swedish government and the county councils, ALF-agreement ( ALFGBG-717231 ) to HUM. ˚ F ˚A received a research grant from ”Stiftelsen för Transplantations- och Cancerforskning”. F ˚A received a research grant from ”Stiftelsen för Transplantations- och Cancerforskning”. Conflicts of interest None declared. 4. Discussion However, the routines for measuring and prescribing TAC, despite aiming for different trough levels, has remained unchanged over time and the sample size is relatively large, which should reduce potential bias. References [32] Asfandiyar S, Abouljoud M, Kim D, et al. Influence of hepatitis C on re- nal function after liver transplantation. Transplant Proc 2006;38(10):3643–5. doi: 10.1016/j.transproceed.2006.10.166 . [13] Yoshida EM, Marotta PJ, Greig PD, et al. Evaluation of renal function in liver transplant recipients receiving daclizumab (Zenapax), mycophenolate mofetil, and a delayed, low-dose tacrolimus regimen vs. a standard-dose tacrolimus and mycophenolate mofetil regimen: a multicenter randomized clinical trial. Liver Transpl 2005;11(9):1064–72. doi: 10.1002/lt.20490 . /j p [33] Durand F, Francoz C, Asrani SK, et al. Acute Kidney Injury After Liver Transplantation. Transplantation 2018;102(10):1636–49. doi: 10.1097/TP. 0 0 0 0 0 0 0 0 0 0 0 02305 . 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La semilla del humanismo. Moralidad y ontología fundamental en Heidegger
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La semilla del humanismo Moralidad y ontología fundamental en Heidegger* Recibido: abril 25 de 2014 | Aprobado: mayo 30 de 2014 Germán Darío Vélez López** gdvelez@eafit.edu.co Resumen A lo largo de todo el análisis preparatorio de la existencia humana (Dasein), Heidegger sostiene la firme convicción de la neutralidad moral de la interpretación fenomenológica. Sin embargo, al momento de determinar el sentido temporal del cuidado (Sorge) como ser del Dasein, parece como si Heidegger no resistiera más la presión del lector y se viera forzado a admitir que, en el paso de la primera a la segunda sección de Ser y tiempo un ideal de existencia, una forma peculiar de humanismo, se hiciera manifiesta y reclamara sus derechos. El propósito de este artículo consiste en mostrar una posible vía para explicar esta problemática continuidad entre ontología fundamental, moral y humanismo, mostrando cómo la finitud del Dasein determina un vínculo necesario entre estos niveles de interpretación de la vida humana. Palabras clave Ontología, apropiación, moral, humanismo, ética, existencia, Dasein. The seed of humanism: morality and fundamental ontology in Heidegger Abstract Throughout the preparatory analysis of human existence (Dasein), Heidegger holds the firm conviction of the moral neutrality of the phenomenological interpretation. However, in determining the temporal sense of care (Sorge) as the being of Dasein, Heidegger seems as if he did not resist the pressure of the reader anymore and was forced to admit that, in the transition from the first to the second section of Being and time, an ideal of existence, a particular form of humanism was made manifest and claim their rights. The purpose of this article is to show a possible way to explain this problematic continuity among fundamental ontology, morality and humanism, showing how the finitude of Dasein, determines a necessary link among these levels of interpretation of the human life. Key words Ontology, ownedness, moral, humanism, ethics, existence, Dasein. * Artículo derivado de la investigación: “La experiencia estética de la apropiación como aplicación de la hermenéutica literaria existencialmente fundada”, financiada por la Dirección de Investigaciones de la Universidad EAFIT. ** Doctor en Filosofía Contemporánea, Universidad París 1-Francia. Profesor titular, Departamento de Humanidades, Universidad EAFIT, MedellínColombia. Revista Co-herencia Vol. 11, No 20 Enero - Junio 2014, pp. 121-139. Medellín, Colombia (ISSN 1794-5887) 121 “Poco a poco se me ha ido manifestando qué es lo que ha sido hasta ahora toda gran filosofía: a saber, la auto-confesión de su autor y una especie de memoires [memorias] no queridas y no advertidas; asimismo, que las intenciones morales (o inmorales) han constituido en toda filosofía el auténtico germen vital del que ha brotado siempre la planta entera. De hecho, para aclarar de qué modo han tenido lugar propiamente las afirmaciones metafísicas más remotas de un filósofo es bueno (e inteligente) comenzar siempre preguntándose: ¿a qué moral quiere esto (quiere él-) llegar?” Nietzsche, Más allá del bien y del mal. 1. La raíz de la raíz Quizás las afirmaciones más remotas de toda la analítica existencial heideggeriana, es decir, de la ontología fundamental, de la más originaria y al mismo tiempo remota ontología, son aquellas con las cuales se caracteriza al ente temático en su especificidad ónticoontológica. El carácter remoto de las mismas nos es comunicado por el propio Heidegger: el Dasein es ónticamente el más próximo, pero ontológicamente el más lejano (Heidegger, 1998: 40/16, 69/43)1. El Dasein es ontológicamente el ente más remoto, y las afirmaciones a propósito de su ser son por ello, las afirmaciones más remotas. Heidegger no las llama afirmaciones metafísicas. Elige un título llamativo y enigmático: indicaciones formales [formalen Anzeige] del Dasein2. Las indicaciones formales son las articulaciones proposicionales más remotas de los “conceptos” ontológicos más fundamentales, es decir, de aquellos en los cuales se determina el modo de ser del Dasein, y que Heidegger denomina “existenciales” [Existenzialien] los cuales, a 1 2 Usaremos el esquema de numeración A/B, donde “A” corresponde al número de página de la edición en español y “B” corresponde a la página de la edición alemana que se usa generalmente como referencia. A partir de la publicación de los seminarios dictados por Heidegger en los años 20, se ha hecho cada vez más notoria la importancia de la “indicación formal” como régimen expresivo de la vida fáctica y como modo de auto-interpretación pre-ontológica. Al respecto conviene consultar la entrada que le dedica J. A. Escudero en su diccionario de Heidegger: “La indicación formal es uno de los elementos esenciales del método hermenéutico-fenomenológico. El término en cuestión aparece inicialmente en las lecciones del semestre de invierno de 1919-1920 y es tematizado en las lecciones del semestre de invierno de 1920-1921, Introducción a la fenomenología de la religión. […] Los indicadores formales expresan conceptualmente la comprensión que la vida fáctica tiene de sí misma; también podría decirse que son categorías fundamentales de la vida […] Los indicadores formales surgen de la necesidad de la vida fáctica de interpretarse a sí misma, mostrando al individuo la situación concreta en que vive. Los indicadores formales son conceptos vacíos y básicamente dinámicos que inician, de manera metódica, una actividad hermenéutica que suministra a la filosofía un punto de partida.” (Escudero, 2009: 90) La semilla del humanismo y ontología fundamental en Heidegger 122 Moralidad Germán Darío Vélez López su vez, “[S]e los debe distinguir rigurosamente de las determinaciones del ser del ente que no tiene la forma de ser del Dasein, a las que damos el nombre de categorías” (70/44). Los indicadores formales fueron ampliamente utilizados por Heidegger en los seminarios que antecedieron a la publicación de Ser y tiempo, su uso en esta obra es, aunque limitado, eficaz. Así, por ejemplo, al inicio del cuarto capítulo, dedicado a investigar quién es cotidianamente el Dasein, Heidegger aclara: “La respuesta a la pregunta acerca de quién es este ente (el Dasein), ya fue aparentemente dada con las indicaciones formales de las determinaciones fundamentales del Dasein (cf. §9). El Dasein es el ente que soy cada vez yo mismo; su ser es siempre el mío. Esta determinación indica [zeigt an] una estructura ontológica, pero sólo eso.” (140/114) Tomemos, pues, por afirmaciones metafísicas remotas las indicaciones formales del Dasein, con las cuales se abre la analítica en su primer capítulo, tal como retrospectivamente alude Heidegger a ellas al inicio del capítulo 4: 1. La “esencia” de este ente consiste en su tener-que-ser. […] La “esencia” del Dasein consiste en su existencia. […] 2. El ser que está en cuestión para este ente en su ser es cada vez el mío. […] Ambos modos de ser, propiedad e impropiedad –estas expresiones han sido adoptadas terminológicamente en su estricto sentido literal–, se fundan en que el Dasein en cuanto tal está determinado por el ser-cada-vez-mío. (67-68/42). El Dasein se comporta con respecto a su ser como con respecto a su posibilidad más propia: puede ser su ser o puede no serlo. Es decir, puede elegirse a sí mismo o puede no hacerlo. En este sentido, puede serle propio su ser, o puede no serle propio. El Dasein se gana o se pierde, porque el ser que está en juego en su existencia es el suyo. Pero quizás hemos enumerado muy rápidamente esta serie de indicaciones formales. Por ejemplo, hemos dicho: “puede ser su ser o puede no ser su ser”. Para el entendimiento corriente, mundano, en absoluto remoto, lo primero más que posible es necesario y lo segundo no es siquiera posible. ¿O es que afirmar que el Dasein puede no ser su ser no es caer en una contradicción? ¿Cómo puede el Dasein, siendo, no ser su ser? O estamos ante la más remota afirmación filosófica o estamos ante la más inmediata contradicción. Pero cabe una tercera opción: tratándose del Dasein, la afirmación “puede no ser su Revista Co-herencia Vol. 11, No 20 Enero - Junio 2014, pp. 121-139. Medellín, Colombia (ISSN 1794-5887) 123 ser” es válida. La analítica existencial abre un campo proposicional en el que entre “el ser es y el no ser no es” cabe afirmar “su ser no es… suyo”. Al Dasein, y sólo a él, su ser puede serle indiferente o no indiferente. Comportándose con respecto a sí mismo como con respecto a su posibilidad más propia, puede llegar a preocuparse por su ser o puede querer olvidarse de él y huir ante él. Volvamos por un momento sobre el fragmento de Más allá del bien y del mal que citamos al inicio. Nietzsche propone de manera sucinta una pauta hermenéutica: para determinar el sentido de una afirmación, nos indica, debemos interrogar el texto con respecto a la moral. Si podemos establecer a qué moral quiere llegar el filósofo podremos determinar adecuadamente el sentido de las afirmaciones metafísicas más remotas3. Admitamos, a continuación, algo que hace parte del planteamiento nietzscheano con respecto a la moral: que una moral es una forma de vida y que esa forma de vida está asociada a una tabla de valores, entendiendo estos últimos de un modo preciso. Los valores son definidos por Nietzsche en la nota 11[73] de los fragmentos póstumos de 1887-1888, como puntos de vista, como perspectivas de la vida: “El punto de vista del “valor” es el punto de vista de las condiciones de conservación y de aumento con respecto a formaciones complejas de relativa duración de la vida en el seno del devenir” (Nietzsche, 2008: 388). Los valores son, pues, condiciones de conservación y aumento de un tipo de vida. Según esto, preguntar con respecto a las afirmaciones metafísicas “¿a qué moral quiere llegar?” equivale a preguntar a qué tipo de vida quiere llegar, qué tipo de vida quieren conservar, promover y fortalecer. Demos un paso más: la vida que tematiza la analítica existencial es la existencia humana. La vida del Dasein, su ser propio, es su existencia. Según esto, preguntar “¿a qué vida quiere llegar?” equivale a preguntar “¿a qué existencia quiere llegar?”. ¿Apuntan las más remotas afirmaciones de la analítica existencial a una existencia preferible, deseable, o formalmente excelente? Con esta pregunta tocamos un lugar frecuentemente visitado de la analítica del Dasein. O para decirlo de manera corriente, ponemos el dedo en la llaga. Esa llaga fue protegida por Heidegger en numerosos pasajes de Ser y tiempo en los que de manera reiterada advertía al lector de no 3 Cabe suponer que este procedimiento hermenéutico mínimo tiene su radio de acción circunscrito a la metafísica, pero quizás no resulte inadecuado extenderlo, como proponemos hacerlo, y aplicarlo a la filosofía en general o en nuestro caso a la filosofía de Heidegger. La semilla del humanismo y ontología fundamental en Heidegger 124 Moralidad Germán Darío Vélez López tomar las afirmaciones de la analítica existencial en términos morales. Esta misma función de advertencia es rastreable en los seminarios previos a la publicación de Ser y tiempo. En ellos tuvo un lugar destacado metodológicamente. Ya hemos anunciado su título: la indicación formal. Esta tiene la función de impedir que los fenómenos sean comprendidos primordialmente a partir de su sentido de contenido. La indicación formal tiene la vocación de liberar el sentido de referencia y de apuntar al sentido de cumplimiento del fenómeno. Su función fue vista por Heidegger a tal punto determinante, que en el seminario sobre Agustín y el neoplatonismo, le asignó el papel de marcar un quiebre en la historia misma del pensamiento. Metafísica es la filosofía que se guía por el sentido de contenido de los fenómenos (el san Agustín neoplatónico), mientras que una ontología fundamental, o mejor, una ciencia pre-teorética de la vida en sí y para sí, es una filosofía que se guía por el sentido de cumplimiento de los fenómenos (el san Agustín que explora en el libro X de las Confesiones los auténticos movimientos existentivos). Así pues, cuando en Ser y tiempo encontramos este tipo de advertencias hechas al lector con respecto a una posible interpretación moralizante de tal o cual concepto, siendo el primero, la matriz de todos ellos, el “concepto” de impropiedad [Uneigentlichkeit], podemos suponer con buenas razones que nos encontramos ante una indicación formal, un anuncio formal, en fin, una advertencia en sentido eminente. Heidegger sostiene a lo largo de todo el análisis preparatorio del Dasein la firme convicción de la neutralidad moral de la interpretación fenomenológica. Consciente, sin embargo, de la tendencia de la vida, y específicamente de la vida filosófica, a caer en la interpretación moral de los fenómenos guiándose por su sentido de contenido específicamente moral, no cedía en su esfuerzo por mantener el análisis libre de estimaciones de valor recordando continuamente el carácter formalmente indicativo de los conceptos existenciales. Sin embargo, en el extremo del más extremo desarrollo de la interpretación fenomenológica del sentido del ser del Dasein, al momento de determinar el sentido temporal del cuidado [Sorge], parece como si Heidegger no resistiera más la presión del lector y se viera forzado a admitir que, en el paso de la primera a la segunda sección –el paso de la existencia impropia a la propiedad de la existencia, y que por esta razón designamos como “apropiación”–, un ideal de existencia se hubiera hecho manifiesto y reclamara sus Revista Co-herencia Vol. 11, No 20 Enero - Junio 2014, pp. 121-139. Medellín, Colombia (ISSN 1794-5887) 125 derechos. Ampliemos un poco más las conjetura: es como si una sospecha mantenida al margen durante el análisis preparatorio del Dasein a lo largo de toda la primera sección de Ser y tiempo, es decir, durante todo el análisis de la estructura existencial de la cotidianidad media del Dasein, no pudiera ser aún mantenida al margen y, tras el desarrollo de la integridad y propiedad del Dasein, Heidegger tuviera que confesar, como a pesar suyo, los supuestos de su indagación, esto es, que a pesar de todo, y aun cuando el punto de partida fenomenológicamente exigible no pudiese ser una concepción predeterminada de la existencia humana, él hubiera partido, sin embargo, de una específica concepción de lo que sería la propiedad de una vida humana. Para confirmar este hecho, que quizás todo lector atento de Ser y tiempo constata con cierta sorpresa no exenta de satisfacción, me permito citar el pasaje en al cual la conjetura apunta. Se trata, pues, de una singular confesión, o para decirlo con Nietzsche, de la autoconfesión del credo moral del autor de la analítica existencial: ¿Pero no hay acaso por debajo de esta interpretación ontológica de la existencia del Dasein una determinada concepción óntica del modo propio de existir, un ideal fáctico del Dasein? Efectivamente es así. Este factum no sólo no debe ser negado ni aceptado a la fuerza, sino que es necesario que se lo conciba en su positiva necesidad, a partir del objeto que constituye el tema de investigación. La filosofía no ha de querer nunca negar sus “supuestos”, pero tampoco deberá contentarse con admitirlos. Ella debe reconocer los supuestos y exponer, en estrecha relación con ellos, aquello para lo que son supuestos. (Heidegger, 1998: 329/310) Podemos, pues, poner en correspondencia directa la pregunta de Nietzsche y la respuesta de Heidegger. Incluso podemos reconocer en la pregunta con la que introduce Heidegger su ideal de fáctico del Dasein, una repetición sui generis de la pregunta nietzscheana: “¿a qué moral quiere esto (quiere él-) llegar? ” equivale a preguntar “¿Qué ideal fáctico del Dasein o qué concepción óntica del modo propio de existir hay por debajo de la interpretación ontológica? ” Dejemos en suspenso por ahora estas preguntas, para concentrarnos en lo que metodológicamente pone al descubierto esta confesión, a saber, que hay “por debajo” de la interpretación ontológica de la existencia del Dasein un supuesto, una concepción óntica, un La semilla del humanismo y ontología fundamental en Heidegger 126 Moralidad Germán Darío Vélez López ideal fáctico. En realidad Heidegger ya había hecho alusión a estos supuestos que están “por debajo” de la interpretación ontológica casi desde el comienzo de su obra. En efecto, encontramos en la introducción un breve pasaje en el cual Heidegger advierte que “la analítica existencial tiene, en última instancia, raíces existentivas, e. d. ónticas.” (36/13). Las raíces son aquello que está justamente por debajo, aquello a partir de lo cual se despliega y se sostiene la interpretación ontológica. Pero, ¿por qué exigiría o necesitaría la interpretación ontológica apoyarse en unas raíces ónticas, y esto por no decir en, justamente, aquellas raíces ónticas del modo propio de existir? Es necesario buscar la respuesta en aquello que hace posible a toda interpretación y no sólo a la interpretación ontológica. Se trata de un conocido aspecto de la hermenéutica fenomenológica heideggeriana, y que dará lugar, en Gadamer, a un despliegue de amplios alcances en lo relativo al problema de la comprensión de la historicidad humana. Dicho brevemente: no hay interpretación ontológica sin supuestos. Si la filosofía se concibe como hermenéutica fenomenológica, entonces hay que admitir que, en este sentido, no hay filosofía sin supuestos. Dentro del proyecto de la interpretación fenomenológica los supuestos garantizan el ingreso en el “círculo hermenéutico” de la comprensión. Toda comprensión supone una pre-comprensión. La estructura de la pre-comprensión está dada por el ver, tener y concebir previos, tal como lo indica claramente Heidegger en el parágrafo 32 de Ser y tiempo. Puesto en términos del conjunto de la analítica existencial, la interpretación ontológica presupone una concepción óntica. Casi un siglo después de la publicación de Ser y tiempo parecemos acostumbrados a admitir esta incómoda intromisión de la hermenéutica en la fenomenología. Pero la voz de Nietzsche nos alcanza, desde un pasado aún más remoto, obligándonos a reconocer en esa incómoda intromisión, la presencia de la moral en la raíz de la ontología fundamental, en la raíz de la raíz. 2. El árbol ideal Llegados a este punto, el diálogo Nietzsche–Heidegger adquiere cierto aspecto tirante y áspero, al mismo tiempo amenaza con irse por las ramas. Más que responder a qué moral quiere llegar, Hei- Revista Co-herencia Vol. 11, No 20 Enero - Junio 2014, pp. 121-139. Medellín, Colombia (ISSN 1794-5887) 127 degger comienza por indicar formalmente de qué situación hermenéutica tiene que partir la interpretación ontológica del Dasein. La analítica existencial tiene que asegurarse un tener previo del Dasein en su integridad y un verlo previo en su propiedad, pues de otro modo la interpretación ontológica será necesariamente incompleta y unilateral. Es en función del propósito hermenéutico de la ontología fundamental que la analítica tiene que intentar superar el análisis de la cotidianidad media y de las estructuras que en ella han sido descubiertas. La analítica del Dasein no llega a un ideal fáctico de existencia porque presuponga tal ideal como un deber ser moral, sino que tiene que presuponer un modo propio de existencia para poder asegurar la originariedad de la manera previa de ver: “Mientras la estructura existencial del poder-ser propio no sea incorporada a la idea de existencia, le faltará originariedad a la manera previa de ver que guía la interpretación existencial.” (253/233). Así pues, en correspondencia con lo que la analítica existencial ha logrado en su primera sección, y de cara a las tareas sucesivas de la interpretación ontológica, declara Heidegger al inicio de la segunda sección de Ser y tiempo, “[U]na cosa se ha vuelto imposible de desconocer: el análisis existencial del Dasein hecho hasta aquí no puede reivindicar para sí la originariedad. En el haber previo estaba siempre tan sólo el ser impropio del Dasein, y éste, en tanto que no entero.” (253/233) Visto de este modo, parece como si la idea de existencia reclamara el ideal. ¿Lo exige en verdad? Esta es la cuestión. En lo que sigue intentaré mostrar que no bien admite Heidegger la presuposición del ideal, su justificación metodológica parece pasar por alto el problema álgido del ideal mismo, concentrándose en la descripción del modo como el Dasein concibe la idea de existencia y de cómo a partir de ella se despliega el análisis preparatorio. Veamos. En primer lugar, parece indiscutible que la idea de existencia está en correspondencia con el ver previo. Cabe suponer que lo que el ver previo ve es la idea, el eidos, el aspecto de la existencia. Esta visión orienta la interpretación. Pero una visión que no contemple de la existencia más que su aspecto impropio o indiferente será una visión sesgada. La originariedad de la visión no puede garantizarse hasta que no se tenga a la vista la idea de la existencia en su propiedad. Los dos primeros capítulos de la segunda sección de Ser y tiempo están dedicados a esta tarea. Al cabo de un análisis existencial de la muerte y de la conciencia moral, puede la analítica existencial La semilla del humanismo y ontología fundamental en Heidegger 128 Moralidad Germán Darío Vélez López garantizar que el Dasein ingresa íntegra y propiamente en el haber previo. La originalidad de la situación hermenéutica está garantizada, de este modo, por el resultado de estos análisis. La fórmula existencial que sirve de título y síntesis de dichos resultados es “resolución precursora” [vorlaufende Entschlossenheit]. Heidegger resume la elaboración que acabamos de referir mediante una escansión singular en el camino de la analítica existencial al comienzo del parágrafo 63: Con el tema de la resolución precursora el Dasein se ha vuelto fenoménicamente visible en lo que respecta a la posibilidad de su propiedad y de su integridad. La situación hermenéutica, hasta ahora insuficiente para la interpretación del sentido de ser del cuidado, ha logrado la necesaria originalidad. El Dasein ha sido puesto en el haber previo en una forma originaria, es decir, en una forma relativa a su modo propio de poder-estar-entero; la orientadora manera previa de ver –la idea de la existencia– ha podido ser determinada mediante la aclaración del poder-ser más propio; con la elaboración concreta de la estructura de ser del Dasein, se ha hecho tan clara su índole ontológica peculiar frente a todo lo que está-ahí, que la manera de entender previa de la existencial del Dasein posee ahora una articulación suficiente como para guiar en forma segura la elaboración conceptual de los existenciales. (329/310) En esta síntesis es posible reconocer los tres elementos característicos de la estructura de la pre-comprensión que determinan la situación hermenéutica. Miremos: mediante la resolución precursora, 1) El Dasein ha sido puesto en el haber previo en forma originaria, es decir, en una forma relativa a su modo propio de poder-estar-entero; 2) La orientadora manera previa de ver –la idea de existencia– ha podido ser determinada mediante la aclaración del poder-ser más propio; 3) para la manera de entender previa se ha hecho clara la índole ontológica peculiar del Dasein con respecto a los otros entes, de modo que la existencialidad del Dasein posee ahora una articulación suficiente como para guiar en forma segura la elaboración conceptual de los existenciales. Este es, como queda dicho, un punto de llegada. Quizás no la meta última de la ontología fundamental, pero sí una meta intermedia, aquella que garantiza la originalidad de la situación hermenéutica. Alcanzar este punto supone, sin embargo, partir de una suposición previa, en la cual podemos localizar los presupuestos realmente Revista Co-herencia Vol. 11, No 20 Enero - Junio 2014, pp. 121-139. Medellín, Colombia (ISSN 1794-5887) 129 básicos, elementales, primeros de la analítica. Se trata de la idea de existencia. ¿De dónde surge esta idea? ¿Cómo llega el Dasein a presuponerla, es decir, a concebirla? Una de las indicaciones más claras con respecto a la procedencia de la idea de existencia la encontramos en el pasaje que estamos comentando detalladamente, situado en el parágrafo quizás metodológicamente más denso, problemático y decisivo de toda la analítica existencial, el parágrafo 63, que por esta razón podemos denominar como la encrucijada metodológica de la segunda sección de Ser y tiempo. Según Heidegger, la idea de existencia es el resultado de una auto-interpretación [Selbstauslegung] propia del ser del Dasein, auto-interpretación pre-ontológica que se desprende de su “comprender óntico” es decir, de su trato cotidiano con el ente intramundano y con el semejante4: “En el descubrimiento circunspecto y ocupado del “mundo” la ocupación misma queda también a la vista. El Dasein se comprende siempre fácticamente en determinadas posibilidades existentivas, aunque los proyectos procedan tan sólo de la comprensión común del uno. Expresa o tácitamente, adecuada o inadecuadamente, de alguna manera la existencia queda concomitantemente comprendida.” (330/312). Por esta razón puede afirmar Heidegger, un poco más adelante, que la idea de existencia, de la cual partió el análisis, estaba justificada, ya que “la indicación formal de la idea de existencia se guiaba por la comprensión del ser que se encuentra en el Dasein mismo” (332/313). Tal indicación formal contiene los siguientes elementos: • “El ente que llamamos Dasein lo soy cada vez yo mismo, y lo soy como un poder-ser en el que está en juego ser este ente” (332/313). • “El Dasein se comprende como estar-en-el-mundo. Y por ser el Dasein de esta manera, comparecen para él entes con el modo de ser de lo a la mano [herramientas, útiles] y de lo que está-ahí [presencia subsistente]” (332/313). • “[El Dasein] no es tan sólo algo que está-ahí, sino que, bien sea en una interpretación mítica y mágica, ya se ha comprendido siempre a sí mismo” (332/313). 4 Esta auto-interpretación del Dasein está relacionada, guardadas las distancias, con la autoconsciencia hegeliana. La experiencia del mundo se “supera” en la conciencia de la experiencia, en la autoconsciencia. Este movimiento reflexivo de la consciencia es el antecedente filosófico decisivo para la autointerpretación existentiva. La semilla del humanismo y ontología fundamental en Heidegger 130 Moralidad Germán Darío Vélez López Estas son, pues, indicaciones formales de la idea de existencia. Aquello que las caracteriza es, paradójicamente, una cierta deficiencia: carecen de “transparencia ontológica” (332/313), de precisión y de conceptualización. En esa medida son pre-suposiciones. Las presuposiciones de la analítica existencial no son premisas de una deducción, ni se encadenan a otras proposiciones sobre el Dasein siguiendo “las reglas formales de la inferencia” (333/314). Las presuposiciones tienen “el carácter de un proyectar comprensor” (333/314). Las indicaciones formales de la existencia dan forma al proyecto comprensor del Dasein. El “proyecto formalmente indicativo” (333/315) abre al Dasein con vistas a su constitución de su ser. Las indicaciones formales constituyen la apertura incipiente del ente temático a partir de la cual puede desarrollarse la comprensión de su ser propio. El desarrollo de la comprensión es la interpretación en la que el ente “toma la palabra con respecto a su ser” (333/315). El tipo de investigación que de este modo se desarrolla tiene una especificidad notable. Queriendo reducirla a la investigación científica y al modo como en ella se elabora el proyecto y se “prueban” las hipótesis, se desconoce lo característico del ente temático, a saber, que su ser consiste en el cuidado, es decir, en la conjunción originaria de proyecto existencial, facticidad de la condición de arrojado, y caída. Nuestro propósito no consiste en explicar en detalle este resultado de la analítica existencial, sino preguntarnos por la dimensión, el alcance y la justificación de los presupuestos que orientan el análisis. Siguiendo la idea de existencia como presupuesto básico que se desarrolla desde el Dasein mismo, la analítica desemboca en esta determinación del ser del Dasein como cuidado. Pero la problemática metodológica que hemos sometido a consideración en este momento intermedio de nuestra elaboración, nos conduce a preguntarnos si el despliegue del cuidado en dirección a alcanzar una situación hermenéutica originaria, no introduce arbitrariamente no una idea sino un ideal de existencia. Heidegger parece haberlo admitido. Pero en la justificación metodológica el ideal parece desaparecer o reducirse a la indicación formal de la idea de existencia. Sin embargo, la idea de existencia, por sí sola, no garantiza la originalidad de la situación hermenéutica, pues justamente para suplir su deficiencia ontológica presupuso Heidegger el ideal. Ahora parece como si al momento de justificarlo quisiera conservar su beneficio sin reconocerle el crédito. Revista Co-herencia Vol. 11, No 20 Enero - Junio 2014, pp. 121-139. Medellín, Colombia (ISSN 1794-5887) 131 Por esta razón, la analítica existencial, siguiendo la metáfora penosamente financiera o contractual, queda en deuda. El ideal pone en rojo el saldo existencial. 3. La savia de la moral Como queda planteado hay, pues, un sutil desplazamiento de la idea de existencia, como presupuesto hermenéutico de la interpretación ontológica, hacia un ideal fáctico del Dasein, como condición de posibilidad de la originalidad de la situación hermenéutica. Hasta el momento sólo hemos aludido, sin embargo, a este ideal. Se trata, actualmente, de mostrarlo. ¿Qué elementos componen el ideal de existencia? Sabemos que dicho ideal está formulado del modo más preciso en el primer capítulo de la segunda sección de Ser y tiempo, capítulo dedicado al análisis existencial de la muerte como posibilidad de un estar entero del Dasein. Una cierta experiencia de la muerte, una comprensión existencial de la misma, garantiza la integridad del haber previo del Dasein. La propiedad o la apropiación de ese haber previo íntegro es exigida por la conciencia que llama al Dasein desde la pérdida en el Uno y lo llama a hacerse cargo de su existencia en su integridad, es decir, en su adelantarse hasta su posibilidad extrema, remota, la más remota: la muerte. Estos son los dos pasos sustanciales de la segunda sección, en los que se decide la posibilidad de la interpretación ontológica, esto es, temporal, del cuidado como sentido de ser del Dasein. ¿Cuál es, entonces, el ideal fáctico del Dasein que Heidegger presupone como base de la interpretación ontológica? El ideal está ligado al concepto existencial de muerte y a la comprensión existencial de la finitud humana. La comprensión de la finitud consiste en adelantarse hasta la posibilidad extrema de la muerte. En ese adelantarse hasta la remotísima posibilidad se constituyen las máximas morales determinantes, la moral a la que quiere llegar la interpretación ontológica. Esta moral determina el modo propio de estar vuelto hacia la muerte, que Heidegger denomina “adelantarse hasta la posibilidad”. Este adelantarse gobierna la producción de la moral correspondiente, tal como puede apreciarse en los dos principios generales siguientes (ver parágrafo 53 de Ser y tiempo): La semilla del humanismo y ontología fundamental en Heidegger 132 Moralidad Germán Darío Vélez López 1. Ideal 1 – El coraje ante la inminencia de la muerte: “El Dasein está constituido por la aperturidad, esto es, por un comprender afectivamente dispuesto. El estar vuelto propiamente hacia la muerte no puede esquivar la posibilidad más propia e irrespectiva, encubriéndola en esta huida y reinterpretándola en función de la comprensión común del uno.” (280/260) 2. Ideal 2 – La serena comprensión de la posibilidad de la muerte: “En el estar vuelto hacia la muerte ésta debe ser comprendida en toda su fuerza como posibilidad, interpretada como posibilidad y, en el comportamiento hacia ella, sobrellevada como posibilidad.” (281/261) 3. Ley de la relación inversa posibilidad/realidad – Afirmación máxima del vivir en la distancia mínima al posible morir: “La máxima proximidad del estar vuelto hacia la muerte en cuanto posibilidad es la máxima lejanía respecto de lo real.” (282/262). Los ideales y la ley que los gobierna delimitan la comprensión que el Dasein puede tener de su poder ser más propio, y son por ello las condiciones de la apropiación. La apropiación, por su parte, no es una especie de comportamiento práctico separado de una comprensión teórica que le serviría de fundamento. El comprender es ya un proyectarse en un modo de existir. El comprender de la posibilidad más propia es por ello, la posibilidad de un proyectar la existencia propia en lo que de suyo le pertenece, es por ello apropiación. Heidegger lo expresa de manera quizás más directa y sencilla: “Es necesario tener presente que comprender no significa primariamente quedarse tan sólo mirando un sentido, sino comprenderse a sí mismo en el poder-ser que se desvela en el proyecto.” (282/263) Someteremos deliberadamente el análisis a una síntesis suplementaria para destacar que este comprender-proyectante, que se despliega a partir de los tres elementos esquemáticamente indicados, tiene una serie de consecuencias que dan origen a algo que podemos ordenar como un conjunto de máximas, las que a falta de un mejor calificativo, podemos designar por ahora como máximas “ontológico-morales” derivadas de la comprensión de la muerte. Este comprender es un adelantarse hasta la posibilidad más propia (1), irrespectiva (2), insuperable (3), cierta (4) e indeterminada (5). Los cinco elementos así dispuestos pueden, pues, ser distribuidos en el siguiente conjunto de máximas: 1. El Dasein puede escapar del uno-mismo y acceder a su sí mismo propio. (Máxima derivada de que la muerte es la posibilidad más Revista Co-herencia Vol. 11, No 20 Enero - Junio 2014, pp. 121-139. Medellín, Colombia (ISSN 1794-5887) 133 propia del Dasein: El Dasein “queda arrebatado al uno, es decir, que, adelantándose puede siempre escapar de él [y de este modo] revela la pérdida en la cotidianidad del uno-mismo que tiene lugar fácticamente.” (282/263)) 2. El Dasein debe hacerse cargo exclusivamente por sí mismo del poder-ser en el que está en juego su ser más propio. (Máxima derivada de que la posibilidad más propia es irrespectiva: “Hace comprender al Dasein que debe hacerse cargo exclusivamente por sí mismo del poder-ser en el que está radicalmente en juego su ser más propio. […] Reivindica [al Dasein] en su singularidad.” (283/263)) 3. El Dasein puede liberarse del estar perdido en las posibilidades que la fortuna le ofrece, comprendiendo y eligiendo las que son propiamente alcanzables. (Máxima derivada de que la posibilidad más propia e irrespectiva es insuperable: “El adelantarse haciéndose libre para la propia muerte libera del estar perdido entre las fortuitas posibilidades que se precipitan sobre nosotros.” (283/264)) (a) Corolario 1: El Dasein puede cambiar. (“El adelantarse le abre a la existencia la posibilidad extrema, la de renunciar a sí misma, quebrantando así toda obstinación respecto de la existencia ya alcanzada. Adelantándose el Dasein se libra de quedar rezagado tras de sí mismo y del poder-ser ya comprendido, y de hacerse así “demasiado viejo para su victoria” (Nietzsche).” (283/264)). (b) Corolario 2: El Dasein puede acceder a una ética coexistencia. (“El Dasein conjura el peligro de desconocer, en virtud de su comprensión finita de su existencia, las posibilidades de existencia de los otros que lo superan, o bien de forzarlas, malinterpretándolas, a entrar en la existencia propia.” (283/264))5. 4. El Dasein puede estar cierto de sí mismo y fundar en esa certeza la “plena propiedad de su existencia” –de un modo más radical que el alcanzado formalmente por la reducción cartesiana. (Máxima derivada de que la posibilidad más propia, irrespectiva e insuperable es cierta: “El tener-por-verdadera la muerte […] muestra una forma distinta y más originaria de certeza que la relativa a un ente que comparece dentro del mundo o a objetos formales […] no reivindica tan sólo un determinado comportamiento del Dasein, sino que atañe a éste en la plena propiedad de su existencia.” (284/265)). 5 Ética del interés común, democracia y anti-totalitarismo. La semilla del humanismo y ontología fundamental en Heidegger 134 Moralidad Germán Darío Vélez López 5. El Dasein debe sostener la certeza de sí mismo en el temple anímico de una incertidumbre amenazante. (Máxima derivada del hecho de que la posibilidad más propia, irrespectiva, insuperable y cierta, es además indeterminada: La indeterminada certeza con respecto al “cuándo”, ocasiona angustia. En nota a pié de página, aclara Heidegger que el temple anímico correlativo de esta amenazante incertidumbre no es sólo angustia, y no lo es como una mera emoción. Así, por ejemplo, poco antes de mencionar en el parágrafo 63 el ideal fáctico, Heidegger nos habla de que “[L]a serena angustia, que nos lleva ante el poder-ser singularizado, se acompaña de la vigorosa alegría por esta posibilidad.” (329/310)). Al cabo de esta reconstrucción de las máximas, encontramos una definición que las resume, y que recoge en un sólo enunciado el núcleo de la moral. En términos de Heidegger, se trata del núcleo del proyecto existencial del modo propio de estar vuelto hacia la muerte: [E]l adelantarse le revela al Dasein su pérdida en el “uno mismo” y lo conduce ante la posibilidad de ser sí mismo sin el apoyo primario de la solicitud ocupada, y de serlo en una libertad apasionada, libre de las ilusiones del uno, libertad fáctica, cierta de sí misma y acosada por la angustia: libertad para la muerte. (285/266). En esta condensación ética, o como quisiera llamarla, en esta savia de la moral, desemboca el proyecto ontológico al cual responden las afirmaciones existenciales más remotas de la interpretación del Dasein. Esta solución o disolución ética de la ontología, esta savia de la moral existencial, no concierne exclusivamente al individuo ni conduce necesariamente a una posición meramente individual, o a un cultivo de sí apartado del otro, sino que también apunta a una política es decir, a la posible modulación social o colectiva de las relaciones de poder entre individuos o grupos de individuos, tal como podría derivarse, de una elaboración pormenorizada de la tercera máxima. ¿Qué tipo de ética es esta? Es necesario, llegados a este punto, guardar todas las distancias necesarias y concluir este segmento de la interpretación con prudencia pero al mismo tiempo con determinación. No se trata, por lo pronto, como advierte Heidegger, en La Revista Co-herencia Vol. 11, No 20 Enero - Junio 2014, pp. 121-139. Medellín, Colombia (ISSN 1794-5887) 135 carta sobre el humanismo (Heidegger, 2001: 273), de una moral secularizada, sino más bien de una ética en régimen formal-indicativo, y esto quiere decir que es una ética que se despliega desde el sentido de cumplimiento de la existencia humana. El sentido de relación de la vida humana consigo misma, con el semejante y con el mundo, está coordinado por el cuidado (Sorge, Fürsorge, Besorgen) mientras que el sentido de contenido está libre. Le corresponde a cada uno determinarlo, es “en cada caso el mío”. Pero este sentido está en cierto modo subordinado al sentido de cumplimiento. No queda, por ello, desprovisto de toda guía, o entregado al capricho de lo que la ocupación propone y la solicitud sustitutiva alivia, sino que es un sentido de cumplimiento orientado por las máximas expuestas. Este sentido tiene por título el término “apropiación”. 4. Lo que fructifica Podemos afirmar, desde un punto de vista general, en una visión que planea sobre esta problemática central de la analítica o sobre esta encrucijada moral, que Heidegger muestra cómo la pregunta genealógica está obligada a darle paso a un planteamiento hermenéutico. Este planteamiento es de carácter hipotético: si se admiten tales supuestos, entonces se sigue esto y aquello. La cuestión para Heidegger no es sólo plantear a qué moral quiere llegar, no lo esconde, como lo haría si su proyecto estuviera gobernado por la cuestión genealógica, antes bien, es una genealogía que ha pasado por el diván de la hermenéutica fenomenológica: Heidegger es consciente, por decirlo así, de sus supuestos. Esa consciencia tiene un límite, sin embargo, como bien descubre Gadamer (1993: 370-377). La propia situación hermenéutica sólo puede ser parcialmente aclarada. Pero la meditación filosófica acompañada de la hermenéutica, puede examinarlos y sopesarlos. Es exigible una destrucción de la analítica existencial en dirección a reconocer las experiencias originarias de las cuales ella surgió. No podemos contentarnos con reconocer que la formalización ontológica ha liberado de contenido a los supuestos, dejando abierto el camino para que el sentido de referencia y cumplimiento, el cuidado, la impropiedad y la propiedad, gobiernen desde una superioridad formal y abstracta la interpretación ontológica y sus consecuencias ónticas. Podemos y acaso tenemos, pues, que preguntar y desplegar en un trabajo hermenéutico los presuLa semilla del humanismo y ontología fundamental en Heidegger 136 Moralidad Germán Darío Vélez López puestos de la savia de la moral y de este modo alcanzar la raíz de la raíz. Heidegger designaba a estos elementos históricos y fácticos básicos las “raíces ónticas” del proyecto ontológico. Establecerlas implica, esquemáticamente, recorrer un camino que sigue una dirección ortogonal con respecto al despliegue genealógico y “deductivo” de la analítica existencial. Es un retorno a los presupuestos y una evaluación de su función, su sentido y sus alcances. Retornando de este modo, llegamos un punto cuyo siguiente paso corresponde mejor a un trabajo arqueológico, pues exige una excavación en el suelo óntico, fáctico, de Heidegger, con el fin de reconocer aquellas raíces que históricamente son las suyas, raíces no formalizadas, no corregidas. ¿Cuáles son? Con la publicación de los primeros seminarios realizados por Heidegger en Friburgo, y con una creciente y cada vez más exhaustiva interpretación de estas ideas germinales de su pensamiento6, es posible establecer cuáles fueron esas raíces, y cuál fue el suelo de experiencia desde el cual creció la planta entera. Se trata, para decirlo brevemente, de la experiencia cristiana primitiva como paradigma del agudizamiento de la experiencia fáctica sobre el mundo propio. En esa experiencia jugaron un papel decisivo San Pablo y San Agustín. La insuficiencia del punto de partida cartesiano, en lo concerniente a una adecuada interpretación del sentido de ser de la vida, del sentido del ser del sum del cogito, conducen a Heidegger a buscar dicha fundamentación en la experiencia paradigmática de las comunidades cristianas primitivas. ¿Por qué justamente en ellas? No podemos desarrollar de manera detallada este aspecto, pero podemos exponer la línea general del argumento: en la interpretación fenomenológica de la primera carta de San Pablo a los tesalonicenses, Heidegger muestra cómo la constitución del propio ser, del llegar a ser, del genesthai de la comunidad de tesalónica y del propio San Pablo es un producto de la relación que establecen recíprocamente mediante la proclamación (Heidegger, 2005: 123 ss.). El ser propio como ser devenido, como llegado a ser de San Pablo y de los tesalonicenses reposa en la relación entre ellos y en la operación por la cual San Pablo remite a los tesalonicenses a su propio saber, obligándolos 6 Esta arqueología, está cada vez mejor documentada en los estudios sobre la obra temprana de Heidegger. Remito al lector, entre otras obras, a los trabajos de J. A. Barash, Th. Kisiel, A. Xolocotzi, Carl F. Gethmann, J. van Buren, B. D. Crowe, J. A. Escudero. Revista Co-herencia Vol. 11, No 20 Enero - Junio 2014, pp. 121-139. Medellín, Colombia (ISSN 1794-5887) 137 en cierto modo a elegir el tipo de vida que quieren vivir, el cómo de la vida. De modo análogo, el llegar a ser de San Agustín no puede separarse de su relación con Dios, del amor a Dios, en la búsqueda de Dios. “¿Quién es él?”, se pregunta San Agustín. Pero no pude responder de un modo plenamente auto-referencial, por medio de una posición absoluta de su yo. El sum del ego agustiniano es un estar en relación con el otro en la búsqueda de Dios. Así pues, tanto en la proclamación como en las Confesiones San Pablo y San Agustín, los dos representantes de las comunidades cristianas primitivas que fueron paradigmáticas para Heidegger, determinan los modos relacionales básicos, la raíz de la raíz desde la que se despliegan las posibilidades de realización, o el sentido de cumplimiento de la vida fáctica. En Ser y tiempo Heidegger sólo hace una breve mención del vínculo de la investigación ontológica con las raíces ónticas del investigador. Pero la tierra, la raíz, la savia y la planta entera están allí, dispuestas a ser interpretadas por el trabajo hermenéutico y por medio del análisis filosófico, separadas del sentido de contenido que caracteriza a la doctrina cristiana, con el propósito de que una política y una ética formalmente indicativas, una ética de la apropiación de sí, de liberadora solicitud ante el otro, y de serena relación con las cosas, fructifiquen y alimenten la vida humana La semilla del humanismo y ontología fundamental en Heidegger 138 Moralidad Germán Darío Vélez López Referencias Barash, J. A. (1988). Heidegger and the historical meaning. Dordrecht: M. Nijhoff. Crowe, B. D. (2006). Heidegger’s reliogious origins. Bloomington: Indiana University Press. Escudero, J. A. (2009). El lenguaje de Heidegger. Barcelona: Herder. Gadamer, H. G. (1997). Verdad y método I. Salamanca: Sígueme. Gethmann, Carl F. (1993). Dasein: Erkennen und Handeln. Berlin: Martin de Gruyter. Greisch, J. (2000). L’arbre de vie et l’arbre du savoir. Paris: Ed. du Cerf. Nietzsche, F. (1995). Más allá del bien y del mal. Madrid: Alianza Editorial. Nietzsche, F. (2008). Fragmentos póstumos. Vol. IV, Madrid: Tecnos. Heidegger, M. (1998). Ser y tiempo. Santiago de Chile: Editorial Universitaria. Heidegger, M (2001). Sein und Zeit. Tübingen: Max Niemeyer. Heidegger, M. (2001). Hitos. Madrid: Alianza Editorial. Heidegger, M. (2003). Estudios sobre mística medieval. México D.F.: Fondo de Cultura Económica. Heidegger, M. (2005). Introducción a la fenomenología de la religión. México: Ediciones Siruela. Kisiel, Th. (1995). The genesis of Heidegger’s Being and Time. Berkeley: University of California Press. Van Buren, J. (1994). The young Heidegger: rumor of the hidden king. Indianapolis: Indiana University Press. Xolocotzi, A. (2004). Fenomenología de la vida fáctica. México D.F.: Universidad Iberoamericana/Plaza y Valdés. Revista Co-herencia Vol. 11, No 20 Enero - Junio 2014, pp. 121-139. Medellín, Colombia (ISSN 1794-5887) 139
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ImageM: a user-friendly interface for the processing of multi-dimensional images with Matlab
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SOFTWARE TOOL ARTICLE ImageM: a user-friendly interface for the processing of multi- dimensional images with Matlab [version 1; peer review: 2 approved with reservations] 1INRAE, INRAE, F-44316 Nantes, France 2INRAE, INRAE, F-44316 Nantes, France 1INRAE, INRAE, F-44316 Nantes, France 2INRAE, INRAE, F-44316 Nantes, France Open Peer Review Approval Status 1 2 version 1 30 Apr 2021 view view John Anthony Jose, De La Salle University, Manila, Philippines Janos Lance Tiberio, De La Salle University Manila, Manila, Philippines 1. Curtis Rueden , Laboratory for Optical and Computational Instrumentation, Center for Quantitative Cell Imaging, University of Wisconsin, Madison, USA 2. Any reports and responses or comments on the article can be found at the end of the article. First published: 30 Apr 2021, 10:333 https://doi.org/10.12688/f1000research.51732.1 Latest published: 30 Apr 2021, 10:333 https://doi.org/10.12688/f1000research.51732.1 v1 SOFTWARE TOOL ARTICLE ImageM: a user-friendly interface for the processing of multi- dimensional images with Matlab [version 1; peer review: 2 approved with reservations] David Legland 1,2, Marie-Françoise Devaux 1 1INRAE, INRAE, F-44316 Nantes, France 2INRAE, INRAE, F-44316 Nantes, France First published: 30 Apr 2021, 10:333 https://doi.org/10.12688/f1000research.51732.1 Latest published: 30 Apr 2021, 10:333 https://doi.org/10.12688/f1000research.51732.1 v1 Abstract Modern imaging devices provide a wealth of data often organized as images with many dimensions, such as 2D/3D, time and channel. Matlab is an efficient software solution for image processing, but it lacks many features facilitating the interactive interpretation of image data, such as a user-friendly image visualization, or the management of image meta-data (e.g. spatial calibration), thus limiting its application to bio-image analysis. The ImageM application proposes an integrated user interface that facilitates the processing and the analysis of multi-dimensional images within the Matlab environment. It provides a user-friendly visualization of multi-dimensional images, a collection of image processing algorithms and methods for analysis of images, the management of spatial calibration, and facilities for the analysis of multi-variate images. ImageM can also be run on the open source alternative software to Matlab, Octave. ImageM is freely distributed on GitHub: https://github.com/mattools/ImageM. Keywords Open Peer Review Approval Status 1 2 version 1 30 Apr 2021 view view John Anthony Jose, De La Salle University, Manila, Philippines Janos Lance Tiberio, De La Salle University Manila, Manila, Philippines 1. Curtis Rueden , Laboratory for Optical and Computational Instrumentation, Center for Quantitative Cell Imaging, University of Wisconsin, Madison, USA 2. Any reports and responses or comments on the article can be found at the end of the article. F1000Research 2021, 10:333 Last updated: 01 SEP 2023 SOFTWARE TOOL ARTICLE ImageM: a user-friendly interface for the processing of multi- dimensional images with Matlab [version 1; peer review: 2 approved with reservations] David Legland 1,2, Marie-Françoise Devaux 1 1INRAE, INRAE, F-44316 Nantes, France 2INRAE, INRAE, F-44316 Nantes, France First published: 30 Apr 2021, 10:333 https://doi.org/10.12688/f1000research.51732.1 Latest published: 30 Apr 2021, 10:333 https://doi.org/10.12688/f1000research.51732.1 v1 Abstract Modern imaging devices provide a wealth of data often organized as images with many dimensions, such as 2D/3D, time and channel. Matlab is an efficient software solution for image processing, but it lacks many features facilitating the interactive interpretation of image data, such as a user-friendly image visualization, or the management of image meta-data (e.g. spatial calibration), thus limiting its application to bio-image analysis. The ImageM application proposes an integrated user interface that facilitates the processing and the analysis of multi-dimensional images within the Matlab environment. It provides a user-friendly visualization of multi-dimensional images, a collection of image processing algorithms and methods for analysis of images, the management of spatial calibration, and facilities for the analysis of multi-variate images. ImageM can also be run on the open source alternative software to Matlab, Octave. ImageM is freely distributed on GitHub: https://github.com/mattools/ImageM. Keywords Open Peer Review Approval Status 1 2 version 1 30 Apr 2021 view view John Anthony Jose, De La Salle University, Manila, Philippines Janos Lance Tiberio, De La Salle University Manila, Manila, Philippines 1. Curtis Rueden , Laboratory for Optical and Computational Instrumentation, Center for Quantitative Cell Imaging, University of Wisconsin, Madison, USA 2. Any reports and responses or comments on the article can be found at the end of the article. F1000Research 2021, 10:333 Last updated: 01 SEP 2023 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 Abstract Modern imaging devices provide a wealth of data often organized as images with many dimensions, such as 2D/3D, time and channel. Matlab is an efficient software solution for image processing, but it lacks many features facilitating the interactive interpretation of image data, such as a user-friendly image visualization, or the management of image meta-data (e.g. spatial calibration), thus limiting its application to bio-image analysis. John Anthony Jose, De La Salle University, 1. The ImageM application proposes an integrated user interface that facilitates the processing and the analysis of multi-dimensional images within the Matlab environment. It provides a user-friendly visualization of multi-dimensional images, a collection of image processing algorithms and methods for analysis of images, the management of spatial calibration, and facilities for the analysis of multi-variate images. ImageM can also be run on the open source alternative software to Matlab, Octave. ImageM is freely distributed on GitHub: https://github.com/mattools/ImageM. Keywords image processing, matlab, multivariate image analysis, segmentation, interactive segmentation Keywords This article is included in the Software and Hardware Engineering gateway. Page 1 of 12 Page 1 of 12 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 This article is included in the NEUBIAS - the Bioimage Analysts Network gateway. Introduction Modern imaging devices provide a wealth of data often organized as images with many dimensions, for instance 2D/3D, time and channel. This leads to new challenges in the exploration, visualization and processing of multi-dimensional data. In particular, multi-spectral images require the application of methods at the intersection of spectroscopy and image processing. Matlab (The Mathworks, Natick, MA), is an efficient software solution for image and signal processing that provides native support for multi-dimensional arrays, a large number of image processing methods, and a great facility for adding custom developments. However, it lacks many features for facilitating the interactive interpretation of image data, such as a user-friendly visualization of multidimensional images, or the management of image meta-data (e.g. spatial calibration), thus limiting its application to bio-image analysis. The ImageJ/Fiji software (Schneider et al., 2012) is an alternative that provides an intuitive graphical user interface for image exploration, and many image processing functions that take into account spatial or channel calibration. The basic functionalities can by extended by a large collection of plugins. The integration of custom developments is possible via macros or plugins, but this often requires advanced programming skills. The ImageM software aims at providing a user-friendly interface for the interactive exploration, processing and analysis of multi-dimensional imaging data within the Matlab environment, while taking into account meta-data and facilitating the integration with custom algorithm developments. This article is included in the NEUBIAS - the Bioimage Analysts Network gateway. Corresponding author: David Legland (david.legland@inrae.fr) Author roles: Legland D: Conceptualization, Methodology, Software, Writing – Original Draft Preparation; Devaux MF: Conceptualization, Investigation, Writing – Original Draft Preparation Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2021 Legland D and Devaux MF. 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 work is properly cited. How to cite this article: Legland D and Devaux MF. ImageM: a user-friendly interface for the processing of multi-dimensional images with Matlab [version 1; peer review: 2 approved with reservations] F1000Research 2021, 10:333 https://doi.org/10.12688/f1000research.51732.1 First published: 30 Apr 2021, 10:333 https://doi.org/10.12688/f1000research.51732.1 p g First published: 30 Apr 2021, 10:333 https://doi.org/10.12688/f1000research.51732.1 Page 2 of 12 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 Operation p To have the whole functionalities, Matlab (v2020a) is required, as well as the Image Processing and the Statistics toolboxes. The Gui Layout Toolbox (Sampson, 2021) is required to benefit from flexible image viewers. ImageM runs on a typical workstation. The application is launched by typing “ImageM” from the command line. Menus provide quick access to common image processing operators, organized in a way to make them easily discoverable by a non-expert user. When necessary, intuitive dialogs allow for setting up parameters. Preview of the results facilitates the choice of relevant settings. Region of interest may be defined interactively to perform interactive measurements. To facilitate usage during scripting and integration with custom developments, images can be easily imported from and exported to the workspace. Applying an operator results in a log entry that can be integrated into a script for quickly generating a workflow from the succession of operations. Segmentation and analysis of regions Octave Octave ImageM can also run within the open-source platform, Octave, after some modification of the code (see Extended data (Legland & Devaux, 2021)). (Legland & Devaux, 2021)). Implementation Implementation p ImageM strongly relies on Matlab’s Object-Oriented Programming. The core feature is an “Image” class that handles arrays up to five dimensions, corresponding to three spatial dimensions, the channels, and the time. It also encapsulates image meta-data such as spatial calibration, image name, or channel names. The user interface relies on various image viewers adapted to the type (grayscale, color, label, multivariate) and the dimensionality of the image (Figure 1a). Interactive tools allow for quick image exploration, for example histograms, line profiles and 3D visualization. The architecture was kept modular to facilitate the inclusion of new features. Figure 1. Result of a typical image processing workflow with ImageM. Original 3D image is visualized via adapted viewer (a). Image processing operators are applied (b), using contextual dialogs (c). Segmentation results are represented by label images (d,g). Analysis of the image results in table frame (f). Menu actions are transcripted into runnable Matlab commands (e). Original image courtesy of K. Belcram (Moukhtar et al., 2019). Figure 1. Result of a typical image processing workflow with ImageM. Original 3D image is visualized via adapted viewer (a). Image processing operators are applied (b), using contextual dialogs (c). Segmentation results are represented by label images (d,g). Analysis of the image results in table frame (f). Menu actions are transcripted into runnable Matlab commands (e). Original image courtesy of K. Belcram (Moukhtar et al., 2019). Page 3 of 12 Page 3 of 12 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 Segmentation and analysis of regions ImageM integrates a large family of image processing operators (linear filtering, mathematical morphology, segmentation). Intuitive dialogs allow tuning of parameters and preview of the result, facilitating the choice of relevant settings (Figure 1c). The type of the result image is automatically inferred to provide the most adequate visualization (Figure 1d and e). Segmentation algorithms result in binary or label images that can be used for quantitative analysis of regions. The spatial calibration of images is considered when available. Features can be visualized as geometric overlays on the image or exported to data tables (Figure 1f). Multivariate image analysis Multivariate image analysis Multi-channel images or microscopy images contain pixels represented of several values. Multivariate image analysis provides a convenient formalism to analyze such images by taking account both the spatial and the spectral dimensions (Geladi & Grahn, 2006). ImageM can convert multi-variate images into data tables, allowing their exploration through multi-variate analysis tools such as Principal Component Analysis or k-means clustering (Figure 2). Resulting tables can be easily back-converted to images to facilitate the visual interpretation. Use case Use case We present here a use case describing image segmentation and region analysis of the 3D image presented in Figure 1. The Extended data (Legland & Devaux, 2021) contains the sample files, and more detailed use cases. First, run the application by typing “ImageM” from Matlab command line. • In the menu, select “File->Open Image …” and choose the file “arabidopsis-embryo.tif”. This opens an image viewer for the 3D image. • In the menu, select “File->Open Image …” and choose the file “arabidopsis-embryo.tif”. This opens an image viewer for the 3D image. Figure 2. Multivariate image analysis of a macrofluorescence image. Left panel: original multivariate image (from Devaux, 2008). Middle panel: application of K-means clustering on principal components. Right panel: color representation of k-means classes. P Figure 2. Multivariate image analysis of a macrofluorescence image. Left panel: original multivariate image (from Devaux, 2008). Middle panel: application of K-means clustering on principal components. Right panel: color representation of k-means classes. Page 4 of 12 Page 4 of 12 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 • Reduce the noise in the image, by selecting “Process->Gaussian Filter …”. This opens a dialog. • Choose value 1 for each of the “sigma” values, and click “OK”. The filtered image appears in a new viewer • Perform 3D segmentation of the cells. Select menu entry “Process->Extended Min Watershed …”. Choose a basin dynamic equal to 6, the “C6” connectivity, and result type as “basins”. This results in a new 3D label image in a new viewer, where each label is displayed with a different color. • Perform 3D segmentation of the cells. Select menu entry “Process->Extended Min Watershed …”. Choose a basin dynamic equal to 6, the “C6” connectivity, and result type as “basins”. This results in a new 3D label image in a new viewer, where each label is displayed with a different color. • Some regions do not correspond to cells. They can be removed manually. Select the menu “Process->Replace Value(s) …” and fill in with “1, 2, 28, 29” (values can be identified by moving the mouse cursor and inspecting the status bar). • Some regions do not correspond to cells. They can be removed manually. Select the menu “Process->Replace Value(s) …” and fill in with “1, 2, 28, 29” (values can be identified by moving the mouse cursor and inspecting the status bar). Use case • Morphometric features can be computed from 3D label images, by selecting the menu “Analyze->Analyze Regions …”. After choosing the features and clicking “OK”, the selected features appear in a new data table frame that can be saved as a text file or exported to the workspace. • The 3D representation of the Figure 1g can be obtained by selecting the menu entry “View -> Show 3D isosurface”, choosing a “smoothing radius” value equal to 1 and checking the options “Reverse Z-axis” and “Rotate Ox”. • The 3D representation of the Figure 1g can be obtained by selecting the menu entry “View -> Show 3D isosurface”, choosing a “smoothing radius” value equal to 1 and checking the options “Reverse Z-axis” and “Rotate Ox”. Summary y The ImageM application proposes a convenient user interface for the visualization and exploration of multi-dimensional images within Matlab. It also provides basic support for regions of interests, the management of data tables. Its modular architecture should facilitate the future incorporation of new functionalities. The current work focuses on a better management of regions of interest, and on the inclusion of user plugins. Software availability Packaged application available for direction download from: https://www.mathworks.com/matlabcentral/fileexchange/ 45847-imagem Source code available from: https://github.com/mattools/ImageM Source code available from: https://github.com/mattools/ImageM Archived source code as at time of publication: http://doi.org/10.5281/zenodo.4674326 (Legland, 2021). License: BSD 2-Clause “Simplified” License License: BSD 2-Clause “Simplified” License License: BSD 2-Clause “Simplified” License Data availability Underlying data Underlying data All data underlying the results are available as part of the article and no additional source data are required. Extended data Zenodo: Extended data for the manuscript “ImageM: a user-friendly interface for the processing of multi-dimensional images with Matlab”, http://doi.org/10.5281/zenodo.4705240 (Legland & Devaux, 2021). This project contains the following extended data: - User manual - Octave compatibility notice - Two detailed use cases - Images used in use cases Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Page 5 of 12 Page 5 of 12 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 Acknowledgements This publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology). Acknowledgements This publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology). F1000Research 2021, 10:333 Last updated: 01 SEP 2023 References Devaux M-F: UV and visible fluorescence images of maize stem: macroscopy and confocal microscopy. [Data set]. Published 2018 via Perscido-Grenoble-Alpes. Reference Source Geladi P, Grahn HF: Multivariate Image Analysis. In: Encyclopedia of Analytical Chemistry (eds Meyers RA, Meyers RA). 2006. Legland D: mattools/ImageM: ImageM v1.3.2.1 (Version v1.3.2.1). Zenodo. 2021, April 9. Publisher Full Text Legland D, Devaux M: Extended data for the manuscript “ImageM: a user-friendly interface for the processing of multi-dimensional images with Matlab” (Version 1.0). Zenodo. 2021, April 20. Publisher Full Text Moukhtar J, Trubuil A, Belcram K, et al.: Cell geometry determines symmetric and asymmetric division plane selection in Arabidopsis early embryos. PLOS Computational Biology. 2019; 15(2): 1–27. PubMed Abstract|Publisher Full Text|Free Full Text Sampson D: GUI Layout Toolbox. MATLAB Central File Exchange. Retrieved April 13, 2021. 2021. Reference Source Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(671-675): 2012. PubMed Abstract|Publisher Full Text|Free Full Text Devaux M-F: UV and visible fluorescence images of maize stem: macroscopy and confocal microscopy. [Data set]. Published 2018 via Perscido-Grenoble-Alpes. Reference Source Geladi P, Grahn HF: Multivariate Image Analysis. In: Encyclopedia of Analytical Chemistry (eds Meyers RA, Meyers RA). 2006. Legland D: mattools/ImageM: ImageM v1.3.2.1 (Version v1.3.2.1). Zenodo. 2021, April 9. Publisher Full Text Legland D, Devaux M: Extended data for the manuscript “ImageM: a user-friendly interface for the processing of multi-dimensional images with Matlab” (Version 1.0). Zenodo. 2021, April 20. Publisher Full Text Moukhtar J, Trubuil A, Belcram K, et al.: Cell geometry determines symmetric and asymmetric division plane selection in Arabidopsis early embryos. PLOS Computational Biology. 2019; 15(2): 1–27. PubMed Abstract|Publisher Full Text|Free Full Text Sampson D: GUI Layout Toolbox. MATLAB Central File Exchange. Retrieved April 13, 2021. 2021. Reference Source Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(671-675): 2012. PubMed Abstract|Publisher Full Text|Free Full Text Page 6 of 12 Page 6 of 12 Version 1 Reviewer Report 09 September 2021 © 2021 Rueden C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. F1000Research 2021, 10:333 Last updated: 01 SEP 2023 Curtis Rueden Fields of view? Fluorescence lifetime? related challenge of scientific imaging. Multiple angles? Fields of view? Fluorescence lifetime? ImageM is limited to 3D+time+channel. Therefore, it does not facilitate handling of any "new challenges" in dimensionality beyond this paradigm. Rather than argue that ImageM addresses new challenges in scientific imaging, the article would be better served explaining that ImageM builds on the wealth of functionality available in MATLAB, enabling more seamless integration of other MATLAB-based tools with its 5D image paradigm. The two use cases, especially use case 2, illustrate this fact well. The lack of discussion of the technical merit of a MATLAB-based implementation of an ImageJ-like program: While this is a short article, it would be ideal—if sufficient space—to mention ImageJ's MATLAB integrations ImageJ-MATLAB (https://imagej.net/scripting/matlab) and MIJ/Miji ( https://imagej.net/plugins/miji), and how ImageM compares to them. This is an important part of the justification for essentially reimplementing ImageJ in MATLAB: what are the downsides of using the existing ImageJ within MATLAB via its built-in JVM? How does ImageM overcome those downsides? I do believe there is technical merit to ImageM. But the article needs to do a clearer job explaining the benefits. > it lacks many features for facilitating the interactive interpretation of image data, such as a user-friendly visualization of multidimensional images or the management of image meta- data (e.g. spatial calibration), thus limiting its application to bio-image analysis. I am not well versed in the ecosystem of MATLAB add-ons, so I cannot evaluate whether this statement is really true, but I am skeptical. Consider rewording to frame things more positively, saying that ImageM provides user-friendly capabilities that build upon, enhance, and complement MATLAB's existing image processing offerings. > The integration of custom developments is possible via macros or plugins, but this often requires advanced programming skills. I disagree about MATLAB programming being easier than ImageJ macro programming. Both are designed to be as accessible as possible, while still being powerful. Instead of arguing that extending ImageJ is too hard, why not instead argue that extending ImageM via MATLAB code enables scripters to easily harness MATLAB's power, flexibility, and extensive suite of toolboxes and add-ons? > Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Curtis Rueden Laboratory for Optical and Computational Instrumentation, Center for Quantitative Cell Imaging, University of Wisconsin, Madison, Wisconsin, USA ImageM is a MATLAB-based application, backed by a modular collection of MATLAB libraries, for performing image processing and analysis on multidimensional scientific images with the common dimensions of X, Y, Z, channel, and time. Modeled closely after ImageJ, ImageM integrates many powerful routines into a central main window, making them more easily accessible, especially to those less versed in writing scripts. The use cases do a nice job of quickly showcasing some of the great things you can do with ImageM. However, the article's introduction and rationale needs work, and the program needs to be better documented and tested. Nonetheless, this software is certainly worth advertising to the scientific community. > Is the rationale for developing the new software tool clearly explained? > Is the rationale for developing the new software tool clearly explained? Partly. I have two problems with the rationale as written: Partly. I have two problems with the rationale as written: The claim that ImageM addresses novel visualization challenges: ○ The claim that ImageM addresses novel visualization challenges: ○ > Modern imaging devices provide a wealth of data often organized as images with many dimensions, for instance 2D/3D, time and channel. This leads to new challenges in the exploration, visualization and processing of multi-dimensional data. In particular, multi- spectral images require the application of methods at the intersection of spectroscopy and image processing. > Modern imaging devices provide a wealth of data often organized as images with many dimensions, for instance 2D/3D, time and channel. This leads to new challenges in the exploration, visualization and processing of multi-dimensional data. In particular, multi- spectral images require the application of methods at the intersection of spectroscopy and image processing. Challenges in visualization of 3D, time and channel are not "new". ImageJ's core gained 5D support in 2007 with version 1.39l, and there were many earlier programs with >=5D support as well (e.g. BioImageXD, VisBio, ImageJ via the Image5D plugins, OME Server). Highlighting multi-spectral as a challenge makes sense, but it is hardly the only dimension- Highlighting multi-spectral as a challenge makes sense, but it is hardly the only dimension- Highlighting multi-spectral as a challenge makes sense, but it is hardly the only dimension- Page 7 of 12 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 related challenge of scientific imaging. Multiple angles? Curtis Rueden > Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes. Use case 1 was fully reproducible for me. Use case 2 was also fully reproducible, but: There appears to be some randomness to the K-Means clustering output. My results, while functionally identical, differed from the screenshot regarding the cluster numbering. ○ Yes. Use case 1 was fully reproducible for me. Use case 2 was also fully reproducible, but: There appears to be some randomness to the K-Means clustering output. My results, while functionally identical, differed from the screenshot regarding the cluster numbering. ○ Page 8 of 12 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 While following the steps, the following error is emitted to the command window: Error using convhull Error computing the convex hull. The points may be collinear. Error in Table/scatterGroup (line 204)                 inds2   = convhull(xdata(inds), ydata(inds)); Error in imagem.actions.table.process.TableKMeans/run (line 88)             scatterGroup(tab(:,1), tab(:,2), k); Error in imagem.gui.FrameMenuBuilder>@(src,evt)action.run(obj.Frame) (line 475)             'Callback', @(src, evt) action.run(obj.Frame));   Error while evaluating Menu Callback. ○ Folding the k-means score table back to an 800x800 image does not emit a line of script for programmatic reproduction. ○ All of that said, the use case of K-means clustering to segment tissue types is very cool! Relatedly, ImageM is hard to install: I could not get the ImageM Octave fork working in Octave. I recommend removing the claim that ImageM works with Octave (Which is a shame because a major problem with ○ Relatedly, ImageM is hard to install: I could not get the ImageM Octave fork working in Octave. I recommend removing the claim that ImageM works with Octave. (Which is a shame, because a major problem with MATLAB is that it is not open source.) ○ I could not get ImageM working in MATLAB Online. This isn't too surprising, but may want to specifically acknowledge that it doesn't work in the online app. ○ I could not get ImageM working in MATLAB Online. This isn't too surprising, but may want to specifically acknowledge that it doesn't work in the online app. ○ I could not get ImageM working in a local MATLAB R2021a after installing it from the Add- On Manager, even after also installing the add-on dependencies MatImage, geom2d, geom3d, and MatStats. Is there really no dependency management system for MATLAB add-ons? Curtis Rueden ○ From the ImageM manual: ○         > An element of an image is usually associated to a square region centered on its coordinat        > Its bounds are therefore ±0.5 around each dimension. A pixel is not a little square. http://alvyray.com/Memos/CG/Microsoft/6_pixel.pdf As mentioned above, I had a hard time getting ImageM installed. Here are the issues I filed on GitHub to give some technical detail: MATLAB  Online – https://github.com/mattools/ImageM/issues/1 ○ Octave – https://github.com/mattools/ImageM/issues/2 ○ MATLAB R2021a – https://github.com/mattools/ImageM/issues/3 ○ Is the rationale for developing the new software tool clearly explained? Partly Is the description of the software tool technically sound? Yes Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Software engineering, software architecture, image visualization, image processing, image analysis. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. add to the menu structure. add to the menu structure. The Help menu should offer links to important resources, especially the online docs. ○ Finally, a couple of miscellaneous comments: The Help menu should offer links to important resources, especially the online docs. ○ Finally, a couple of miscellaneous comments: Mathworks named the program MATLAB in all caps ("MATLAB"), not title case ("Matlab"). ○ > An element of an image is usually associated to a square region centered on its coordinates. > Its bounds are therefore ±0.5 around each dimension. Is the rationale for developing the new software tool clearly explained? Partly Is the description of the software tool technically sound? Yes Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Curtis Rueden Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Software engineering, software architecture, image visualization, image processing, image analysis. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Is the rationale for developing the new software tool clearly explained? Partly Is the description of the software tool technically sound? Yes Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes Competing Interests: No competing interests were disclosed. Curtis Rueden If not, that is a huge downside of using MATLAB compared to Java, Python, JavaScript, Ruby, or other popular language ecosystem. ○ I was finally able to get ImageM to work by opening the ImageM-1.3.2.1-full.mlappinstall bundle downloaded from the GitHub releases page. It added a working ImageM icon to MATLAB's APPS tab. The documentation—especially the GitHub repository's README.md and ImageM page on MATLAB Central—needs to explain in much more detail how to install this software. ○ I was finally able to get ImageM to work by opening the ImageM-1.3.2.1-full.mlappinstall bundle downloaded from the GitHub releases page. It added a working ImageM icon to MATLAB's APPS tab. The documentation—especially the GitHub repository's README.md and ImageM page on MATLAB Central—needs to explain in much more detail how to install this software. ○ I continued to receive startup errors about the MATLAB path due to backslashes in the path strings. Based on the errors I received trying to run it with backslashes in paths, it seems like ImageM is only tested on Windows. I was testing ImageM on Ubuntu Linux 20.04 LTS. ○ More generally, ImageM's documentation is lacking: I continued to receive startup errors about the MATLAB path due to backslashes in the path strings. Based on the errors I received trying to run it with backslashes in paths, it seems like ImageM is only tested on Windows. I was testing ImageM on Ubuntu Linux 20.04 LTS. ○ ll d i i l ki More generally, ImageM's documentation is lacking: Why is the manual only a PDF? Why not online as HTML or Markdown? ○ More generally, ImageM s documentation is lacking: Why is the manual only a PDF? Why not online as HTML or Markdown? ○ There is no way to quick-launch commands from the menu. A command finder or search bar would be very helpful, especially as people start developing ImageM extensions that ○ There is no way to quick-launch commands from the menu. A command finder or search bar would be very helpful, especially as people start developing ImageM extensions that ○ Page 9 of 12 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 add to the menu structure. The Help menu should offer links to important resources, especially the online docs. ○ Finally, a couple of miscellaneous comments: Mathworks named the program MATLAB in all caps ("MATLAB"), not title case ("Matlab"). F1000Research 2021, 10:333 Last updated: 01 SEP 2023 Reviewer Report 23 July 2021 Reviewer Report 23 July 2021 https://doi.org/10.5256/f1000research.54926.r89355 © 2021 Jose J et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. John Anthony Jose Electronics and Communications Enginnering Department, De La Salle University, Manila, Philippines Janos Lance Tiberio De La Salle University Manila, Manila, Philippines John Anthony Jose De La Salle University Manila, Manila, Philippines The authors are able to clearly explain the problem and contribution of the paper. However, the method of how they validated their contribution is quite limited. Specifically, they mentioned that the problem they would like to solve is that the existing approaches on visualizing and exploring multi-dimensional images lacks features in interactive interpretation. Concretely, it is about the lack of user-friendly visualization and management of image meta-data. However, their results didn’t provide any validation that their contribution is has better user-friendly approaches compared to the existing one. The authors can improve on their article by concretely discussing on how their results was able to directly address the problem that they are trying to solve. Additionally, it will be helpful if they provide comparisons with existing software for multi-dimensional image analysis. Competing Interests: No competing interests were disclosed. Page 10 of 12 Is the description of the software tool technically sound? Yes Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly Competing Interests: No competing interests were disclosed. Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Page 11 of 12 F1000Research 2021, 10:333 Last updated: 01 SEP 2023 Reviewer Expertise: Computer Vision, Image Processing We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com F1000Research 2021, 10:333 Last updated: 01 SEP 2023 Reviewer Expertise: Computer Vision, Image Processing We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com Reviewer Expertise: Computer Vision, Image Processing We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. The benefits of publishing with F1000Research: Page 12 of 12
https://openalex.org/W616698103
https://www.pure.ed.ac.uk/ws/files/23198336/1_s2.0_S2213596015001178_main.pdf
English
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Analysis of transcript changes in a heme-deficient mutant of Escherichia coli in response to CORM-3 [Ru(CO)3Cl(glycinate)]
Genomics data
2,015
cc-by
4,760
General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Edinburgh Research Explorer Edinburgh Research Explorer Analysis of transcript changes in a heme-deficient mutant of Escherichia coli in response to CORM-3 [Ru(CO)3Cl(glycinate)] Citation for published version: Wilson, JL, Mclean, S, Begg, R, Sanguinetti, G & Poole, RK 2015, 'Analysis of transcript changes in a heme-deficient mutant of Escherichia coli in response to CORM-3 [Ru(CO)3Cl(glycinate)]', Genomics Data, vol. 5, pp. 231-234. https://doi.org/10.1016/j.gdata.2015.06.008 a r t i c l e i n f o This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Specifications Organisms Escherichia coli K12 MG1655 (wild-type strain) Escherichia coli K12 W3310 hemA::KmR (hemA mutant strain; Keio collection (1,2)). Sex N/A Data acquisition Transcriptomic data were acquired using a ‘Two-Color Microarray-Based Prokaryote Analysis (FairPlay III Label- ing)’ by Agilent Technologies (Inc. 2009) and an Agilent DNA microarray scanner (G2505) controlled by Agilent Scan Control software (v8.5). Data format Raw Experimental factors The hemA mutant allele was P1-transduced from E. coli strain W3310 into E. coli strain MG1655. Global transcriptomic analyses were employed to compare changes (continued) Specifications in the transcriptome of the hemA mutant versus the wild-type following treatment with CORM-3. Experimental features Wild-type and hemA strains of E. coli MG1655 were grown under anaerobiosis in batch culture in custom-made, stirred in 250 ml mini-fermenter vessels at 37 °C during continuous sparging with 100% nitrogen gas. Cells were grown in a defined minimal medium containing 0.5% glucose as the sole and limiting source of energy and carbon. The medium was supplemented with 0.1% casamino acids and 5% LB to support the growth of the hemA mutant; kanamycin (50 μg/ml) was added to cultures of the heme-deficient mutant only. At an OD600 of 0.2, CORM-3 (100 μM) was added to wild-type and mutant cultures, and iCORM-3 (100 μM) was added to mutant cultures only. Culture samples were taken immediately prior to the addition of compounds and at 10, 20, 40, 60 and 120 min. Cells were harvested directly into phenol:ethanol and total RNA was purified using Qiagen's (continued on next page) ⁎ Corresponding author at: INSERM U955 Equipe 12, 3rd Floor Room 3053/3060, Faculté de Médecine, Université Paris-Est, 8 rue du Général Sarrail, 94010 Créteil, France. E-mail address: jayne-louise.wilson@inserm.fr (J.L. Wilson). 1 Current affiliation: School of Life Sciences, University of Nottingham, Nottingham NR7 2RD, UK. in the transcriptome of the hemA mutant versus the wild-type following treatment with CORM-3. Wild-type and hemA strains of E. coli MG1655 were grown under anaerobiosis in batch culture in custom-made, stirred in 250 ml mini-fermenter vessels at 37 °C during continuous sparging with 100% nitrogen gas. Cells were grown in a defined minimal medium containing 0.5% glucose as the sole and limiting source of energy and carbon. Analysis of transcript changes in a heme-deficient mutant of Escherichia coli in response to CORM-3 [Ru(CO)3Cl(glycinate)] Jayne Louise Wilson a,⁎, Samantha McLean a,1, Ronald Begg b, Guido Sanguinetti b, Robert K. Poole a a Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK b School of Informatics, The University of Edinburgh, Edinburgh EH8 9AB, UK Jayne Louise Wilson a,⁎, Samantha McLean a,1, Ronald Begg b, Guido Sanguinetti b, Robert K. Poole a a Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK b School of Informatics, The University of Edinburgh, Edinburgh EH8 9AB, UK Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Genomics Data 5 (2015) 231–234 Genomics Data 5 (2015) 231–234 Contents lists available at ScienceDirect Genomics Data journal homepage: http://www.journals.elsevier.com/genomics-data/ Contents lists available at ScienceDirect Contents lists available at ScienceDirect journal homepage: http://www.journals.elsevier.com/genomics-data/ journal homepage: http://www.journals.elsevier.com/genomics-data/ a r t i c l e i n f o Article history: Received 1 June 2015 Accepted 7 June 2015 Available online 13 June 2015 Keywords: Escherichia coli Heme deficient mutant CO-releasing molecule Transcriptomics Statistical modelling Article history: Received 1 June 2015 Accepted 7 June 2015 Available online 13 June 2015 Keywords: Escherichia coli Heme deficient mutant CO-releasing molecule Transcriptomics Statistical modelling This article describes in extended detail the methodology applied for acquisition of transcriptomic data, and sub- sequent statistical data modelling, published by Wilson et al. (2015) in a study of the effects of carbon monoxide- releasing molecule-3 (CORM-3 [Ru(CO)3Cl(glycinate)]) on heme-deficient bacteria. The objective was to identify non-heme targets of CORM action. Carbon monoxide (CO) interacts with heme-containing proteins, in particular respiratory cytochromes; however, CORMs have been shown to elicit multifaceted effects in bacteria, suggesting that the compounds may have additional targets. We therefore sought to elucidate the activity of CORM-3, the first water-soluble CORM and one of the most characterised CORMs to date, in bacteria devoid of heme synthesis. Importantly, we also tested inactive CORM-3 (iCORM-3), a ruthenium co-ligand fragment that does not release CO, in order to differentiate between CO- and compound-related effects. A well-established hemA mutant of Escherichia coli was used for the study and, for comparison, parallel experiments were performed on the corre- sponding wild-type strain. Global transcriptomic changes induced by CORM-3 and iCORM-3 were evaluated using a Two-Color Microarray-Based Prokaryote Analysis (FairPlay III Labeling) by Agilent Technologies (Inc. 2009). Data acquisition was carried out using Agilent Feature Extraction software (v6.5) and data normalisation, as well as information about gene products and their function was obtained from GeneSpring GX v7.3 (Agilent Technologies). Functional category lists were created using KEGG (Kyoto Encyclopedia of Genes and Genomes). Relevant regulatory proteins for each gene were identified, where available, using regulonDB and EcoCyc (World Wide Web). Statistical data modelling was performed on the gene expression data to infer transcription factor activities. The transcriptomic data can be accessed through NCBI's Gene Expression Omnibus (GEO): series acces- sion number GSE55097 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55097). © 2015 The Authors Published by Elsevier Inc This is an open access article under the CC BY license Article history: Received 1 June 2015 Accepted 7 June 2015 Available online 13 June 2015 Keywords: Escherichia coli Heme deficient mutant CO-releasing molecule Transcriptomics Statistical modelling Keywords: Escherichia coli Heme deficient mutant CO-releasing molecule Transcriptomics Statistical modelling © 2015 The Authors. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.gdata.2015.06.008 2213-5960/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 2.6. cDNA synthesis RNA (16 μg) for each sample was incubated with 5 μg of random primers (Invitrogen) at 72 °C for 10 min then chilled on ice for 10 min. cDNA synthesis was initiated by the addition of a reaction mix consisting of 6 μl 5× First Strand (FS) buffer (Invitrogen), 3 μl 0.1 M DTT (Invitrogen), 0.6 μl 50× dNTP master mix (0.1 mM dATP, dGTP, dTTP and 0.05 mM dCTP) (Roche) and 2.9 μl nuclease-free water (Qiagen). Samples were treated with either 2 μl Cy3 or 2 μl Cy5 (Invitrogen). SuperScript III (1.5 μl 200 U μl−1, Invitrogen) was added to each sample followed by 5 min incubation at 25 °C then an overnight incubation at 50 °C. Samples were hydrolysed by the addition of 15 μl 0.1 M NaOH and incubation at 72 °C for 10 min. To neutralise, 15 μl 0.1 M HCl was added. The samples were cleaned up using a QIAquick PCR purification kit (Qiagen) according to the manufacturer's 2.2. Preparation of P1 lysates for transduction of the hemA mutant allele in- to E. coli strain MG1655 Due to poor growth of the heme-deficient mutant of E. coli, the pub- lished protocol [5] was adjusted to enable transduction of the hemA muta- tion. Lysates were produced by growing the donor strain (W3110 hemA) overnight at 37 °C, during shaking at 200 rpm, in 5 ml rich broth [3] sup- plemented with 25 μM δ-ALA and 5 mM CaCl2. The culture was concentrat- ed to 1 ml in supplemented rich broth and 0.05 ml added to 0.1 ml of the wild-type MG1655 P1 lysate (2 × 109 plaque forming units/ml) diluted as follows: 10−5, 10−4, 10−3 and 10−2. The mix was incubated at 37 °C for 20 min. Pre-warmed terrific broth [TB; tryptone (8 g/l) and NaCl (5 g/l), pH 7] (1 ml; supplemented with 0.5% glucose), 25 μM δ-ALA and 1.5 ml molten terrific broth soft agar (TBSA; TB containing 7 g/l of agar) were added to the bacteria/phage cultures, mixed and poured on top of pre-warmed (37 °C) phage lysate plates (tryptone (8 g/l), yeast extract (5 g/l), NaCl (5 g/l), glucose (2 g/l) and agar (12 g/l), and after autoclaving, 10 ml 0.5 M CaCl2, 10 ml 1 M MgCl2·6H2O and 1 ml 10 mM FeCl3 were added) [5]. Once solidified, the plates were incubated at 37 °C in a moist atmosphere until plaques were nearly confluent. The plates were then chilled at 4 °C for 30 min be- fore being overlaid with 5 ml of phage dilution buffer and left over- night at 4 °C. The overlaying liquid was removed and filtered through a sterile 45 μm nitrocellulose filter into a cryovial (Nalgene) and stored at 4 °C under chloroform. 2.1. Bacterial culture conditions Starter colonies of wild-type E. coli K-12 MG1655 and E. coli K-12 MG1655 hemA were grown on nutrient agar and rich broth agar plates [3], respectively, and incubated overnight at 37 °C. For transcriptomic analysis, anaerobic liquid cultures were grown in 250 ml defined medi- um [3] in mini-fermenter vessels [4] continually sparged with nitrogen, during stirring at 200 rpm. A constant temperature of 37 °C was main- tained in the growth vessel using a water jacket from a remote water bath. Cultures were inoculated with 5% v/v of overnight starter cultures grown in rich broth [3] and then harvested and re-suspended in defined medium prior to inoculation. Optical density measurements were made using a Jenway 7315 spectrophotometer. 1. Direct link to deposited data At an OD600 of 0.2, a control sample was taken from the untreated cultures of the E. coli wild-type and hemA mutant strains, immediately followed by the addition of 100 μM CORM-3, or equimolar iCORM-3. Five further samples were taken at 10, 20, 40, 60 and 120 min post- addition of compound. At each time-point, culture samples of 20 ml were removed from minifermenter vessels (anaerobic), added to a chilled mix of 125 μl phenol and 2.38 ml ethanol and vortexed immedi- ately for 5 s, incubated on ice for 5 min followed by centrifugation for 5 min at 5500 rpm, 4 °C. The raw data have been deposited in NCBI's Gene Expression Omni- bus and are accessible through GEO series accession number GSE55097 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55097). 2.5. RNA isolation and determination RNA was isolated using a Qiagen ‘RNeasy Mini Kit (50)’ according to the manufacturer's instructions. Sample pellets were resuspended in 200 μl of TE buffer containing lysozyme (15 mg/ml) and vortexed for 10 s followed by incubation at room temperature for 5 min. Samples were vortexed every minute during the 5 min incubation. RLT buffer (700 μl) containing β mercaptoethanol (10 μl/ml) was added followed by vigorous vortexing and the addition of 500 μl 96% ethanol. Samples were mixed by gentle swirling and the resultant lysates applied to an RNeasy Mini column, centrifuged for 30 s at 10,000 rpm and the flow- through liquid discarded. Buffer RW1 (350 μl) was added to the column followed by centrifugation for 30 s at 10,000 rpm. After removal of the flow-through liquid, a mixture of 10 μl DNase I with 70 μl buffer RDD was transferred directly onto the RNeasy silicagel membrane and incu- bated at room temperature for 15 min to allow for DNase digestion. Buffer RW1 (350 μl) was then added to the column followed by a further 5 min incubation at room temperature and centrifugation for 30 s at 10,000 rpm. The column was then washed twice with 500 μl buffer RPE. To elute any remaining ethanol, the column was centrifuged for an additional 30 s at 10,000 rpm. RNase-free water (30 μl) was used for the elution of RNA by centrifugation for 1 min at 10,000 rpm. To in- crease the yield of RNA, the 30 μl was reapplied to the column and elut- ed again via centrifugation. The concentration of RNA was determined spectrophotometrically using a Beckman DU 650 UV/Vis spectropho- tometer against a DEPC-treated milli-Q H2O blank. One A260 unit is equal to 40 μg RNA per ml. The quality of the RNA was determined by running the samples on a 0.8% agarose gel in 1× TBE. Samples producing clear bands corresponding to 16S and 23S species were used for micro- array analysis. a r t i c l e i n f o The medium was supplemented with 0.1% casamino acids and 5% LB to support the growth of the hemA mutant; kanamycin (50 μg/ml) was added to cultures of the heme-deficient mutant only. At an OD600 of 0.2, CORM-3 (100 μM) was added to wild-type and mutant cultures, and iCORM-3 (100 μM) was added to mutant cultures only. Culture samples were taken immediately prior to the addition of compounds and at 10, 20, 40, 60 and 120 min. Cells were harvested directly into phenol:ethanol and total RNA was purified using Qiagen's ⁎ Corresponding author at: INSERM U955 Equipe 12, 3rd Floor Room 3053/3060, Faculté de Médecine, Université Paris-Est, 8 rue du Général Sarrail, 94010 Créteil, France. E-mail address: jayne-louise.wilson@inserm.fr (J.L. Wilson). (continued on next page) (continued on next page) http://dx.doi.org/10.1016/j.gdata.2015.06.008 2213-5960/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 016/j.gdata.2015.06.008 e Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). J.L. Wilson et al. / Genomics Data 5 (2015) 231–234 232 (continued) Specifications RNeasy Mini Kit. Two independent biological experiments were performed for each condition with two technical (dye swap) repeats per biological experiment. Consent N/A Sample/data source location The University of Sheffield, Sheffield, United Kingdom. rich broth agar [3] supplemented with 0.125 mM Na4P2O7 (an efficient Ca2+ chelator) and spread with 25 μM δ-ALA. After overnight incuba- tion at 37 °C, any putative transductant colonies were restreaked and verified by: 1) streaking a colony onto mutant validation defined medi- um agar plates containing succinate instead of glucose, with and with- out δ-ALA, and; 2) cytochrome analysis to confirm cytochrome deficiency. (continued) (continued) 2.3. P1 transduction of the recipient strain An overnight culture of the recipient strain (wild-type E. coli MG1655) was grown in 5 ml of TY broth [tryptone (16 g/l), yeast extract (10 g/l) and NaCl (10 g/l)] supplemented with 5 mM CaCl2. Aliquots (0.1 ml) of the recipient culture were mixed with 0.1 ml of the W3110 hemA lysate at the following dilutions: 100, 10−1 and 10−2. The mix was incubated for 20 min at 37 °C. The entire mixture was plated on J.L. Wilson et al. / Genomics Data 5 (2015) 231–234 233 instructions. Buffer PB was added to the samples at a ratio of 5:1 volumes. The mix was transferred into a spin column and centrifuged for 1 min at 10,000 rpm. The flow-through was discarded and the column was washed twice with 750 μl buffer PE. To ensure removal of all liquids, the column was centrifuged for an additional 1 min at 10,000 rpm. Nuclease-free water (50 μl) was used for the elution of cDNA by centrifu- gation for 1 min at 10,000 rpm. To measure the concentration of single- stranded cDNA, the labelled cDNA was denatured by heating to 95 °C for 5 min and then quantified using a NanoDrop ND-1000 UV–VIS spectro- photometer version 3.2.1 against a nuclease-free water blank. The following equations were used to calculate the yield of cDNA and its specific activity, respectively. and 40 μl of sample was slowly dispensed onto a gasket well in a ‘drag and dispense’ manner, avoiding contact between the pipette tip or hybridisation solution and the gasket walls. An array slide was placed ‘active side’-down onto the SureHyb gasket slide and the two slides held in place by the SurehHyb chamber cover and the clamp. The assem- bled chamber was vertically rotated to wet the gasket and the samples were allowed to hybridise for 17 h at 65 °C during gentle rotation. Following incubation, the microarray wash procedure for Agilent's two-colour platform was carried out according to the manufacturer's in- structions. Two gene expression (GE) wash buffers (Agilent), supple- mented with 0.005% Triton X-102 (Agilent) prior to first use, were used in this protocol. GE wash buffer #2 was preheated to 37 °C over- night. After hybridisation, the array slides were washed in GE wash buff- er #1 at room temperature for 1 min on a stirring platform set to medium speed. 2.3. P1 transduction of the recipient strain The slides were then submerged in GE wash buffer #2 and washed for 1 min then scanned immediately using an Agilent DNA microarray scanner (Agilent Technologies, G2505) controlled by Agilent Scan Control software (v8.5). Output twocolour.tiff image files were produced according to the scanning instructions in the Fairplay III microarray protocol (Agilent Technologies, 252009). cDNA ng ð Þ ¼ A260  330 ng=μl  50 μl sample volume pmol Cy3 or Cy5 per μg cDNA ¼ Cy3 ½  or Cy5 ½  ð Þ= cDNA ½  ð Þ  1000: cDNA ng ð Þ ¼ A260  330 ng=μl  50 μl sample volume pmol Cy3 or Cy5 per μg cDNA ¼ Cy3 ½  or Cy5 ½  ð Þ= cDNA ½  ð Þ  1000: cDNA ng ð Þ ¼ A260  330 ng=μl  50 μl sample volume pmol Cy3 or Cy5 per μg cDNA ¼ Cy3 ½  or Cy5 ½  ð Þ= cDNA ½  ð Þ  1000: Samples were suitable for hybridisation if the yield was N825 ng and the specific activity was N8 pmol Cy3 or Cy5 per μg DNA. 2.7. Hybridisation, washing and scanning procedures 2.8. Analysis of microarray data For each reaction, Cy3-labelled cDNA and Cy5-labelled cDNA were diluted in nuclease-free water to give a final concentration of 400 ng in a total volume of 20 μl. The diluted cDNA was boiled at 100 °C for 2 min, chilled on ice for 2 min then incubated at room temperature for a further 2 min. Blocking agent (5 μl of 10× stock) and 25 μl of 2× GEx hybridisation buffer HI-RPM were added to each reaction tube and the mix was centrifuged for 1 min at 13,000 rpm. Samples were loaded onto the array slides immediately. The Agilent microarray hy- bridization assembly consists of an Agilent SureHyb chamber, a gasket slide, an array slide and a clamp. To assemble the chamber, a clean gasket slide was inserted into the Agilent SureHyb chamber base Data acquisition was carried out using Agilent Feature Extraction software (v6.5), which allows measurement of the Cy3 and Cy5 fluores- cence of each feature in the scanned microarray image. Data were nor- malised using GeneSpring GX v7.3 (Agilent Technologies) by dividing the experimental channel by the control channel and applying a global LOWESS normalisation, which removes dye intensity-dependent arte- facts caused by non-linearity of Cy5 and Cy3 fluorescence at low levels. Identification of statistically significant gene expression changes was achieved by applying a t-test with a 2-fold cut-off and p b 0.05. Four Fig. 1. Differential expression of notable genes altered in hemA mutant or wild-type bacteria in response to CORM-3 or iCORM-3. The colour-scale bar shows mean fold changes in indi- vidual genes of the hemA mutant of E. coli and the corresponding wild-type grown anaerobically in a defined medium after the addition of 100 μM CORM-3 or, for the mutant only, 100 μM iCORM-3. Unless otherwise stated, p values were ≤0.05; * indicates a p value that exceeds 0.05. Fig. 1. Differential expression of notable genes altered in hemA mutant or wild-type bacteria in response to CORM-3 or iCORM-3. The colour-scale bar shows mean fold changes in indi- vidual genes of the hemA mutant of E. coli and the corresponding wild-type grown anaerobically in a defined medium after the addition of 100 μM CORM-3 or, for the mutant only, 100 μM iCORM-3. Unless otherwise stated, p values were ≤0.05; * indicates a p value that exceeds 0.05. J.L. Wilson et al. / Genomics Data 5 (2015) 231–234 234 could be inferred for that particular TF. 2.9. Statistical modelling of transcriptional responses 2.9. Statistical modelling of transcriptional responses Statistical data modelling was used to infer transcription factor (TF) activities based on the gene expression time-series generated from the microarray analyses. We used a probabilistic model [6], which inte- grates gene expression data with TF-target information (obtained from data bases such as EcoCyc) to determine the optimal TF activity profiles that can explain the expression data and compatibly with the constraints imposed by the network structure. The model adopts a log-linear approximation, expressing gene expression (log) changes as a weighted linear combination of changes in the activity of the TFs that regulate the genes in the network. A schematic representation of the model is given in Fig. 2; the approach is freely available as open- source software in the TFInfer tool [7]. Although the log-linear approxi- mation is a simplification of the dynamics of transcription, its simplicity permits efficient, large-scale statistical inference, so that one may obtain data-driven estimates of many TF activities simultaneously. The approach has already been extensively adopted for bacterial tran- scriptomics, leading to numerous novel insights [8]. The authors declare that there are no competing interests. We then interrogated the results of the TFInfer analysis to deduce differences in TF response between various stimuli. To do so, we computed absolute Pearson correlation coefficients between mean TF profiles from different TFInfer runs, e.g. the hemA mutant exposed to CORM-3 versus iCORM-3. For each condition, transcription factor profiles were discarded if a constant time-series could fit within the error bars given by TFInfer, indicating that no significant response 3. Conclusions This work adopted robust and established methods for the prepara- tion and growth of bacterial strains, RNA isolation and microarray anal- yses, coupled with innovative statistical modelling to identify relevant transcription factors and their roles in bacterial adaptation to CORM-3. Our study showed that CORM-3 is a potent bactericidal molecule, even against bacteria that do not contain heme, evoking general stress responses as well as disrupting the cell membrane, iron acquisition and utilisation mechanisms and zinc management processes. Acknowledgements The authors would like to thank all of the contributors to the original research article [3]. This work was supported in part by the Biotechnology and Biological Sciences Research Council (UK, BBSRC) (BB/H016805/1), a Postgraduate Studentship to JLW and the Leverhulme Trust (RPG-2013-041). G.S. acknowledges support from the European Research Council through grant MLCS 306999. Fig. 2. Schematic representation of the TFInfer modelling framework. The conceptual model underpinning TFInfer is that external stimulation elicits transcriptional responses through changes in the activity of transcription factors (TFs). Hence, a stimulus (left- hand side) will determine a change in TF activity (middle layer) which will then result in observable changes in gene expression (right panel). The changes in gene expression depend on the TF activity changes and the wiring diagram of the regulatory network, determining which TF regulates which gene(s). TFInfer adopts a log-linear approximation to model TF-gene interactions in order to solve the inverse problem. 2.8. Analysis of microarray data Profiles that were discarded from one condition, but not from the other, were given a ‘place-holder’ value of 2. The absolute value of the Pearson correlation coefficient was calculated for the remaining pairs of TF profiles, enabling us to score TFs which behaved consistently/differently in the two conditions. A value between 0 and 1 is given, where a value close to 0 represents a low cor- relation between transcription factor profiles and a value close to 1 rep- resents transcription factor profiles that are highly correlated. Taking the absolute value means that positive correlations are scored as high as negative correlations. This is done because TFInfer does not know a priori the sign of TF-gene interactions, which means that transcription factor profiles generated from TFInfer could be inverted. Only after the data have been received from the modellers can this be corrected by comparing inferred signs with information from databases on transcrip- tion factor activity and flipping a transcription factor profile, as well as the signs of its interactions, where necessary. Flipping a transcription factor profile will not affect the absolute value of the correlation coefficient. replicates were obtained for each condition tested: two biological re- peats of CORM-3- or iCORM-3-treated samples hybridised against an untreated control, each with two technical (dye-swap) repeats. Infor- mation about gene products and their function was obtained from GeneSpring GX v7.3 (Agilent Technologies). Functional category lists were created using KEGG (Kyoto Encyclopedia of Genes and Genomes) [1,2]. Relevant regulatory proteins for each gene were identified, where available, using regulonDB and EcoCyc (World Wide Web). The func- tional categories that contained the most highly altered genes are pre- sented in Fig. 1: differential expression and the function of notable genes within these categories are also shown, along with the transcrip- tion factors (TFs) involved in their regulation. 2.9. Statistical modelling of transcriptional responses Fig. 2. Schematic representation of the TFInfer modelling framework. The conceptual model underpinning TFInfer is that external stimulation elicits transcriptional responses through changes in the activity of transcription factors (TFs). Hence, a stimulus (left- hand side) will determine a change in TF activity (middle layer) which will then result in observable changes in gene expression (right panel). The changes in gene expression depend on the TF activity changes and the wiring diagram of the regulatory network, determining which TF regulates which gene(s). TFInfer adopts a log-linear approximation to model TF-gene interactions in order to solve the inverse problem. ( ) p g [4] L.J. Lee, J.A. Barrett, R.K. Poole, Genome-wide transcriptional response of chemostat- cultured Escherichia coli to zinc. J. Bacteriol. 187 (2005) 1124–1134. References [1] T. Baba, T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K.A. Datsenko, M. Tomita, B.L. Wanner, H. Mori, Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2 (2006) (2006 0008). [2] N. Yamamoto, K. Nakahigashi, T. Nakamichi, M. Yoshino, Y. Takai, Y. Touda, A. Furubayashi, S. Kinjyo, H. Dose, M. Hasegawa, K.A. Datsenko, T. Nakayashiki, M. Tomita, B.L. Wanner, H. Mori, Update on the Keio collection of Escherichia coli single-gene deletion mutants. Mol. Syst. Biol. 5 (2009) 335. [3] J.L. Wilson, L.K. Wareham, S. McLean, R. Begg, S. Greaves, B.E. Mann, G. Sanguinetti, R.K. Poole, CO-releasing molecules have nonheme targets in bacteria: transcriptomic, mathematical modeling and biochemical analyses of CORM-3 [Ru(CO)Cl(glycinate)] actions on a heme-deficient mutant of Escherichia coli. Antioxid. Redox Signal. (2015) http://dx.doi.org/10.1089/ars.2014.6151. [4] L.J. Lee, J.A. Barrett, R.K. Poole, Genome-wide transcriptional response of chemostat- cultured Escherichia coli to zinc. J. Bacteriol. 187 (2005) 1124–1134. [5] J.H. Miller, Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press N.Y., 1972 [6] G. Sanguinetti, N.D. Lawrence, M. Rattray, Probabilistic inference of transcription fac- tor concentrations and gene-specific regulatory activities. Bioinformatics 22 (2006) 2775–2781. [7] H.M. Asif, M.D. Rolfe, J. Green, N.D. Lawrence, M. Rattray, G. Sanguinetti, TFInfer: a tool for probabilistic inference of transcription factor activities. Bioinformatics 26 (2010) 2635–2636. Fig. 2. Schematic representation of the TFInfer modelling framework. The conceptual model underpinning TFInfer is that external stimulation elicits transcriptional responses through changes in the activity of transcription factors (TFs). Hence, a stimulus (left- hand side) will determine a change in TF activity (middle layer) which will then result in observable changes in gene expression (right panel). The changes in gene expression depend on the TF activity changes and the wiring diagram of the regulatory network, determining which TF regulates which gene(s). TFInfer adopts a log-linear approximation to model TF-gene interactions in order to solve the inverse problem. [8] A.I. Graham, G. Sanguinetti, N. Bramall, C.W. McLeod, R.K. Poole, Dynamics of a starvation-to-surfeit shift: a transcriptomic and modelling analysis of the bacterial response to zinc reveals transient behaviour of the Fur and SoxS regulators. Microbiology 158 (2012) 284–292.
https://openalex.org/W4392456010
https://link.springer.com/content/pdf/10.1007/s00415-024-12270-y.pdf
English
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Sex impacts treatment decisions in multiple sclerosis
Journal of neurology
2,024
cc-by
8,908
Abstract Abstract Background  Individual disease-modifying treatment (DMT) decisions might differ between female and male people with MS (pwMS). ntify sex-related differences in DMT strategies over the past decades in a real-world setting. j yf g p g Methods  In this cohort study, data from the Austrian Multiple Sclerosis Treatment Registry (AMSTR), a nationwide pro- spectively collected registry mandatory for reimbursement, were retrospectively analyzed. Of 4840 pwMS, those with relaps- ing–remitting MS, aged at least 18 years, who started DMT and had at least two clinical visits, were identified. At baseline, demographics, Expanded Disability Status Scale (EDSS) score, annualized relapse rate (ARR) in the prior 12 months and MRI lesion load were assessed. At follow-up, ARR, EDSS scores, and DMT were determined. Results  A total of 4224 pwMS were included into the study and had a median of 10 (IQR 5–18) clinical visits over an observation period of 3.5 (IQR 1.5–6.1) years. Multivariable Cox regression analysis revealed that the probability of DMT escalation due to relapse activity was lower in female than male pwMS (HR 4.1 vs. 8.3 per ARR). Probability of discontinu- ing moderate-effective DMT was higher in female pwMS when they were younger (HR 1.03 per year), and lower in male pwMS at higher age (HR 0.92). Similarly, female pwMS were more likely to stop highly effective DMT than male pwMS (HR 1.7). Among others, the most frequent reason for DMT discontinuation was family planning in female pwMS. All sex- related effects were independent of disease activity, such as MRI lesion load, baseline ARR or EDSS. Conclusions  Real-world treatment decisions are influenced by sex-related aspects. Awareness of these associations should prevent unwarranted differences in MS care. f Methods  In this cohort study, data from the Austrian Multiple Sclerosis Treatment Registry (AMSTR), a nationwide pro- spectively collected registry mandatory for reimbursement, were retrospectively analyzed. Of 4840 pwMS, those with relaps- ing–remitting MS, aged at least 18 years, who started DMT and had at least two clinical visits, were identified. At baseline, demographics, Expanded Disability Status Scale (EDSS) score, annualized relapse rate (ARR) in the prior 12 months and MRI lesion load were assessed. At follow-up, ARR, EDSS scores, and DMT were determined. p Results  A total of 4224 pwMS were included into the study and had a median of 10 (IQR 5–18) clinical visits over an observation period of 3.5 (IQR 1.5–6.1) years. Journal of Neurology (2024) 271:3256–3267 https://doi.org/10.1007/s00415-024-12270-y Journal of Neurology (2024) 271:3256–3267 https://doi.org/10.1007/s00415-024-12270-y ORIGINAL COMMUNICATION Harald Hegen1 · Klaus Berek1 · Florian Deisenhammer1 · Thomas Berger2,3 · Christian Enzinger4 · Michael Guger5,6 · Jörg Kraus7,8 · Janette Walde9 · Franziska Di Pauli1 Received: 24 October 2023 / Revised: 24 January 2024 / Accepted: 19 February 2024 / Published online: 5 March 2024 © The Author(s) 2024 Abstract Multivariable Cox regression analysis revealed that the probability of DMT escalation due to relapse activity was lower in female than male pwMS (HR 4.1 vs. 8.3 per ARR). Probability of discontinu- ing moderate-effective DMT was higher in female pwMS when they were younger (HR 1.03 per year), and lower in male pwMS at higher age (HR 0.92). Similarly, female pwMS were more likely to stop highly effective DMT than male pwMS (HR 1.7). Among others, the most frequent reason for DMT discontinuation was family planning in female pwMS. All sex- related effects were independent of disease activity, such as MRI lesion load, baseline ARR or EDSS. Conclusions  Real-world treatment decisions are influenced by sex-related aspects. Awareness of these associations should pre ent n arranted differences in MS care f Conclusions  Real-world treatment decisions are influenced by sex-related aspects. Awareness of these associations should prevent unwarranted differences in MS care. Keywords  Multiple sclerosis · Gender · Sex · Treatment · Registry Vol:.(1234567890) Keywords  Multiple sclerosis · Gender · Sex · Treatment · Registry Abbreviations AE Adverse events ALZ Alemtuzumab AMSTR Austrian Multiple Sclerosis Treatment Registry ARR​ Annualized relapse rate BL Baseline CLB Cladribine DMF Dimethyl fumarate DMT Disease-modifying treatment EDSS Expanded Disability Status Scale Harald Hegen and Klaus Berek have contributed equally to this work. * Franziska Di Pauli franziska.dipauli@i-med.ac.at 1 Department of Neurology, Medical University of Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria 2 Department of Neurology, Medical University of Vienna, Vienna, Austria 3 Comprehensive Center for Clinical Neurosciences and Mental Health, Medical University of Vienna, Vienna, Austria 4 Department of Neurology, Medical University of Graz, Graz, Austria 5 Department of Neurology, Pyhrn-Eisenwurzen Hospital Steyr, Steyr, Austria 6 Medical Faculty, Johannes Kepler University Linz, Linz, Austria 7 Department of Laboratory Medicine, Paracelsus Medical University and Salzburger Landeskliniken, Salzburg, Austria 8 Department of Neurology, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany 9 Department of Statistics, Faculty of Economics and Statistics, University of Innsbruck, Innsbruck, Austria Study design In this cohort study, we retrospectively analyzed demo- graphic, clinical, and para-clinical data from the Austrian Multiple Sclerosis Treatment registry (AMSTR). For details of the registry, we refer to Guger et al. [10]. Briefly, the AMSTR records data which have been prospectively entered into this database since 2006 during routine clinical visits from MS centers throughout Austria. This includes, at baseline, date of clinical onset of MS, number of relapses in the prior 12 months, Expanded Disability Status Scale (EDSS) score, load of hyperintense lesions on T2-weighted MRI (> 9, ≤ 9), and the use of previous DMT. At follow-up visits, occurrence of relapses, EDSS score, DMT, and adverse events (AE) are required to be documented every 3–6 months. In case of DMT change, reasons are provided (e.g., family planning or MRI activ- ity). The dataset contains records from 89 centers, dated from August 3, 2006 to November 10, 2020. Patients’ inclusion criteria Patients were eligible for inclusion if they had a diagno- sis of relapsing–remitting MS [11–13], were aged at least 18 years at start of DMT, with a minimum of 2 consecutive visits. Eligible pwMS were categorized as starting high- efficacy DMT (hDMT), i.e. ocrelizumab (OCR), alem- tuzumab (ALZ), natalizumab (NTZ), fingolimod (FTY), cladribine (CLB), or moderate-efficacy DMT (mDMT), i.e. dimethyl fumarate or teriflunomide [14–17]. All DMTs were given according to their label. Family planning, in particular pregnancy and lactation, around the use of DMT is an important issue in the care of people with MS (pwMS), especially in female pwMS, as the disease starts in young adulthood and, thus, at the age with childbearing potential [8, 9]. Maternal risk of dis- ease worsening needs to be weighed against the fetal and new-born risk due to DMT exposure. While some DMTs are contraindicated during pregnancy and lactation, and re- activation of inflammatory MS disease activity might occur in case of DMT discontinuation, evidence increased in the last years for optimized strategies particular in women with high disease activity to reduce under-treatment and preg- nancy-associated MS morbidity [9]. In addition, more safety data about fetal and new-born risk were gained. Introduction A female predominance in relapsing multiple sclerosis (RMS) is well known with a female: male ratio of 2.3–3.5:1 which even showed an increase in the last decades [1, 2]. To date, several differences in MS disease course depend- ing on the biological sex were described, e.g., women are younger at disease onset [3], show a higher relapse activity [4], develop secondary progressive MS (SPMS) later during the disease course [5], and have slower disability progres- sion [6]. Only a limited number of clinical trials performed sex-specific subgroup analysis regarding the efficacy of disease-modifying treatments (DMT), even though a recent meta-analysis found no clear sex-based difference of DMT response with regard to clinical outcomes [7]. Abbreviations Journal of Neurology (2024) 271:3256–3267 3257 FTY Fingolimod FU Follow-up GLAT Glatiramer acetate GLM Generalized linear model hDMT High-efficacy DMT IFN Interferon-beta mDMT Moderate-efficacy DMT MRI Magnetic resonance imaging NTZ Natalizumab OCR Ocrelizumab pwMS People with multiple sclerosis RMS Relapsing multiple sclerosis T2LL T2 lesions load TER Teriflunomide FTY Fingolimod FU Follow-up GLAT Glatiramer acetate GLM Generalized linear model hDMT High-efficacy DMT IFN Interferon-beta mDMT Moderate-efficacy DMT MRI Magnetic resonance imaging NTZ Natalizumab OCR Ocrelizumab pwMS People with multiple sclerosis RMS Relapsing multiple sclerosis T2LL T2 lesions load TER Teriflunomide Results We used statistical analyses to identify the impact of sex on treatment strategies adjusting for age (years), disease duration (years), DMT (categorical variable), prior ARR, baseline EDSS, baseline MRI T2L load (> 9, ≤ 9), pre-treat- ment (yes, no), EDSS progression (yes, no), and ARR on DMT. For the binary variable, mDMT vs. hDMT, logistic regression was used. For time to treatment initiation (time between disease onset and start of registry treatment), a gen- eralized linear model (GLM) with gamma distribution and log link was used taking into account the skewness of the dependent variable distribution. Cox regression was used for time to escalation (Question 1), time to de-escalation (Ques- tion 3), and time to treatment discontinuation (Question 2 and 4). For the Cox regression with the dependent variable time to escalation, we used MRI activity during DMT as additional co-variable.ff Of 4840 pwMS available in the AMSTR, a total of 4224 (87%) were included in this study and had at median of 10 (IQR 5–18) visits over an observation period of 3.5 (IQR 1.5–6.1) years. At baseline, 2792 pwMS received hDMT, while 1432 pwMS received mDMT (Fig. 1). Frequency of mDMT and hDMT were similarly distributed between women and men (Table e-1, Table e-2, Fig. 2). Time to start of DMT was associated with various disease activity measures. Female pwMS had a longer time to DMT start independent of age, clinical disease activity (including ARR before and EDSS score at treatment start), MRI lesion load, and the prior administration of DMT (Table e-3, Fig. e-1). Definition of disease activity A relapse was defined as patient-reported symptoms and objectively confirmed neurological signs typical for an acute central nervous system inflammatory demyelinat- ing event with duration of at least 24 h in the absence of fever or infection and separated from the last relapse by at least 30 days [11]. Annualized relapse rate (ARR) was calculated as the sum of relapses per observation period in years. Besides that, knowledge of patient management (reason- able or not) in real-world cohorts is of importance, as it creates awareness of daily clinical routine and may prevent unwarranted differences in MS care. Study results on sex- related differences in DMT prescriptions and treatment strat- egies in pwMS are scarce which is why we performed the present study. Disability (EDSS) progression was defined as an increase of EDSS score from baseline of at least 1.5 points if baseline EDSS was 0, 1.0 point if baseline EDSS was ≥ 1.0 and ≤ 5.0, and 0.5 points if baseline EDSS was ≥ 5.5 [18].i MRI activity was defined as new or enlarging T2 lesions [19]. Journal of Neurology (2024) 271:3256–3267 3258 information criterion (AIC) [20]. If the interaction effects were not improving the AIC, they were not included in the final model. Statistical analysis Continuous variables were displayed as median and inter- quartile range (IQR). Categorical variables were shown as frequencies and percentages. Objective We aimed to identify differences in treatment between female and male pwMS including differences in treatment escalation, de-escalation, and discontinuation. A posteriori power analyses for the above-mentioned multivariable models with binary and non-binary covariates were computed. We fixed the type one error rate at 5% and used effect sizes as revealed by the models. Either we used the actual sample size to calculate the a posteriori power, or set the power to 0.8 to compute the necessary sample size.i Definition of treatment strategies DMT escalation was defined as the patients’ first switch from mDMT to hDMT. DMT de-escalation was the patients’ first switch from hDMT to mDMT. DMT discontinuation included patients who stopped either mDMT or hDMT. i We checked all models for multicollinearity with the vari- ance inflation factor[21]. Coefficients ( 훽 ) and 95% confiden- tial intervals (CI) were presented as the main output of these models. As quality measure for the GLMs the Cox–Snell pseudo R2 measure is provided, for the Cox regressions the R-squared measure based on the partial likelihood ratio statistic [22]. Additionally, we performed sensitivity analy- ses. We ran regression analyses (for the primary outcomes) depending on DMT start, where we split our cohort into a part covering the last 5 years vs. before. Ethics Secondary outcomes subsumed i) time to DMT initiation and ii) initiation of mDMT versus hDMT. The AMSTR is approved by the ethical committee of the Medical University of Vienna (Approval number 2096/2013) and the Medical University of Innsbruck (Approval number 1235/2020). Primary outcomes The primary outcomes were time to DMT escalation (‘Ques- tion 1’), time to discontinuation of mDMT (‘Question 2’), time to DMT de-escalation (‘Question 3’), and time to dis- continuation of hDMT (‘Question 4’). These time periods were calculated as the difference between baseline (start of first registry treatment) and treatment change. A p value < 0.05 was considered statistically significant. Bonferroni–Holm correction for multiple testing was per- formed [23]. All analyses were done using the statistical software R [24] with the “powerSurvEpi” package [25]. Relapses in women are a weaker trigger for DMT escalation. Our focus was on the sex effect, so interaction effects of sex with the independent variables were selected for all models in addition to the main effects via Akaike Of 1211 pwMS receiving mDMT (Fig. 1), 149 (12.3%) were escalated to hDMT (Table e-4) due to various rea- sons, 88 (59%) had relapses, 16 (11%) isolated MRI Journal of Neurology (2024) 271:3256–3267 3259 Fig. 1   Patient selected by various inclusion criteria. ALZ, Alemtu- zumab; AMST, Austrian Multiple Sclerosis Treatment; CLA, cladrib- ine; DMF, dimethyl fumarate; DMT, disease-modifying treatment; FTY, fingolimod; FU, follow-up; GLAT, glatiramer acetate; hDMT, high-efficacy DMT; mDMT, moderate-efficacy DMT; IFN, inter- feron-beta; NTZ, natalizumab; OCR, ocrelizumab; TER, teriflunomide Fig. 1   Patient selected by various inclusion criteria. ALZ, Alemtu- zumab; AMST, Austrian Multiple Sclerosis Treatment; CLA, cladrib- ine; DMF, dimethyl fumarate; DMT, disease-modifying treatment; FTY, fingolimod; FU, follow-up; GLAT, glatiramer acetate; hDMT, high-efficacy DMT; mDMT, moderate-efficacy DMT; IFN, inter- feron-beta; NTZ, natalizumab; OCR, ocrelizumab; TER, teriflunomide DMT de‑escalation at similar frequencies in women and men with MS Of 1836 pwMS receiving NTZ or FTY over a period of at least 12 months, 78 (4.2%) were deescalated to a mDMT. Patients receiving ALZ, CLB, or OCR were not included in the analy- sis due to lack of de-escalating patients (Fig. 1). Twenty-nine (37%) switched from FTY and 49 (63%) from NTZ (Table e-9). Distribution of demographic and clinical characteristics between female and male pwMS is given in Table e-10. Most frequent reasons for de-escalation were JCV positivity (n = 38; 49%) and adverse events (n = 27; 36%) followed by pwMS’ request (n = 8; 11%), transition to SPMS (n = 4; 4%), and other reasons (n = 1; 1%). Multivariable Cox regression analysis showed that pwMS with lack of EDSS progression (β = − 0.7, [− 1.2, − 0.2]) was more likely de-escalated (Table 3). No interaction effects with sex were statistically significant. Moderate‑efficacy DMT were more likely discontinued in women at younger age. activity and 6 (4%) patients had isolated EDSS progres- sion. Twenty-nine (19%) patients reported AEs and 10 (7%) patients other causes for DMT escalation. Distribu- tion of demographic and clinical characteristics between female and male pwMS is given in Table e-5. Of 862 pwMS who were on mDMT for at least 12 months (Fig. 1), 73 (8.5%) stopped DMT (Table e-6). Distribution of demographic and clinical characteristics between female and male pwMS is given in Table e-7. Multivariable Cox regression analysis showed that var- ious disease activity-related variables, such as ARR dur- ing treatment (β = 2.1, 95% CI [1.8, 2.5]), EDSS progres- sion (β = 0.4, [0.1, 0.8]) and occurrence of MRI activity (β = 3.2, [2.5, 3.8]), were predictors of DMT escalation. Besides that, also a sex-related effect on the time to DMT escalation was observed. While an increase in ARR by 1 means an increased hazard ratio (HR) of approximately 8 to escalate DMT in men (β = 2.1, [1.8, 2.5]), the increased HR was only approximately 4 in women (β = 1.4, [1.2, 1.6]) (Table 1, Fig. 3). Multivariable Cox regression analysis showed that lower ARR (β = − 1.7, [− 3.3, − 0.1]) as well as EDSS progression (β = 0.8, [0.1–1.4,]) was associated with treatment termination. Female pwMS had a higher HR of approximately 3 for stopping treatment (β = 1.2, [0.4, 2.0]). Additionally, there was a different impact of age on the probability for treatment discontinuation depending on sex. The younger female pwMS were, the higher the HR for treatment discontinuation (β = − 0.03, [− 0.06, 0.0]). Journal of Neurology (2024) 271:3256–3267 3260 Fig. 2   Different DMT in women and men at baseline. ALZ, Alem- tuzumab; CLA, cladribine; DMF, dimethyl fumarate; DMT, disease- modifying treatment; NTZ, natalizumab; OCR, ocrelizumab; S1P, sphingosine-1-phosphate receptor modulator; TER, teriflunomide Women with MS more frequently stopped high‑efficacy DMT Of 1941 pwMS with hDMT (Fig. 1), 231 (12%) stopped DMT (Table e-11). Distribution of demographic and clinical char- acteristics between female and male pwMS is given in Table e-12. Multivariable Cox regression revealed that EDSS progres- sion (β = 0.6, [0.3, 0.9]) during DMT was associated with DMT stop. Higher relapse rate was associated with lower HR for DMT stop (β = − 1.0, [− 1.7, − 0.2]); however, this effect was reversed in a subgroup of pwMS who requested DMT stop due to various reasons including adverse events, family planning or specific patient request (Table 4). Overall, females had an increased HR of 1.7 to quit hDMT (β = 0.5, [0.2, 0.9]). The most frequent reasons for stopping hDMT were JCV positivity (n = 32; 14%), adverse events (n = 31; 13%), pro- gression (n = 31; 13%), pwMS’ request (n = 43; 19%), family planning (n = 74; 32%), neutralizing antibodies (n = 1; 0.4%), progressive multifocal leukoencephalopathy (n = 7; 3%), sta- bility (n = 3; 1%), and other reasons (n = 9; 4%). Compared to the average female predominance of approximately 70%, family planning was the reason for hDMT stopping clearly dominated by a sex effect, as all of these individuals were female (Table e-13). Fig. 2   Different DMT in women and men at baseline. ALZ, Alem- tuzumab; CLA, cladribine; DMF, dimethyl fumarate; DMT, disease- modifying treatment; NTZ, natalizumab; OCR, ocrelizumab; S1P, sphingosine-1-phosphate receptor modulator; TER, teriflunomide Contrary, male pwMS had overall a lower HR for stop- ping treatment, but the HR increased with increasing age (β = 0.09, [0.03, 0.14]) (Table 2, Fig. 4). Reasons for stopping mDMT were family planning (n = 24; 33%), patient`s request (n = 19; 26%) disease sta- bility (n = 8; 11%), and disease progression (n = 5; 7%). In 17 pwMS (26%), DMT was stopped due to AEs. Compared to the average female predominance of approximately 70%, family planning as reason for mDMT stopping was dominated by female pwMS (100%), while disease pro- gression by male sex (80%) (Table e-8). Discussion Real-world data regarding sex-related differences in MS treatment strategies are scarce. The results of our study should create awareness of potential sex-related treatment differences and avoid unwarranted differences in MS care. 3261 Journal of Neurology (2024) 271:3256–3267 Table 1   Cox regression analysis identifying predictors of DMT escalation (Question 1) Coefficient SE P value 95% CI Hazard ratio Sex (female, ref: male) 0.446 0.232 0.055 − 0.010 0.902 1.562 Age [years] − 0.021 0.010 0.036 − 0.041 − 0.001 0.979 Disease duration [years] − 0.025 0.015 0.099 − 0.056 0.005 0.975 Pre-ARR​† 0.004 0.107 0.973 − 0.206 0.214 1.004 EDSS§ 0.144 0.072 0.046 0.003 0.286 1.155 MRI ­T2LL§ (> 9, ref: ≤ 9) − 0.021 0.235 0.930 − 0.482 0.441 0.979 Pre-treatment¶ (yes, ref: no) 0.017 0.180 0.926 − 0.337 0.370 1.017 DMT (DMF, ref: TER) − 0.043 0.191 0.824 − 0.417 0.332 0.958 ARR on DMT 2.117 0.172  < 0.001 1.779 2.455 8.309 EDSS progression (yes, ref: no) 0.417 0.177 0.019 0.069 0.765 1.517 MRI activity during DMT (yes, ref: no) 3.166 0.316  < 0.001 2.546 3.786 23.709 Sex : ARR on DMT − 0.708 0.186  < 0.001 − 1.073 − 0.344 0.493 Bold p values hold with Bonferroni–Holm correction ARR​, annualized relapse rate; CI, confidence interval; DMF, dimethyl fumarate; DMT, disease-modifying treatment; EDSS, Expanded Disability Status Scale; MRI, magnetic resonance imaging; ref, reference; SE, standard error; T2LL, T2 lesions load; TER, teriflunomide ARR​, annualized relapse rate; CI, confidence interval; DMF, dimethyl fumarate; DMT, disease-modifying treatment; EDSS, Expanded Disability Status Scale; MRI, magnetic resonance imaging; ref, reference; SE, standard error; T2LL, T2 lesions load; TER, teriflunomide † ARR was determined in the 12 months prior to baseline ¶  Pre-treatment included interferon-beta and/ or glatiramer acetate [] shows units and () indicates reference categories “:” denotes interaction effects between variables Fig. 3   Time to DMT escalation depending on patients’ sex. To visu- alize the interaction effect of sex and ARR on DMT on the probabil- ity of DMT escalation, we computed the estimated Cox regression survival probabilities separately for male and female pwMS accord- ing to the occurrence of relapse (no relapse versus relapse > 1). In addition, DMT was set to “DMF”, pre-treatment “yes”, baseline MRI T2 lesion load “ > 9” and EDSS progression “no”. All other param- eters (age, disease duration, pre-ARR, baseline EDSS) were set to their median values. Discussion ARR​, annualized relapse rate; DMT, disease- modifying treatment; EDSS, Expanded Disability Status Scale; DMF, dimethyl fumarate Journal of Neurology (2024) 271:3256–3267 3262 R d b d th ti l lik lih d ti t ti ti d th C d l 0 453 Coefficient SE P value 95% CI Hazard ratio Sex (female, ref: male) 1.214 0.405 0.003 0.421 2.007 3.366 Age^ [years] 0.087 0.028 0.002 0.032 0.142 1.091 Disease duration [years] − 0.031 0.020 0.131 − 0.071 0.009 0.970 Pre-ARR​† − 0.027 0.159 0.864 − 0.338 0.284 0.973 EDSS§ 0.132 0.097 0.172 − 0.058 0.322 1.141 MRI ­T2LL§ (> 9, ref: ≤ 9) − 0.032 0.329 0.922 − 0.678 0.613 0.968 Pre-treatment¶ (yes, ref: no) 0.337 0.252 0.181 − 0.157 0.830 1.400 DMT (DMF, ref: TER) 0.617 0.316 0.051 − 0.003 1.237 1.853 ARR on DMT − 1.674 0.823 0.042 − 3.287 − 0.060 0.188 EDSS progression (yes, ref: no) 0.773 0.328  < 0.001 0.131 1.416 2.167 Sex : Age − 0.112 0.029  < 0.001 − 0.170 − 0.055 0.894 Table 2   Cox regression analysis identifying predictors of early moderate-efficacy DMT discontinuation (Question 2) R-squared based on the partial likelihood ratio statistic under the Cox model: 0.453 Bold p-values hold with Bonferroni–Holm correctioni ARR​, annualized relapse rate; CI, confidence interval; DMF, dimethyl fumarate; DMT, disease-modifying treatment; EDSS, Expanded Disability Status Scale; MRI, magnetic resonance imaging; ref, reference; SE, standard error; T2LL, T2 lesions load; TER, teriflunomide ARR​, annualized relapse rate; CI, confidence interval; DMF, dimethyl fumarate; DMT, disease-modifying treatment; EDSS, Expanded Disability Status Scale; MRI, magnetic resonance imaging; ref, reference; SE, standard error; T2LL, T2 lesions load; TER, teriflunomide † ARR was determined in the 12 months prior to baseline ¶ Pre-treatment included interferon-beta and/ or glatiramer acetate ^Due to multicollinearity with the interaction effect age was demeaned [] shows units and () indicates reference categoriesf “:” denotes interaction effects between variables f Fig. 4   Time to mDMT discontinuation depending on patients’ sex and age. To visualize the interaction effect of sex and age on the probability of treatment discontinuation, we computed the estimated Cox regression survival probabilities for male (left panel) and female pwMS (right panel), each separately for mature (age set at 55 years) and young pwMS (age set at 25 years). In addition, DMT was set to “DMF”, pre-treatment “yes”, baseline MRI T2 lesion load “ > 9”, EDSS progression “no” and ARR on DMT “0”. Discussion ARR​, annualized relapse rate; DMT, disease- modifying treatment; EDSS, Expanded Disability Status Scale; DMF, dimethyl fumarate Fig. 3   Time to DMT escalation depending on patients’ sex. To visu- alize the interaction effect of sex and ARR on DMT on the probabil- ity of DMT escalation, we computed the estimated Cox regression survival probabilities separately for male and female pwMS accord- ing to the occurrence of relapse (no relapse versus relapse > 1). In addition, DMT was set to “DMF”, pre-treatment “yes”, baseline MRI T2 lesion load “ > 9” and EDSS progression “no”. All other param- eters (age, disease duration, pre-ARR, baseline EDSS) were set to their median values. Discussion In addition, DMT was set to Journal of Neurology (2024) 271:3256–3267 3263 In our study, we observed that female pwMS had a longer approximately 8 times higher probability to escalate DMT in Table 3   Cox regression analysis identifying predictors of DMT de-escalation (Question 3) R square: 0.160 Bold p values hold with Bonferroni–Holm correction ARR​, annualized relapse rate; CI, confidence interval; DMT, disease-modifying treatment; EDSS, Expanded Disability Status Scale; FTY, fingolimod; MRI, magnetic resonance imaging; NTZ, natalizumab; ref, reference; SE, standard error; T2LL, T2 lesions load § These variables were assessed at baseline † ARR was determined in the 12 months prior to baseline ¶ Pre-treatment included interferon-beta and/or glatiramer acetate [] shows units and () indicates reference categories Coefficient SE P value 95%-CI Hazard ratio Sex (female, ref: male) − 0.385 0.235 0.101 − 0.845 0.075 0.680 Age [years] 0.014 0.014 0.326 − 0.014 0.042 1.014 Disease duration [years] 0.008 0.020 0.709 − 0.032 0.047 1.008 Pre-ARR​† − 0.060 0.102 0.558 − 0.260 0.140 0.942 EDSS§ − 0.069 0.090 0.443 − 0.246 0.107 0.933 MRI ­T2LL§ (> 9, ref: ≤ 9) 0.076 0.466 0.871 − 0.838 0.990 1.079 Pre-treatment¶ (yes, ref: no) 0.119 0.379 0.754 − 0.624 0.862 1.126 DMT (NTZ, ref: FTY) − 0.058 0.273 0.831 − 0.593 0.477 0.943 ARR on DMT 0.292 0.437 0.504 − 0.565 1.148 1.339 EDSS progression (yes, ref: no) − 0.719 0.266 0.007 − 1.240 − 0.197 0.487 Table 4   Cox regression analysis identifying predictors of early hDMT discontinuation (Question 4) R squared: 0.596 Bold p values hold with Bonferroni–Holm correction ARR​, annualized relapse rate; DMT, disease-modifying treatment; CI, confidence interval; EDSS, Expanded Disability Status Scale; FTY, fingolimod; MRI, magnetic resonance imaging; NTZ, natalizumab; ref, reference; SE, standard error; T2LL, T2 lesions load § These variables were assessed at baseline † ARR was determined in the 12 months prior to baseline ¶ Pre-treatment included interferon-beta and/ or glatiramer acetate # Additionally, interaction between the variable “ARR on DMT” and different “reasons for DMT stop” (family planning, adverse events, patient request) had to be considered due to confounding [] shows units and () indicates reference categories Coefficient SE P value 95% CI Hazard ratio Sex (female, ref: male) 0.542 0.169 0.001 0.211 0.873 1.719 Age [years] 0.001 0.008 0.927 − 0.015 0.017 1.001 Disease duration [years] − 0.021 0.012 0.090 − 0.045 0.003 0.979 Pre-ARR​† − 0.034 0.061 0.569 − 0.153 0.084 0.966 EDSS§ 0.163 0.047  < 0.001 0.070 0.255 1.176 MRI ­T2LL§ (> 9, ref: ≤ 9) 0.030 0.280 0.914 − 0.518 0.579 1.031 Pre-treatment¶ (yes, ref: no) − 0.236 0.190 0.217 − 0.608 0.138 0.791 DMT (NTZ, ref: FTY) − 0.009 0.160 0.956 − 0.322 0.304 0.991 ARR on ­DMT# − 0.957 0.377 0.011 − 1.697 − 0.217 0.384 EDSS progression (yes, ref: no) 0.634 0.152  < 0.001 0.337 0.932 1.886 Table 3   Cox regression analysis identifying predictors of DMT de-escalation (Question 3) R square: 0.160 Coefficient SE P value 95%-CI Hazard ratio Sex (female, ref: male) − 0.385 0.235 0.101 − 0.845 0.075 0.680 Age [years] 0.014 0.014 0.326 − 0.014 0.042 1.014 Disease duration [years] 0.008 0.020 0.709 − 0.032 0.047 1.008 Pre-ARR​† − 0.060 0.102 0.558 − 0.260 0.140 0.942 EDSS§ − 0.069 0.090 0.443 − 0.246 0.107 0.933 MRI ­T2LL§ (> 9, ref: ≤ 9) 0.076 0.466 0.871 − 0.838 0.990 1.079 Pre-treatment¶ (yes, ref: no) 0.119 0.379 0.754 − 0.624 0.862 1.126 DMT (NTZ, ref: FTY) − 0.058 0.273 0.831 − 0.593 0.477 0.943 ARR on DMT 0.292 0.437 0.504 − 0.565 1.148 1.339 EDSS progression (yes, ref: no) − 0.719 0.266 0.007 − 1.240 − 0.197 0.487 Bold p values hold with Bonferroni–Holm correctioni p ARR​, annualized relapse rate; CI, confidence interval; DMT, disease-modifying treatment; EDSS, Expanded Disability Status Scale; FTY, fingolimod; MRI, magnetic resonance imaging; NTZ, natalizumab; ref, reference; SE, standard error; T2LL, T2 lesions load § These variables were assessed at baseline † ARR was determined in the 12 months prior to baseline ¶ Pre-treatment included interferon-beta and/or glatiramer acetate [] shows units and () indicates reference categories Table 4   Cox regression analysis identifying predictors of early hDMT discontinuation (Question 4) R squared: 0.596 Coefficient SE P value 95% CI Hazard ratio Sex (female, ref: male) 0.542 0.169 0.001 0.211 0.873 1.719 Age [years] 0.001 0.008 0.927 − 0.015 0.017 1.001 Disease duration [years] − 0.021 0.012 0.090 − 0.045 0.003 0.979 Pre-ARR​† − 0.034 0.061 0.569 − 0.153 0.084 0.966 EDSS§ 0.163 0.047  < 0.001 0.070 0.255 1.176 MRI ­T2LL§ (> 9, ref: ≤ 9) 0.030 0.280 0.914 − 0.518 0.579 1.031 Pre-treatment¶ (yes, ref: no) − 0.236 0.190 0.217 − 0.608 0.138 0.791 DMT (NTZ, ref: FTY) − 0.009 0.160 0.956 − 0.322 0.304 0.991 ARR on ­DMT# − 0.957 0.377 0.011 − 1.697 − 0.217 0.384 EDSS progression (yes, ref: no) 0.634 0.152  < 0.001 0.337 0.932 1.886 Bold p values hold with Bonferroni–Holm correction Bold p values hold with Bonferroni–Holm correction ARR​, annualized relapse rate; DMT, disease-modifying treatment; CI, confidence interval; EDSS, Expanded Disability Status Scale; FTY, fingolimod; MRI, magnetic resonance imaging; NTZ, natalizumab; ref, reference; SE, standard error; T2LL, T2 lesions load § These variables were assessed at baseline † ARR was determined in the 12 months prior to baseline ¶ Pre-treatment included interferon-beta and/ or glatiramer acetate # Additionally, interaction between the variable “ARR on DMT” and different “reasons for DMT stop” (family planning, adverse events, patient request) had to be considered due to confounding [] shows units and () indicates reference categories § These variables were assessed at baseline ¶ Pre-treatment included interferon-beta and/ or glatiramer acetate # Additionally, interaction between the variable “ARR on DMT” and different “reasons for DMT stop” (family planning, adverse events, patient request) had to be considered due to confounding In our study, we observed that female pwMS had a longer time to DMT start independent of age, clinical disease activ- ity (ARR, EDSS) and MRI lesion load. Table 4   Cox regression analysis identifying predictors of early hDMT discontinuation (Question 4) Discussion All other parameters (disease duration, pre-ARR, baseline EDSS) were set to their median values. ARR​, annualized relapse rate; DMF, dimethyl fumarate; DMT, disease-modifying treatment; EDSS, Expanded Disability Status Scale; mDMT, moderate-efficacy DMT “DMF”, pre-treatment “yes”, baseline MRI T2 lesion load “ > 9”, EDSS progression “no” and ARR on DMT “0”. All other parameters (disease duration, pre-ARR, baseline EDSS) were set to their median values. ARR​, annualized relapse rate; DMF, dimethyl fumarate; DMT, disease-modifying treatment; EDSS, Expanded Disability Status Scale; mDMT, moderate-efficacy DMT Fig. 4   Time to mDMT discontinuation depending on patients’ sex and age. To visualize the interaction effect of sex and age on the probability of treatment discontinuation, we computed the estimated Cox regression survival probabilities for male (left panel) and female pwMS (right panel), each separately for mature (age set at 55 years) and young pwMS (age set at 25 years). Discussion Female pwMS also had delayed treatment escalation despite relapses com- pared to male pwMS. An increase in ARR by 1 means an approximately 8 times higher probability to escalate DMT in men, but only 4 times higher probability in women. In addi- tion, discontinuation of mDMT was more likely in females, especially when they were younger, and the probability to stop hDMT was higher in female compared to male pwMS. Journal of Neurology (2024) 271:3256–3267 3264 clinical trials, strict eligibility criteria such as consent to contraception during the study period may not reflect the diverse patient population encountered in routine clinical practice. In addition to clinical trials, an increasing number of real-world studies are available, but these mainly focus on different treatment strategies such as the early use of highly effective therapies versus their late use or the relapse rate after stopping DMT. Since the propensity score method is often used in these studies to reduce sex-selection bias through matching, they do not allow any statement in regard of sex-related differences in treatment strategies. On the other hand, the real-world register data shown here enables a representative overview of a sex-bias in the treatment of MS. Especially since an entry in the registry is required for reimbursement and a special quality-related feature of the AMSTR is that data where external and independent moni- tored. This guarantees improved acquisition and complete- ness of the data, and the representativeness of the cohort. Similar results were found in the Danish Multiple Scle- rosis Registry [26]. Men were more likely to receive hDMT right from the start (odds ratio 1.53) and were more likely to be escalated to hDMT (odds ratio 2.03) than women. Sex differences in treatment discontinuation or de-escalation were not examined in this study. Supporting the results of our study, further work reported that women received hDMT less frequently than men (odds ratio 0.92) [27]. In addition, consistent with our results, this study showed that younger age, higher relapse rate, and higher EDSS scores were also associated with a higher likelihood of hDMT [27]. Fur- thermore, in 499 pwMS receiving interferon-beta as first DMT, interferon-beta discontinuation was more frequent in female pwMS than in male pwMS (HR 1.42) [28].These results were confirmed by another study, which showed that women were more likely to stop interferon-beta or glati- ramer acetate initiated after MS diagnosis than men [29]. Discussion The limitation of these studies is that there are no data for hDMT discontinuation.ff There are some limitations to this study. The first limita- tion of the study is that the AMSTR has existed since the first hDMT was approved and therefore previously approved therapies (interferon-beta preparations, glatiramer acetate) are not captured in this registry. This also explains the high percentage (66%) of hDMT in the AMSTR, as many patients were pre-treated with interferon-beta or glatiramer acetate. Also, the data on the discontinuation of newer treatments such as CD20 antibodies or the de-escalation of these are therefore very limited. Furthermore, in case of treatment dis- continuation, no further follow-up data are available, unless patients re-start any treatment again in future. Although we included a set of relevant co-variables in the multivariable analyses, other potential confounders might not have been covered, e.g. MRI data were only available in patients with DMT escalation, and not available in patients before DMT discontinuation or de-escalation. Such information might impact on treatment decision making. Also, after identify- ing a sex-related effect, e.g., on DMT stopping, we could only describe the possible causes, such as family planning as a main reason in approximately 30% of DMT stoppers and that all of them were females. This observation provides an exploratory hypothesis for the observed sex-effect, but could not be included in the multivariable analysis, as this information was not available for pwMS continuing DMT. However, subgroup analyses confirmed this hypothesis, as after exclusion of female pwMS with family planning, the sex-related effect was lost (Table e-14 and Table e-15). Fur- thermore, the reason “family planning” was not available for all research questions (e.g., time to DMT start), and a differentiation between wish for pregnancy, pregnancy and lactation was not possible due to lack of data. One reason for different treatment strategies and differ- ent weighting of disease activity is family planning. In our study, family planning was one of the most common reasons to discontinue a DMT. The AMSTR started in 2006 [10]. At that time and in subsequent years, there was insufficient evidence of fetal risk and use of DMT. Thus, all DMT had a contraindication in pregnancy and were discontinued before conception or in early pregnancy [30]. Due to increasing data from pregnancy registries, it is now possible to continue certain therapies depending on the activity of the disease and individual benefit–risk evaluation [9]. Discussion CE received funding for travel and speaker honoraria from Biogen, Bayer, Celgene, Merck, Novartis, Roche, Shire, Gen- zyme, and Teva Pharmaceutical Industries Ltd./sanofi-aventis; re- search support from Merck Serono, Biogen, and Teva Pharmaceutical Industries Ltd./sanofi-aventis; serving on scientific advisory boards for Bayer, Biogen, Celgene, Merck, Novartis, Roche and Teva Pharma- ceutical Industries Ltd./sanofi-aventis. MG received support and hono- raria for research, consultation, lectures and education from Alexion, Almirall, Bayer, Biogen, Bristol-Myers-Squibb, Celgene, Genzyme, Horizon, Janssen-Cilag, MedDay, Merck, Novartis, Roche, Sanofi Aventis and TEVA ratiopharm. JK received consulting and/or research funding and/ or educational support from Almirall, Bayer, Biogen, Celgene/ Bristol Myers Squibb, MedDay, Medtronic, Merck, Novartis, Roche, Sanofi- Aventis, Shire, and TEVA ratiopharm. JW has nothing to disclose. FDP has participated in meetings sponsored by, received honoraria (lectures, advisory boards, consultations) or travel fund- ing from Bayer, Biogen, Celgene, Merck, Novartis, Sanofi-Genzyme, Roche and Teva. i.e., statistically significant effects of co-variables remained qualitatively the same (for the primary outcomes). There was only one exception. After 2015, the frequency of pwMS discontinuing hDMT was lower (14% vs. 7%). This might be attributed to different treatment strategies in the last years due to the risk of rebound phenomenon. Whether the observed sex-related effect (main effect) remains after 2015, evidence is not unambiguous yet. A larger time series would be necessary.f Besides the sex-related effects as discussed above, there might be additional sex-related effects that were not uncov- ered in our study due to low statistical power, e.g., the sex effect on time to DMT de-escalation (showing a clinically relevant coefficient of − 0.4 and a posteriori power of 0.5). There is one exception. The lacking effect of sex on time to DMT escalation is indeed supported by the power analysis. A posteriori power analyses for the multivariable models are given in Table e-20, e-21, e-22 and e-23. Since several studies have shown an increasing risk of disability due to relapses during pregnancy, awareness of the conscious or partly unconscious different treatment of women and men is of great importance for the treating neu- rologist. Our study results should increase the awareness of sex-related treatment differences, thus, prevent unwarranted treatment decisions and eventually prevent further disease activity and morbidity especially in women. Discussion Despite the clear results of our study, a replication in a different cohort would be desirable, where also some of the above-men- tioned limitations could be addressed, e.g., where patients on immunodepleting agents such as CD20 antibodies are included. Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Supplementary Information  The online version contains supplemen- tary material available at https://​doi.​org/​10.​1007/​s00415-​024-​12270-y. Discussion i E.g., in the case of NTZ treatment, bearing a high risk of disease reactivation or even rebound activity, treatment interruption should be avoided to prevent women from fur- ther, potentially debilitating disease activity [31]. Whereas in pwMS treated with NTZ (depending on the activity of the disease), it is possible to continue the therapy with a different treatment interval, S1P receptor modulators (where rebound or a high risk of disease reactivation is also described [32]) must be discontinued due to their potential teratogenic effect [33]. Few data are available regarding treatment with CD20 antibodies and pregnancy. Although continuous administra- tion of CD20 antibodies is necessary to suppress disease activity, no excessive disease activity after discontinuation has been observed so far [34]. In contrast to hDMT, discon- tinuation of mDMT in women with a stable course of disease without clinical or radiological disease activity is considered relatively safe [35].fff Until now, a different effectiveness of the different DMT depending on sex could not be proven and does not justify different treatment [2, 36]. However, in clinical studies, sex subgroup analyses are still rarely done and a definitive state- ment regarding a different treatment response in female and male pwMS is not possible yet with certainty [7, 37]. In We also performed sensitivity analyses (Table e-16, e-17, e-18 and e-19). We did not observe a cohort effect, when we compared pwMS starting DMT before or after 2015, 3265 Journal of Neurology (2024) 271:3256–3267 i.e., statistically significant effects of co-variables remained qualitatively the same (for the primary outcomes). There was only one exception. After 2015, the frequency of pwMS discontinuing hDMT was lower (14% vs. 7%). 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Relationship of Topology, Multiscale Phase Synchronization, and State Transitions in Human Brain Networks
Frontiers in computational neuroscience
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Relationship of Topology, Multiscale Phase Synchronization, and State Transitions in Human Brain Networks Minkyung Kim 1, 2, Seunghwan Kim 1, George A. Mashour 2, 3 and UnCheol Lee 2, 3* 1 Department of Physics, Pohang University of Science and Technology, Pohang, South Korea, 2 Center for Consciousness Science, University of Michigan Medical School, Ann Arbor, MI, United States, 3 Department of Anesthesiology, University of Michigan Medical School, Ann Arbor, MI, United States How the brain reconstitutes consciousness and cognition after a major perturbation like general anesthesia is an important question with significant neuroscientific and clinical implications. Recent empirical studies in animals and humans suggest that the recovery of consciousness after anesthesia is not random but ordered. Emergence patterns have been classified as progressive and abrupt transitions from anesthesia to consciousness, with associated differences in duration and electroencephalogram (EEG) properties. We hypothesized that the progressive and abrupt emergence patterns from the unconscious state are associated with, respectively, continuous and discontinuous synchronization transitions in functional brain networks. The discontinuous transition is explainable with the concept of explosive synchronization, which has been studied almost exclusively in network science. We used the Kuramato model, a simple oscillatory network model, to simulate progressive and abrupt transitions in anatomical human brain networks acquired from diffusion tensor imaging (DTI) of 82 brain regions. To facilitate explosive synchronization, distinct frequencies for hub nodes with a large frequency disassortativity (i.e., higher frequency nodes linking with lower frequency nodes, or vice versa) were applied to the brain network. In this simulation study, we demonstrated that both progressive and abrupt transitions follow distinct synchronization processes at the individual node, cluster, and global network levels. The characteristic synchronization patterns of brain regions that are “progressive and earlier” or “abrupt but delayed” account for previously reported behavioral responses of gradual and abrupt emergence from the unconscious state. The characteristic network synchronization processes observed at different scales provide new insights into how regional brain functions are reconstituted during progressive and abrupt emergence from the unconscious state. This theoretical approach also offers a principled explanation of how the brain reconstitutes consciousness and cognitive functions after physiologic (sleep), pharmacologic (anesthesia), and pathologic (coma) perturbations. Keywords: emergence, explosive synchronization, state transition, anesthesia, brain network, consciousness, Kuramoto model ORIGINAL RESEARCH published: 30 June 2017 doi: 10.3389/fncom.2017.00055 Edited by: Jamie Sleigh, University of Auckland, New Zealand Edited by: Jamie Sleigh, University of Auckland, New Zealand Reviewed by: Jesus M. Cortes, BioCruces Health Research Institute, Spain Zhenhu Liang, Yanshan University, China *Correspondence: UnCheol Lee uclee@med.umich.edu Received: 26 January 2017 Accepted: 07 June 2017 Published: 30 June 2017 Citation: Kim M, Kim S, Mashour GA and Lee U (2017) Relationship of Topology, Multiscale Phase Synchronization, and State Transitions in Human Brain Networks. Front. Comput. Neurosci. 11:55. doi: 10.3389/fncom.2017.00055 Received: 26 January 2017 Accepted: 07 June 2017 Published: 30 June 2017 INTRODUCTION synchronization patterns in computational models based on neuroanatomically-derived human brain networks. We implemented the Kuramoto model, a simple oscillatory model, in a human brain network with 82 nodes (including cortical and subcortical areas) to simulate the dynamic interactions among brain regions. To facilitate the delayed but abrupt transition, we applied the principle of explosive synchronization, derived from network science, as a potential mechanism for the discontinuous transition from a desynchronized to synchronized state (Gómez- Gardeñes et al., 2011). High frequency disassortativity (Leyva et al., 2013b; Zhu et al., 2013; Skardal and Arenas, 2014) was applied to the human brain network in order to suppress giant synchronization cluster formation (Zhang et al., 2014, 2015). This network configuration primarily prohibits hubs from synchronizing, which leads to a delay in synchronization that reaches a critical point of abrupt global synchronization. How does the brain reconstitute the capacity for consciousness and cognition after a major perturbation like general anesthesia? What determines reversibility in some states (e.g., sleep) and irreversibility in others (e.g., coma)? The underlying mechanism of the reconstitution of brain function is poorly understood despite significant neuroscientific and clinical implications. Anesthesia has been used as a tool to inhibit spontaneous brain activities and reversibly suppress consciousness but more recently has been used to investigate the recovery process from unconsciousness. Recent empirical studies demonstrated that brain recovery from anesthetic-induced unconsciousness is not random, but ordered. Hudson et al. found that during the emergence from anesthesia, brain dynamics pass through an ordered sequence of states that is different from a random walk (Hudson et al., 2014). Furthermore, diverse emergence patterns have been observed from the electroencephalogram (EEG) of humans. For example, Hight et al. reported two distinct emergence patterns in general anesthesia (Hight et al., 2014). One showed progressive spectral changes of EEG before the response, while the other showed no explicit change of EEG spectral properties before the abrupt return of responsiveness. Chander et al. classified the emergence patterns into four types based on the spectral behaviors of EEG such as delta (1–4 Hz) and alpha/spindle (8–14 Hz), as well as different levels of pain (Chander et al., 2014). These emergence patterns can be qualitatively described as “progressive and earlier state transition” and “abrupt but delayed state transition.” However, previous studies examined local field potentials and frontal EEG rather than global brain activities. Lee et al. Citation: June 2017 | Volume 11 | Article 55 Frontiers in Computational Neuroscience | www.frontiersin.org 1 Synchronization of Brain Networks Kim et al. INTRODUCTION identified network recovery properties in healthy individuals emerging from anesthesia that followed similar patterns, suggesting the possibility that differential network principles account for various behavioral phenotypes (Lee et al., 2011). We furthermore compared distinct synchronization processes between progressive and abrupt transitions on the scale of the individual node, clusters, and global network structure. We demonstrate that the distinctive synchronization processes are significantly determined by the underlying brain network structure with a given frequency configuration. This approach could provide a principled explanation of how brain networks reconstitute regional activities during progressive and abrupt emergence at a network level, which could be applied to recovery from physiologic (sleep), pharmacologic (anesthesia), and pathologic (coma) states of unconsciousness. Network Model We used a simple phase oscillator model, the Kuramoto model (Kuramoto, 1984), in a group-averaged anatomical brain network to simulate the dynamic behavior of two different types of emergence patterns. The Kuramoto model is defined as the following: The synchronization process has been studied with cat and human brain networks investigating the role of hub, modular structure, and global network structure (Honey and Sporns, 2008; Breakspear et al., 2010; Gómez-Gardeñes et al., 2010; Cabral et al., 2011; Villegas et al., 2014; Hellyer et al., 2015; Schmidt et al., 2015; Váša et al., 2015; Finger et al., 2016). However, these studies were limited to progressive synchronization and did not address delayed or abrupt synchronization, which is potentially applicable to delayed anesthetic emergence. In this study we compare, for the first time, the distinct synchronization processes in a human brain network under progressive and abrupt synchronization conditions. Our main objective is to understand distinct emergence patterns in terms of network synchronization rather than model state-specific EEG signatures, per-se. Because temporal coordination is a necessary condition for information integration and transmission across brain regions, the recovery pattern of the brain network synchronization may reflect the recovery pattern of consciousness. θi = ωi + λ XN j=1 Aij sin(θj −θi), i = 1, 2, . . . N (1) (1) Here, θi is the phase, ωi is the initial angular frequency of ith oscillator, and λ is the coupling strength between all connected nodes. N is the total number of nodes and Aij is the adjacency matrix, which is an anatomical brain network structure. The anatomical brain network was acquired from group-averaged diffusion tensor imaging (DTI) with 82 nodes, including cortical and subcortical areas (Van Den Heuvel and Sporns, 2011). Frontiers in Computational Neuroscience | www.frontiersin.org Network Configuration We also calculated the frequency disassortativity (ρf), defined as a Pearson correlation between node frequency and the average frequency of neighbor nodes, in order to achieve a more robust occurrence of abrupt transitions (Li et al., 2013). Large frequency disassortativity enhances the frequency mismatches between neighbor nodes and makes it possible to overcome the homogeneity of the network structure itself (Boccaletti et al., 2016). We generated 100 frequency configurations with large values of frequency disassortativity (ρf < −0.3) to analyze the characteristics of abrupt transition. Gaussian distribution with mean 10 Hz and variance 0.2 Hz with large frequency disassortativity (ρf < −0.3) and various frequency configurations with diverse frequency disassortativity values were also simulated for a comparison of Network Configuration Initial phases randomly distributed between (−π, π) and specific frequency distributions for progressive and abrupt transition types were assigned to the nodes. In this simulation, we assume that different initial frequency distributions reflect different regional brain dynamics. According to explosive synchronization, the initial frequency distribution within the network topology may determine the synchronization path from the desynchronized state. We used a Gaussian distribution with the mean 10 Hz and variance 0.2 Hz to simulate the In this simulation study, we modeled the potential network mechanisms for these archetypal emergence patterns (“progressive and earlier” and “abrupt but delayed”) by assessing June 2017 | Volume 11 | Article 55 Frontiers in Computational Neuroscience | www.frontiersin.org 2 Synchronization of Brain Networks Kim et al. TABLE 1 | Glossary of terms. Keywords Descriptions Node Degree The number of edges/links connected to a node in a network. Hub In this study, a node that has a high degree is defined as a hub node. Hub structure plays a crucial role in communication and information transmission in the brain. First-order phase transition Discrete changes from incoherent to synchronized state or vice versa, as the coupling strength of coupled oscillators increases or decreases, respectively. A more continuous change is referred to as a “second-order phase transition.” Explosive (or abrupt) synchronization A phenomenon characterized by first-order phase transition between incoherent and synchronized states in a network of coupled oscillators. The key mechanism of explosive synchronization is to suppress the formation of a giant synchronization cluster in a network, mainly inhibiting the hub synchronizations. Progressive synchronization A phenomenon characterized by a second-order phase transition between incoherent and synchronized states in a network of coupled oscillators. The hub node dominates the synchronization process by entraining the neighbor nodes. Frequency disassortativity A tendency for nodes oscillating at higher frequencies to connect with nodes at lower frequencies, or vice versa. Large frequency disassortativity contributes to generating the network conditions for explosive synchronization. In this study, a node that has a high degree is defined as a hub node. Hub structure plays a crucial role in communication and information transmission in the brain. Discrete changes from incoherent to synchronized state or vice versa, as the coupling strength of coupled oscillators increases or decreases, respectively. Network Configuration We also calculated the frequency disassortativity (ρf), defined as a Pearson correlation between node frequency and the average frequency of neighbor nodes, in order to achieve a more robust occurrence of abrupt transitions (Li et al., 2013). Large frequency disassortativity enhances the frequency mismatches between neighbor nodes and makes it possible to overcome the homogeneity of the network structure itself (Boccaletti et al., 2016). We generated 100 frequency configurations with large values of frequency disassortativity (ρf < −0.3) to analyze the characteristics of abrupt transition. Gaussian distribution with mean 10 Hz and variance 0.2 Hz with large frequency disassortativity (ρf < −0.3) and various frequency configurations with diverse frequency disassortativity values were also simulated for a comparison of the robustness (Figure S1). See Table 1 for an explanation of network terminology. alpha bandwidth of human EEG activity (Moon et al., 2015). Here, we considered only the alpha frequency band (9 to 11 Hz), because the alpha frequency band shows significant and consistent global connectivity changes along with state changes induced by diverse anesthetics (Lee H. et al., 2013; Blain-Moraes et al., 2014; Kim et al., 2016). We generated 100 frequency configurations of Gaussian distribution to observe canonical behaviors of progressive transition. To simulate the abrupt transition, we first selected 15 nodes (18% of all nodes) with high degrees (i.e., high number of connections) as hub nodes on the basis of rich club organization using degree k = 21 (Van Den Heuvel and Sporns, 2011). These hub nodes include thalamus, hippocampus, putamen, superior frontal, superior parietal, precuneus, and insula of both hemispheres, which are potentially related to consciousness (Bogen, 1995; Martuzzi et al., 2010; Ku et al., 2011; Spoormaker et al., 2012) and important for inter-modular synchronization in human brain networks (Schmidt et al., 2015). We used different frequency distributions for the hub nodes (Gaussian distribution with mean 10.3 Hz and variance 0.05 Hz for 6 nodes; Gaussian distribution with mean 9.7 Hz and variance 0.05 Hz for 9 nodes), which can be one way to suppress the formation of a giant synchronization cluster in the system by inducing large frequency mismatches between high degree nodes (Zhu et al., 2013). In particular, we assigned the relationship between node degree and frequency a V-shape, which is similar to a previous study of explosive synchronization (Leyva et al., 2013b). Synchronization Measures and Computation We numerically solved the differential equations of the Kuramoto model using the 4th order Runge-Kutta method with 1,000 discretization steps. The first half of the time series was discarded and the last 15 of 30 s were used for each simulation. The sampling rate was 1,000 Hz and the coupling strength λ increases from 0 to 0.4 with δλ = 0.002. In order to observe the dynamics of the functional network of each λ, we calculated the average pairwise synchrony between node i and j, Dij, defined as Dij = Aij 1 1t Xτ+1t τ ei[θi(t)−θj(t)] (2) (2) which is a symmetric phase synchronization matrix. Using the Dij, we can obtain an order parameter to estimate the level of global synchronization, rlink, rlink = 1 2Nl X i,j Dij (3) (3) where Nl is the total number of links. We also examined the synchronization level of each node, with the local order parameter represented as, ri = 1 2ni X j ∈nni Dij (4) (4) where ni is the number of links connected with node i. We used median values for global and local order parameters of 100 configurations for the analysis in order to avoid the confound of outliers. With ri, we compared synchronization processes of two transitions at the individual node level. Network Configuration A more continuous change is referred to as a “second-order phase transition.” A phenomenon characterized by first-order phase transition between incoherent and synchronized states in a network of coupled oscillators. The key mechanism of explosive synchronization is to suppress the formation of a giant synchronization cluster in a network, mainly inhibiting the hub synchronizations. A phenomenon characterized by a second-order phase transition between incoherent and synchronized states in a network of coupled oscillators. The hub node dominates the synchronization process by entraining the neighbor nodes. A tendency for nodes oscillating at higher frequencies to connect with nodes at lower frequencies, or vice versa. Large frequency disassortativity contributes to generating the network conditions for explosive synchronization. alpha bandwidth of human EEG activity (Moon et al., 2015). Here, we considered only the alpha frequency band (9 to 11 Hz), because the alpha frequency band shows significant and consistent global connectivity changes along with state changes induced by diverse anesthetics (Lee H. et al., 2013; Blain-Moraes et al., 2014; Kim et al., 2016). We generated 100 frequency configurations of Gaussian distribution to observe canonical behaviors of progressive transition. To simulate the abrupt transition, we first selected 15 nodes (18% of all nodes) with high degrees (i.e., high number of connections) as hub nodes on the basis of rich club organization using degree k = 21 (Van Den Heuvel and Sporns, 2011). These hub nodes include thalamus, hippocampus, putamen, superior frontal, superior parietal, precuneus, and insula of both hemispheres, which are potentially related to consciousness (Bogen, 1995; Martuzzi et al., 2010; Ku et al., 2011; Spoormaker et al., 2012) and important for inter-modular synchronization in human brain networks (Schmidt et al., 2015). We used different frequency distributions for the hub nodes (Gaussian distribution with mean 10.3 Hz and variance 0.05 Hz for 6 nodes; Gaussian distribution with mean 9.7 Hz and variance 0.05 Hz for 9 nodes), which can be one way to suppress the formation of a giant synchronization cluster in the system by inducing large frequency mismatches between high degree nodes (Zhu et al., 2013). In particular, we assigned the relationship between node degree and frequency a V-shape, which is similar to a previous study of explosive synchronization (Leyva et al., 2013b). Frontiers in Computational Neuroscience | www.frontiersin.org Reconstitution Order of Brain Region Reconstitution Order of Brain Region We calculated the difference between first coupling strength values λri>0.2 and λri>0.8 satisfying ri ≥ 0.2 and ri ≥ 0.8 to investigate the reconstitution order of brain regions. The difference between the coupling strengths of each node was deemed to be the integration duration τ from a low synchronization level in an unconscious state to a high synchronization level in a conscious state. The given thresholds are empirically observed synchronization levels (Kim et al., 2016). Network Configurations Define Progressive and Abrupt Transitions Progressive and Abrupt Transitions Figure 1A presents two exemplary cases of progressive and abrupt transitions in a brain network as coupling strength increases. The distinctive patterns, progressive (blue), and abrupt (red), of global order parameters, rlink, for both transitions are clear. The rlink of the progressive transition continuously increases from an unsynchronized to synchronized state, but the rlink of the abrupt transition jumps discontinuously at λ = 0.148, which is suggestive of a process involving explosive synchronization. Figures 1B–E shows the different initial network configurations for the progressive and abrupt transitions. The relationships between initial frequencies and degrees for 82 nodes are shown in Figures 1B,C. For the abrupt transition, we assigned a V-shape to the relationship between frequency and node degree, in accordance with a previous study (Leyva et al., 2013b). The V-shape relationship yields large frequency mismatches between high degree nodes, which inhibit the formation of giant synchronization clusters. The relationships between frequencies and the average neighbor frequencies are illustrated in Figures 1D,E. The frequency disassortativities are −0.098 and −0.430 for progressive and abrupt transitions. The large frequency disassortativity (ρf < −0.3) generates a tendency for a higher frequency node to have lower frequency neighbor nodes. These frequency mismatches applied to the nodes are more likely to produce abrupt synchronization in a network (Li et al., 2013). Synchronization Cluster Analysis After exploring the behaviors of synchronization processes at the individual node level, we next investigated the synchronization process at the cluster level. One of the significant differences June 2017 | Volume 11 | Article 55 Frontiers in Computational Neuroscience | www.frontiersin.org 3 Synchronization of Brain Networks Kim et al. Kim et al. between progressive and abrupt transitions is the process of cluster merging. Therefore, we examined the process of synchronization cluster formation in the two transition types. We constructed the binary synchronization matrix Sij defined as corrections. For the comparison of reconstitution orders of each region between two transitions, we used Wilcoxon rank sum test and defined the nodes with p < 0.05 as the brain regions with significantly different reconstitution orders between progressive and abrupt transitions. From this analysis, we could predict the regional recovery process in a transition as well as the differences of the global network recovery process between progressive and abrupt emergence patterns. Sij = 1, if Dij > 0.95 Sij = 0, otherwise (5) (5) We considered two nodes i and j as synchronized if Dij is larger than 0.95 (Zhang et al., 2014). The synchronization threshold of 0.95 between connected links in the human brain network demonstrated distinctive changes in terms of the number of clusters and the size of a giant cluster for both transition patterns, and revealed the critical states for each transition pattern. With the matrix Sij for each coupling strength λ, we calculated the number of synchronization clusters, NC, and the size of a giant synchronization cluster, GC, which is the largest synchronization cluster among all clusters. We then took the median of 100 configurations for each transition. We considered two nodes i and j as synchronized if Dij is larger than 0.95 (Zhang et al., 2014). The synchronization threshold of 0.95 between connected links in the human brain network demonstrated distinctive changes in terms of the number of clusters and the size of a giant cluster for both transition patterns, and revealed the critical states for each transition pattern. With the matrix Sij for each coupling strength λ, we calculated the number of synchronization clusters, NC, and the size of a giant synchronization cluster, GC, which is the largest synchronization cluster among all clusters. We then took the median of 100 configurations for each transition. Relationship between Structure and Dynamics y We examined the global relationship between structure and dynamics during both transitions to grasp the detailed synchronization process within the network topology. We first calculated the Spearman correlation between degree and median local order parameter over 100 configurations of each λ. We then divided the brain network into 5 subgroups with degree order; (1) 4 ≤k < 11, (2) 11 ≤k < 14, (3) 14 ≤k < 18, (4) 18 ≤k < 22, (5) 22 ≤k < 32 to understand the dynamics of hub and peripheral nodes within the network structure. Each subgroup has at least 15 nodes. In one configuration, the synchronization level of each subgroup (rs) was acquired by taking the average of ri within a subgroup. We then took the median of the average values over the repeated 100 configurations. We compared the synchronization level and rank among subgroups to reveal further details of the synchronization process for every λ associated with topology. Global and Local Synchronization Processes for Progressive and Abrupt Transitions The relationships between initial frequencies and node degrees for e and (C) abrupt transitions are presented. The abrupt transition has a V-shape in the relationship. The relationships between initial frequencies and encies of the nearest neighbor nodes are also presented for (D) progressive transition (ρf =−0.098) and (E) abrupt transition (ρf =−0.430). obal and local synchronization for progressive and abrupt transitions. (A) Median global order parameter of progressive (blue) and abrupt (red) 100 frequency configurations. Colored area indicates the 25–75% values of rlink of 100 configurations. Local order parameter ri of (B) progressive and sition. The nodes are aligned with descending order of degree from top to bottom. Color indicates the value of ri. FIGURE 1 | Distinct phase synchronization patterns in computational models of the human brain. (A) Exemplary cases for progressive (blue) and abrupt (red) transitions. The abrupt transition shows a sharp increase of the order parameter at λ = 0.148 . The relationships between initial frequencies and node degrees for (B) progressive and (C) abrupt transitions are presented. The abrupt transition has a V-shape in the relationship. The relationships between initial frequencies and average frequencies of the nearest neighbor nodes are also presented for (D) progressive transition (ρf =−0.098) and (E) abrupt transition (ρf =−0.430). FIGURE 1 | Distinct phase synchronization patterns in computational models of the human brain. (A) Exemplary cases for progressive (blue) and abrupt (red) transitions. The abrupt transition shows a sharp increase of the order parameter at λ = 0.148 . The relationships between initial frequencies and node degrees for (B) progressive and (C) abrupt transitions are presented. The abrupt transition has a V-shape in the relationship. The relationships between initial frequencies and average frequencies of the nearest neighbor nodes are also presented for (D) progressive transition (ρf =−0.098) and (E) abrupt transition (ρf =−0.430). FIGURE 2 | Global and local synchronization for progressive and abrupt transitions. (A) Median global order parameter of progressive (blue) and abrupt (red) transitions for 100 frequency configurations. Colored area indicates the 25–75% values of rlink of 100 configurations. Local order parameter ri of (B) progressive and (C) abrupt transition. The nodes are aligned with descending order of degree from top to bottom. Color indicates the value of ri. FIGURE 2 | Global and local synchronization for progressive and abrupt transitions. (A) Median global order parameter of progressive (blue) and abrupt (red) transitions for 100 frequency configurations. Global and Local Synchronization Processes for Progressive and Abrupt Transitions We calculated the difference between first coupling strength values λri>0.2 and λri>0.8 satisfying ri ≥ 0.2 and ri ≥ 0.8 to investigate the reconstitution order of brain regions. The difference between the coupling strengths of each node was deemed to be the integration duration τ from a low synchronization level in an unconscious state to a high synchronization level in a conscious state. The given thresholds are empirically observed synchronization levels (Kim et al., 2016). We took the median of rlink for 100 frequency configurations to observe the canonical behaviors for each transition type. The median rlink as a function of λ is shown in Figure 2A. Under the progressive transition condition (blue), the rlink increases gradually in all steps of λ. In the abrupt transition (red), the global synchronization is relatively delayed for a long period before a major change, followed by the steep increase of median rlink within a short range of coupling strength (around λ = 0.16). This delay was expected from the initial network configurations of the abrupt transition, which prohibits the network from being globally synchronized. Figures 2B,C shows how the higher and lower degree nodes were differentially synchronized during progressive and abrupt transitions. If we define a node with the local order parameter ri = 0.8 as synchronized, the data τi = λri>0.8 −λri>0.2 (6) (6) We then ranked the 82 regions in terms of τi and repeated it over 100 network configurations. Within each transition, we compared the reconstitution orders among 82 regions and among 10 sub-regions (Table S1). We performed the Kruskal- Wallis test with multiple comparisons considering p < 0.05 as a significant difference among the regions with Bonferroni We then ranked the 82 regions in terms of τi and repeated it over 100 network configurations. Within each transition, we compared the reconstitution orders among 82 regions and among 10 sub-regions (Table S1). We performed the Kruskal- Wallis test with multiple comparisons considering p < 0.05 as a significant difference among the regions with Bonferroni June 2017 | Volume 11 | Article 55 Frontiers in Computational Neuroscience | www.frontiersin.org Synchronization of Brain Networks Kim et al. Kim et al. stinct phase synchronization patterns in computational models of the human brain. (A) Exemplary cases for progressive (blue) and abrupt (red) e abrupt transition shows a sharp increase of the order parameter at λ = 0.148 . Synchronization Cluster Formations for Progressive and Abrupt Transitions well as how it changes along with increasing coupling strength for progressive and abrupt transitions. The Spearman correlation between node degree and median ri over 100 configurations was calculated to elucidate the global relationship. We investigated synchronization processes in the brain network at the level of clusters for progressive and abrupt transitions. The number of synchronization clusters, NC, and the size of giant synchronization cluster, GC, were examined to study how the clusters merge and how the size of the largest cluster develops during progressive and abrupt transitions (Figures 3A,B). As shown in Figure 3A, the evolution of NC as a function of λ for two transitions is similar when λ is small (<0.086), but they show different cluster merging behaviors at higher values. In the progressive transition (blue), the NC decreases slowly, whereas the NC of the abrupt transition (red) is relatively preserved until λ ∼= 0.15 and thereafter sharply drops. The evolution of size of giant synchronization cluster, GC, for the two transitions is illustrated in Figure 3B. The size of GC in the progressive transition (blue) grows gradually, while the size of giant synchronization clusters of the abrupt transition (red) grows faster with constraints that make the largest synchronization cluster size bigger than 15 before λ = 0.148. Thus, the progressive transition follows the general synchronization path, in which a cluster is first centered around hub nodes attracting circumjacent peripheral nodes, and gradually grows into a dominant giant cluster (Gómez-Gardeñes et al., 2007). By contrast, for the abrupt transition, several smaller sized clusters are formed, but do not merge together until a certain coupling strength. At a critical point, they abruptly coalesce into big clusters (Zhang et al., 2014). In comparison to the progressive transition, the abrupt transition consistently demonstrates the delay of the major change and the sharp drop of the NC with abrupt growth of the size of abrupt growth of the size of the GC. The overall correlation values between node degrees and local order parameters of the progressive transition are higher than the abrupt transition in Figure 4A. During the progressive transition, the correlation increases until it has a maximum value at λ = 0.1. After that point, the correlation decreases until it has almost zero value. Dynamics of Hub and Peripheral Nodes Dynamics of Hub and Peripheral Nodes We analyzed how hub and peripheral structures in the brain network are reorganized during the progressive and abrupt transitions as coupling strength increases (Figures 5A,B). We ranked the degree-classified subgroups in terms of the median rs over 100 configurations for each subgroup (Figures 5C,D). The initial ranks were randomly given by the initial network configurations for both transitions. However, the ranks of subgroups are reorganized in distinctive ways as the coupling strength increases, depending on the type of transition. The reorganization among the subgroups takes place in a low and short coupling strength range (λ: 0.03–0.07) for the progressive transition, whereas it occurs in a relatively high and broad coupling strength range (λ: 0.064–0.168) for the abrupt transition. During the progressive transition, the subgroups of higher degree go up to higher ranks of synchronization, while the subgroups of lower degree descend to the lower ranks of synchronization. At the end of the short and random reorganization process, the five subgroups have been arranged Synchronization Cluster Formations for Progressive and Abrupt Transitions The relatively higher correlations of the progressive transition imply that the synchronization strengths of the brain regions during the progressive transition are more predictable and reflected at the individual node level. The correlation of the abrupt transition reaches a maximum (=0.70), which is delayed compared to the progressive transition and with a lower value than the progressive transition (=0.87). Global and Local Synchronization Processes for Progressive and Abrupt Transitions Colored area indicates the 25–75% values of rlink of 100 configurations. Local order parameter ri of (B) progressive and (C) abrupt transition. The nodes are aligned with descending order of degree from top to bottom. Color indicates the value of ri. topology itself, represented as the node degree, has influence on the synchronization level in the progressive transition, whereas the effect of network topology is suppressed by the frequency configurations during the abrupt transition before a critical level. Thus, distinctive synchronization processes for two types of transition are observed at the individual node level of the brain network. demonstrate that, in the progressive transition, the sequence of the synchronization process correlates with the node degree. In other words, highly-connected hub nodes are synchronized earlier than less-connected peripheral nodes. By contrast, in the abrupt transition, the local synchronization of nodes across the network takes place suddenly when a critical threshold of coupling strength is crossed. This implies that the network June 2017 | Volume 11 | Article 55 Frontiers in Computational Neuroscience | www.frontiersin.org 5 Synchronization of Brain Networks Kim et al. FIGURE 3 | Analysis of synchronization clusters. (A) Change in the median number of synchronization clusters (NC) as a function of λ in progressive (blue) and abrupt (red) transitions. Colored area indicates the 25–75% values. (B) Change in the median size of a giant synchronization cluster (GC) as a function of λ in progressive (blue) and abrupt (red) transition. Colored area indicates the 25–75% values. FIGURE 3 | Analysis of synchronization clusters. (A) Change in the median number of synchronization clusters (NC) as a function of λ in progressive (blue) and abrupt (red) transitions. Colored area indicates the 25–75% values. (B) Change in the median size of a giant synchronization cluster (GC) as a function of λ in progressive (blue) and abrupt (red) transition. Colored area indicates the 25–75% values. Synchronization Cluster Formations for Progressive and Abrupt Transitions General Relationship between Structure and Dynamics FIGURE 4 | Relationship between degree and local order parameter ri for coupling strength λ. (A) Spearman correlation between degree and median local order parameter ri for coupling strength λ . Blue line indicates progressive transition and red line indicates abrupt transition. Black dotted vertical lines indicate λ = 0.02, 0.1, 0.16, and 0.22. Four representations of degree vs. ri for λ = 0.02, 0.1, 0.16, and 0.22 (from left to right) in (B) progressive and (C) abrupt transition (Spearman correlation = 0.55, 0.87, 0.53, and 0.14 for gradual; Spearman correlation = 0.24, 0.50, 0.70, and 0.12 for abrupt transition). FIGURE 5 | Synchronization level and synchronization rank for subgroups classified according to degree. Global synchronization monitored by median Dij for all nodes i of each subgroup in (A) progressive and (B) abrupt transitions. Each colored line indicates a synchronization level of each subgroup classified with node degree k (black: 4 ≤k < 11, blue: 11 ≤k < 14, yellow: 14 ≤k < 18, green: 18 ≤k < 22, and red: 22 ≤k < 32). Synchronization rank among subgroups in (C) progressive and (D) abrupt transitions. Each color is the same as denoted in (A,B). In the progressive transition, subgroups with a higher degree are synchronized earlier. In the abrupt transition, the synchronization of hub nodes is suppressed below a certain value λ, but sharply increases after the critical point is reached. FIGURE 5 | Synchronization level and synchronization rank for subgroups classified according to degree. Global synchronization monitored by median Dij for all nodes i of each subgroup in (A) progressive and (B) abrupt transitions. Each colored line indicates a synchronization level of each subgroup classified with node degree k (black: 4 ≤k < 11, blue: 11 ≤k < 14, yellow: 14 ≤k < 18, green: 18 ≤k < 22, and red: 22 ≤k < 32). Synchronization rank among subgroups in (C) progressive and (D) abrupt transitions. Each color is the same as denoted in (A,B). In the progressive transition, subgroups with a higher degree are synchronized earlier. In the abrupt transition, the synchronization of hub nodes is suppressed below a certain value λ, but sharply increases after the critical point is reached. in descending order of degree with the descending order of synchronization. General Relationship between Structure and Dynamics We examined the global relationship between network structure (node degree) and node dynamics (local order parameter) as June 2017 | Volume 11 | Article 55 Frontiers in Computational Neuroscience | www.frontiersin.org 6 Synchronization of Brain Networks Kim et al. Kim et al. FIGURE 4 | Relationship between degree and local order parameter ri for coupling strength λ. (A) Spearman correlation between degree and median local order parameter ri for coupling strength λ . Blue line indicates progressive transition and red line indicates abrupt transition. Black dotted vertical lines indicate λ = 0.02, 0.1, 0.16, and 0.22. Four representations of degree vs. ri for λ = 0.02, 0.1, 0.16, and 0.22 (from left to right) in (B) progressive and (C) abrupt transition (Spearman correlation = 0.55, 0.87, 0.53, and 0.14 for gradual; Spearman correlation = 0.24, 0.50, 0.70, and 0.12 for abrupt transition). FIGURE 5 | Synchronization level and synchronization rank for subgroups classified according to degree. Global synchronization monitored by median Dij for all nodes i of each subgroup in (A) progressive and (B) abrupt transitions. Each colored line indicates a synchronization level of each subgroup classified with node degree k (black: 4 ≤k < 11, blue: 11 ≤k < 14, yellow: 14 ≤k < 18, green: 18 ≤k < 22, and red: 22 ≤k < 32). Synchronization rank among subgroups in (C) progressive and (D) abrupt transitions. Each color is the same as denoted in (A,B). In the progressive transition, subgroups with a higher degree are synchronized earlier. In the abrupt transition, the synchronization of hub nodes is suppressed below a certain value λ, but sharply increases after the critical point is reached. i d di d f d i h h d di d l i b d d d l l d FIGURE 4 | Relationship between degree and local order parameter ri for coupling strength λ. (A) Spearman correlation between degree and median local order parameter ri for coupling strength λ . Blue line indicates progressive transition and red line indicates abrupt transition. Black dotted vertical lines indicate λ = 0.02, 0.1, 0.16, and 0.22. Four representations of degree vs. ri for λ = 0.02, 0.1, 0.16, and 0.22 (from left to right) in (B) progressive and (C) abrupt transition (Spearman correlation = 0.55, 0.87, 0.53, and 0.14 for gradual; Spearman correlation = 0.24, 0.50, 0.70, and 0.12 for abrupt transition). General Relationship between Structure and Dynamics Median rank of integration durations over 100 configurations was used for ordering. Black squares in 4th line mark the regions that have statistically different reconstitution orders between progressive and abrupt transitions (Wilcoxon rank sum test; p < 0.05 considered significant). Name of brain regions was abbreviated for convenience (See Table S1 for full name). The suppression of synchronization due to the initial network configuration makes the full ordered ranks delayed until λ = 0.168. The reorganization follows a systematic process, going up or down only one rank, rather than by discontinuous jumps of multiple steps. Interestingly, before the highest degree group occupies the top rank, the other subgroups already have established and maintained the ordered ranks (λ < 0.122). During this period, the highest degree group remains in the bottom rank, then rises to the higher rank in a step-wise fashion. The coupling strength at which the five subgroups were fully reorganized in ordered rank is similar to the coupling strength of the maximum correlation between node degrees and local order parameters in Figure 4A. The suppression of synchronization due to the initial network configuration makes the full ordered ranks delayed until λ = 0.168. The reorganization follows a systematic process, going up or down only one rank, rather than by discontinuous jumps of multiple steps. Interestingly, before the highest degree group occupies the top rank, the other subgroups already have established and maintained the ordered ranks (λ < 0.122). During this period, the highest degree group remains in the bottom rank, then rises to the higher rank in a step-wise fashion. The coupling strength at which the five subgroups were fully reorganized in ordered rank is similar to the coupling strength of the maximum correlation between node degrees and local order parameters in Figure 4A. regions in progressive transition are relatively integrated at the same stages (rank = 15 and 17 for bilateral sup. fron., and rank = 12 and 11 for bilateral sup. pari.) but there is a significant difference of reconstitution order between them in the abrupt transition (rank = 4 and 8 for bilateral sup. fron., and rank = 30 and 24 for bilateral sup. pari., p < 0.05). Average ranks of sub-regions are provided in Figure S3. General Relationship between Structure and Dynamics This reorganization process takes place before the major increase of the global order parameter of the brain network (Figure 2A), and before reaching the maximum correlation between node degrees and local order parameters (λ = 0.1) in Figure 3A. The abrupt transition demonstrates a significantly different reorganization process compared to the progressive transition. Frontiers in Computational Neuroscience | www.frontiersin.org June 2017 | Volume 11 | Article 55 7 Kim et al. Synchronization of Brain Networks FIGURE 6 | Reconstitution orders of regional brain structures. Squares in 1st line show node degrees of the region. Squares in 2nd (3rd) line are the reconstitution orders for progressive (abrupt) transition. Warmer (cooler) color indicates a higher (lower) degree or faster (slower) integration. Difference between first coupling strength values reaching local order parameter ri > 0.2 and ri > 0.8 was defined as the integration duration for each region. Median rank of integration durations over 100 configurations was used for ordering. Black squares in 4th line mark the regions that have statistically different reconstitution orders between progressive and abrupt transitions (Wilcoxon rank sum test; p < 0.05 considered significant). Name of brain regions was abbreviated for convenience (See Table S1 for full name). FIGURE 6 | Reconstitution orders of regional brain structures. Squares in 1st line show node degrees of the region. Squares in 2nd (3rd) line are the reconstitution orders for progressive (abrupt) transition. Warmer (cooler) color indicates a higher (lower) degree or faster (slower) integration. Difference between first coupling strength values reaching local order parameter ri > 0.2 and ri > 0.8 was defined as the integration duration for each region. Median rank of integration durations over 100 configurations was used for ordering. Black squares in 4th line mark the regions that have statistically different reconstitution orders between progressive and abrupt transitions (Wilcoxon rank sum test; p < 0.05 considered significant). Name of brain regions was abbreviated for convenience (See Table S1 for full name). FIGURE 6 | Reconstitution orders of regional brain structures. Squares in 1st line show node degrees of the region. Squares in 2nd (3rd) line are the reconstitution orders for progressive (abrupt) transition. Warmer (cooler) color indicates a higher (lower) degree or faster (slower) integration. Difference between first coupling strength values reaching local order parameter ri > 0.2 and ri > 0.8 was defined as the integration duration for each region. General Relationship between Structure and Dynamics Nodes with significantly different reconstitution order between progressive and abrupt transition are marked as black squares in the last line in Figure 6 (Wilcoxon rank sum test; p < 0.05). Recovery of bilateral putamen and left thalamus is faster in the abrupt transition. Reconstitution of the prefrontal area in the progressive transition seems to occur faster than the abrupt transition with a significant difference in right medial orbitofrontal cortex. Bilateral superior frontal regions are integrated faster in the abrupt transition whereas bilateral superior parietal regions are integrated faster in the progressive transition. Bilateral insular and bilateral post-central (primary somatosensory cortex) regions are relatively reconstituted at early stage in abrupt transition. Reconstitution of Brain Regions for Progressive and Abrupt Transitions In order to investigate the reconstitution processes in actual brain structures, we compared the ranks of τi of brain regions within each transition and for both transitions (Figure 6). In accordance with the synchronization process we identified above (hub nodes are synchronized earlier than peripheral nodes), the higher degree brain regions recover faster compared with the lower degree regions in both transitions (Spearman correlation = −0.51, p < 0.001 for progressive transition; Spearman correlation = −0.59 and p < 0.001 for abrupt transition). This pattern is maintained even if the reconstitution duration of the abrupt transition is relatively shorter than the progressive transition. Most subcortical regions—including thalamus, caudate, putamen, pallidum, hippocampus, and accumbens—recover at early stages (reconstitution order <20, Table S1) in both transitions. Ranks of all regions for 100 configurations are presented in Figure S2. For progressive transitions, nodes in subcortical, prefrontal, and occipital regions reconstitute earlier than other regions (p < 0.05, significantly earlier than at least 5 regions). Recovery of central and insular regions is faster than the other regions in the abrupt transition (p < 0.05, significantly earlier than at least 5 regions). Hub nodes in frontal (bilateral sup. fron.) and parietal (bilateral sup. pari.) Frontiers in Computational Neuroscience | www.frontiersin.org DISCUSSION Synchronization of neural activities is an important condition for efficient information transmission among neural populations (Varela et al., 2001; Tononi, 2004; Melloni et al., 2007; Uhlhaas et al., 2009; Wang, 2010; Hipp et al., 2011; Plankar et al., 2013; Bressler and Richter, 2015). Anesthesia induces unconsciousness, fragmenting functional brain networks, and disrupting efficient information integration (Alkire et al., 2008; Lee et al., 2009; Boveroux et al., 2010; Ku et al., 2011; Schrouffet al., 2011; Schröter et al., 2012; Casali et al., 2013; Jordan et al., 2013; Lee U. et al., 2013; MacDonald et al., 2015), which is usually accompanied by global spatiotemporal desynchronization of the brain (Imas et al., 2006; Lee H. et al., 2013; Blain-Moraes et al., 2014; Liang et al., 2015; Palanca et al., 2015; Huang et al., 2016; Kim et al., 2016). However, after discontinuation of general anesthetics, the brain restores its activity spontaneously Frontiers in Computational Neuroscience | www.frontiersin.org June 2017 | Volume 11 | Article 55 8 Kim et al. Synchronization of Brain Networks and with diverse patterns (Lee et al., 2011; Chander et al., 2014; Hight et al., 2014). Previous empirical data analysis has demonstrated distinctive evolution patterns of EEG for progressive/earlier or abrupt/delayed emergence from anesthesia. The characteristic evolution patterns of empirical EEG can potentially be explained by the patterns of progressive and abrupt synchronization transitions that were identified in this study of a neuroanatomically-informed model of the human brain. Notably, the scope of the current study is to simulate the macroscopic network of the whole-brain level. It should be differentiated from the previous state transition studies in mesoscopic networks at the neural population level (Steyn-Ross et al., 1999, 2001, 2004). During progressive synchronization, the brain network is synchronized gradually from hub nodes attracting peripheral nodes and the synchronization cascade is triggered earlier with a lower coupling strength. The reorganization process at the subgroup level is completed and remains stable before major global change, which indicates that the brain network is sub-structurally already well-organized at the early stage of synchronization. y By contrast, during explosive patterns of synchronization, a network is synchronized discontinuously at a critical point with the delay of global integration (Zhang et al., 2014). DISCUSSION For the abrupt transition, we used the V-shape relationship between node and frequency (i.e., hub nodes have higher and lower frequencies than central frequency at the same time) with large frequency disassortativity (higher frequency nodes tend to link with lower frequency nodes, or vice versa) to inhibit the synchronization of hub nodes (Leyva et al., 2013b; Li et al., 2013). Consequently, the suppression of hub synchronization prohibits the formation of giant clusters, allowing many small but disconnected clusters to grow until the network reaches the critical threshold where a small perturbation triggers the abrupt transition to global synchronization. Therefore, the global synchronization is delayed, but all clusters are combined at once in a single explosive unification of the brain network. In the reorganization process at the subgroup level, the delayed and slow reorganization of hub groups induces delayed synchronization throughout the brain. The reorganization of hub groups occurs with the change of individual node and global network levels, which means the brain network is not prepared to be organized at the sub-structural level in comparison with the gradual transition. The network configuration for the abrupt transition might mirror the different dose-dependent effects of anesthetic drugs on brain regions (Detsch et al., 1999; Liu et al., 2013; Sellers et al., 2013; Hutchison et al., 2014; Lv et al., 2016), a hypothesis that requires empirical confirmation. Progressive synchronization has been studied extensively with a focus on the effects of hub, modular structure, and global network topology in model and brain networks (Honey and Sporns, 2008; Kitzbichler et al., 2009; Breakspear et al., 2010; Gómez-Gardeñes et al., 2010; Cabral et al., 2011; Villegas et al., 2014; Hellyer et al., 2015; Schmidt et al., 2015; Váša et al., 2015; Finger et al., 2016). The hubs in scale-free networks dominate the synchronization process, whereas in random networks (which lack hub nodes), many individual nodes are synchronized earlier (Gómez-Gardeñes et al., 2007). In human brain networks, the hub-to-hub connections are critical for inter-modular synchronization and the perturbation of the rich club hubs significantly suppresses synchronization among the functional modules (Schmidt et al., 2015). Moreover, the location of each node in a network determines the temporal order of the synchronization process. For instance, the connector hub, which mediates several modular structures in a network, is synchronized at the last moment (Arenas et al., 2006). DISCUSSION This is true across species; as one example, the hub areas in the cat brain consistently played a critical role in the synchronization process (Gómez-Gardeñes et al., 2010). In contrast to the many studies of progressive synchronization, abrupt synchronization in a network has been investigated only recently with a series of studies focused on explosive synchronization (Gómez-Gardeñes et al., 2011; Leyva et al., 2013a,b; Li et al., 2013; Zhang et al., 2014, 2015). Since the key mechanism of explosive synchronization is to suppress the synchronization of hub nodes, we were able to predict the hub dominance in progressive synchronization. If hub structure is disrupted, there can be a significant change in the synchronization process. Another novel finding in this study was that, in the correlation between node degree and local order parameter, progressive transitions have a larger correlation than abrupt transitions. This implies that the local dynamics of progressive synchronization processes are more predictable based on the brain network structure within a broad range of coupling strengths. The maximum peaks of correlation in both transitions indicate that the local order parameters are linearly arranged along with the node degrees before the formation of a giant synchronization cluster (Figures 3A, 4B,C). Notably, the maximum correlations between network structure and local dynamics during both transitions might reveal critical states in which the networks balance functional integration and segregation for the given conditions. Although the network reconfiguration processes are distinctive between progressive and abrupt transition, the principle of a higher degree node leading to higher local synchronization seems to be a necessary condition for triggering the global synchronization process in both transitions. In this study, we simulated emergence patterns in brain networks using the Kuramoto phase oscillator model applied to a human brain network. Altering the network configurations allowed us to model transitions at the individual node, cluster, and global network levels. We identified distinct patterns of progressive and abrupt synchronization transitions (progressive/early, abrupt/delayed). These synchronization phenotypes are consistent with behavioral phenotypes and related EEG patterns identified during progressive and abrupt emergence from unconsciousness (Lee et al., 2011; Chander et al., 2014; Hight et al., 2014). In addition, the simulation study sheds light on how regional brain functions reconstitute during progressive and abrupt emergence from anesthesia. The simulations performed, based only on network principles of the two synchronization processes, yielded results that are consistent with the reconstitution of human brain functions from anesthesia. Frontiers in Computational Neuroscience | www.frontiersin.org SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fncom. 2017.00055/full#supplementary-material AUTHOR CONTRIBUTIONS MK performed research and wrote the paper. SK and GM interpreted data and contributed to writing the paper. UL conceived of the study, interpreted the data, and wrote the paper. CONCLUSIONS This model study has many limitations. First, a simple coupled oscillatory model like Kuramoto limits interpretation because it can only capture the coarse-grained, large-scale synchronization process. Second, there are regional and temporal patterns of EEG in various frequency ranges during anesthesia and we are unable to explain complex EEG patterns with this model. We only took into account the alpha frequency band of EEG around 10 Hz to simulate the regional brain dynamics, but there are numerous other oscillations of relevance to consciousness and unconsciousness. More detailed models exhibiting a broad range of frequency spectrum will be important for further study. Third, the human brain network is neither a random, nor a scale-free network. It has a complex structure through which information transmission can be efficiently achieved with many types of hubs (Van Den Heuvel and Sporns, 2013). With this complicated structure, it is difficult to find the exact conditions for explosive synchronization. Therefore, the V-shape relationship between node degrees and frequencies as well as the large frequency disassortativity that we used are not unique methods to suppress the synchronization of hub nodes. Another possible network configuration suppressing the synchronization of hub nodes could alter the synchronization process. Fourth, despite our primary focus on modeling progressive and abrupt emergence, a mixed pattern of progressive and abrupt transitions empirically exists. Future study would be required to generalize our models toward a combined version of progressive and abrupt transitions. Fifth, we determined the hub nodes based only on the anatomical brain network structure. However, the effects of nodes on synchronization, even with the same anatomical node degree, could be different depending on their local network structure. Sixth, we used an anatomical human brain network parceled out This model study demonstrated that progressive and abrupt synchronization transitions in a human brain network can occur based on network principles alone. Distinctive characteristics of network synchronization processes appear to match progressive and abrupt emergence patterns from unconsciousness based on behavior and EEG patterns. The characteristic network reconstitution processes observed at the individual node, cluster, and global network levels suggest underlying mechanisms for how regional brain functions are reconstituted during the progressive and abrupt emergence from the unconscious state, providing a theoretical foundation for further studies. ACKNOWLEDGMENTS This work is supported by the National Institutes of Health R01 GM098578 (PIs: GM and UL), and the James S. McDonnell Foundation (PI: GM). DISCUSSION In both transitions, there was an early recovery June 2017 | Volume 11 | Article 55 Frontiers in Computational Neuroscience | www.frontiersin.org 9 Synchronization of Brain Networks Kim et al. into 82 nodes including cortical and subcortical regions. The finite-size effect of the network could have affected our results. To mitigate the finite size effect, we repeated the simulation 100 times and considered the averaged feature. Finally, we have established only loose associations between network principles of synchronization and behavioral or EEG phenotypes of recovery observed in humans recovering from anesthesia. Further work that studies synchronization processes in humans during the reconstitution of consciousness and cognition will be important to validate these findings. of subcortical areas and a relatively late recovery of cortical areas, especially frontal and parietal areas. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Tononi, G. (2004). An information integration theory of consciousness. BMC Neurosci. 5:42. doi: 10.1186/1471-2202-5-42 Uhlhaas, P., Pipa, G., Lima, B., Melloni, L., Neuenschwander, S., Nikoli´c, D., et al. (2009). Neural synchrony in cortical networks: history, concept and current status. Front. Integr. Neurosci. 3:17. doi: 10.3389/neuro.07.017.2009 June 2017 | Volume 11 | Article 55 Frontiers in Computational Neuroscience | www.frontiersin.org 12
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Mitochondrial oxidative stress activates COX-2/mPGES-1/PGE2 cascade induced by albumin in renal proximal tubular cells
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ABSTRACT COX-2/mPGES-1/PGE2 cascade is of importance in the pathogenesis of kidney injury. Meanwhile, recent studies documented a detrimental role of mitochondrial oxidative stress in kidney diseases. The present study was undertaken to investigate the role of mitochondrial oxidative stress in albumin-induced activation of COX-2/ mPGES-1/PGE2 cascade in renal proximal tubular cells. Following albumin overload in mice, we observed a significant increase of oxidative stress and mitochondrial abnormality determined by transmission electron microscope, which was attenuated by the administration of MnTBAP, a mitochondrial SOD2 mimic. More interestingly, albumin overload-induced upregulation of COX-2 and mPGES-1 at mRNA and protein levels was largely abolished by MnTBAP treatment in mice. Meanwhile, urinary PGE2 excretion was also blocked by MnTBAP treatment. Furthermore, mouse proximal tubule epithelial cells (mPTCs) were treated with albumin. Similarly, COX-2/mPGES-1/ PGE2 cascade was significantly activated by albumin in dose- and time-dependent manners, which was abolished by MnTBAP treatment in parallel with a blockade of oxidative stress. Collectively, the findings from current study demonstrated that mitochondrial oxidative stress could activate COX-2/mPGES-1/PGE2 cascade in proximal tubular cells under the proteinuria condition. Mitochondrial oxidative stress/ COX-2/mPGES-1/PGE2 could serve as the important targets for the treatment of proteinuria-associated kidney injury. Mitochondrial oxidative stress activates COX-2/mPGES-1/PGE2 cascade induced by albumin in renal proximal tubular cells Yibo Zhuang1,2,3,*, Chenhu Wang1,2,3,*, Chunfeng Wu1,2,3, Dan Ding1,2,3, Fei Zhao1,2,3, Caiyu Hu1,2,3, Wei Gong1,2,3, Guixia Ding1,2,3, Yue Zhang1,2,3, Lihong Chen1,2,3, Guangrui Yang1,2,3, Chunhua Zhu1,2,3, Aihua Zhang1,2,3, Zhanjun Jia1,2,3 and Songming Huang1,2,3 1Department of Nephrology, Children’s Hospital of Nanjing Medical University, Nanjing 210008, China 2Jiangsu Key Laboratory of Pediatrics, Nanjing 210029, China 3Nanjing Key Laboratory of Pediatrics, Nanjing 210008, China *These authors equally contributed to this work Correspondence to: Songming Huang, email: smhuang@njmu.edu.cn Keywords: albumin; mitochondrial oxidative stress; COX-2; PGE2; proximal tubular cells Received: November 19, 2017        Accepted: January 04, 2018        Published: January 12, 2018 Copyright: Zhuang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. www.impactjournals.com/oncotarget/ Oncotarget, 2018, Vol. 9, (No. 10), pp: 9235-9245 Research Paper This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Mitochondrial oxidative stress activates COX-2/mPGES-1/PGE2 cascade induced by albumin in renal proximal tubular cells Yibo Zhuang1,2,3,*, Chenhu Wang1,2,3,*, Chunfeng Wu1,2,3, Dan Ding1,2,3, Fei Zhao1,2,3, Caiyu Hu1,2,3, Wei Gong1,2,3, Guixia Ding1,2,3, Yue Zhang1,2,3, Lihong Chen1,2,3, Guangrui Yang1,2,3, Chunhua Zhu1,2,3, Aihua Zhang1,2,3, Zhanjun Jia1,2,3 and Songming Huang1,2,3 1Department of Nephrology, Children’s Hospital of Nanjing Medical University, Nanjing 210008, China 2Jiangsu Key Laboratory of Pediatrics, Nanjing 210029, China 3Nanjing Key Laboratory of Pediatrics, Nanjing 210008, China *These authors equally contributed to this work Correspondence to: Songming Huang, email: smhuang@njmu.edu.cn Keywords: albumin; mitochondrial oxidative stress; COX-2; PGE2; proximal tubular cells Received: November 19, 2017        Accepted: January 04, 2018        Published: January 12, 2018 Copyright: Zhuang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. www.impactjournals.com/oncotarget/ Oncotarget, 2018, Vol. 9, (No. 10), pp: 9235-9245 Research Paper Mitochondrial oxidative stress activates COX-2/mPGES-1/PGE2 cascade induced by albumin in renal proximal tubular cells Yibo Zhuang1,2,3,*, Chenhu Wang1,2,3,*, Chunfeng Wu1,2,3, Dan Ding1,2,3, Fei Zhao1,2,3, Caiyu Hu1,2,3, Wei Gong1,2,3, Guixia Ding1,2,3, Yue Zhang1,2,3, Lihong Chen1,2,3, Guangrui Yang1,2,3, Chunhua Zhu1,2,3, Aihua Zhang1,2,3, Zhanjun Jia1,2,3 and Songming Huang1,2,3 1Department of Nephrology, Children’s Hospital of Nanjing Medical University, Nanjing 210008, China 2Jiangsu Key Laboratory of Pediatrics, Nanjing 210029, China 3Nanjing Key Laboratory of Pediatrics, Nanjing 210008, China *These authors equally contributed to this work Correspondence to: Songming Huang, email: smhuang@njmu.edu.cn Keywords: albumin; mitochondrial oxidative stress; COX-2; PGE2; proximal tubular cells Received: November 19, 2017        Accepted: January 04, 2018        Published: January 12, 2018 Copyright: Zhuang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. www.impactjournals.com/oncotarget/ Oncotarget, 2018, Vol. 9, (No. 10), pp: 9235-9245 Research Paper Mitochondrial oxidative stress activates COX-2/mPGES-1/PGE2 cascade induced by albumin in renal proximal tubular cells Yibo Zhuang1,2,3,*, Chenhu Wang1,2,3,*, Chunfeng Wu1,2,3, Dan Ding1,2,3, Fei Zhao1,2,3, Caiyu Hu1,2,3, Wei Gong1,2,3, Guixia Ding1,2,3, Yue Zhang1,2,3, Lihong Chen1,2,3, Guangrui Yang1,2,3, Chunhua Zhu1,2,3, Aihua Zhang1,2,3, Zhanjun Jia1,2,3 and Songming Huang1,2,3 1Department of Nephrology, Children’s Hospital of Nanjing Medical University, Nanjing 210008, China 2Jiangsu Key Laboratory of Pediatrics, Nanjing 210029, China 3Nanjing Key Laboratory of Pediatrics, Nanjing 210008, China *These authors equally contributed to this work Correspondence to: Songming Huang, email: smhuang@njmu.edu.cn Keywords: albumin; mitochondrial oxidative stress; COX-2; PGE2; proximal tubular cells Received: November 19, 2017        Accepted: January 04, 2018        Published: January 12, 2018 Copyright: Zhuang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. www.impactjournals.com/oncotarget/ Oncotarget, 2018, Vol. 9, (No. 10), pp: 9235-9245 Research Paper www.impactjournals.com/oncotarget/ Copyright: Zhuang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: Zhuang et al. Albumin directly stimulated COX-2/mPGES-1/ PGE2 cascade in renal proximal tubular cells To investigate whether albumin could directly stimulate COX-2/mPGES-1/PGE2 cascade in vitro, the mPTCs were treated with albumin, and the regulation of PGE2 synthetic enzymes including COX-2, COX-1, mPGES-1, mPGES-2, and cPGES was determined by qRT-PCR and Western blotting. As shown by the data, albumin dose-dependently induced COX-2, mPGES-1 mRNA (Figure 4A–4E) and protein (Figure 5A–5C) expressions without affecting the other PGE2 synthetic enzymes. Meanwhile, a time-dependent stimulation of this COX-2/mPGES-1 pathway was also observed at mRNA (Figure 6A–6E) and protein (Figure 7A–7C) levels. In line with the stimulation of the COX-2/mPGES-1 axis, the PGE2 release in the cell culture medium was also dose- and time-dependently elevated (Figure 5D and Figure7D). All of these data indicated a direct effect of albumin on the stimulation of COX-2/mPGES-1/PGE2 cascade in renal proximal tubular cells. Mitochondrial abnormality has been found in albumin-treated human proximal tubule cells [21] and CKDs [22, 23]. Moreover, our previous studies also demonstrated that mitochondrial oxidative stress contributes to the kidney injury induced by the aldosterone [24, 25]. These findings highly suggested a detrimental role of mitochondrial dysfunction in proteinuric kidney diseases. Thus, in the present study, we investigated the role of mitochondrial oxidative stress in albuminuria-stimulated activation of COX-2/mPGES-1/PGE2 cascade in vivo and in vitro. INTRODUCTION PGE2 is generated through a cyclooxygenases (COXs)/PGE2 synthases (PGESs) cascade [5]. To date, three PGE2 synthases including mPGES-1, mPGES-2, and cPGES were cloned with the best characterization of mPGES-1. The genetic deletion of mPGES-2 and cPGES did not reduce PGE2 production under basal or stress conditions [6, 7]. Kidney is a major source of prostaglandins (PGs) including PGE2, PGD2, PGI2, PGF2α, and thromboxane A2 (TXA2). PGE2 is of importance in fluid metabolism regulation [8–11] and Proteinuria plays an established role in mediating renal tubular injury and is viewed as a causative factor in promoting the progression of kidney diseases [1, 2]. Evidence from numerous studies suggested that the cellular phenotypic changes and apoptotic response may serve as the underlying mechanisms of proteinuria- induced kidney injury [3, 4]. However, detailed molecular mechanisms remain elusive. www.impactjournals.com/oncotarget Oncotarget 9235 might serve as a key factor leading to the stimulation of COX-2/mPGES-1/PGE2 cascade. kidney injuries [12–14]. PGI2 and TXA2 are documented to be important in modulating renal hemodynamics [15, 16]. The roles of PGD2 and PGF2α in kidney remain uncertain. Our previous reports demonstrated that COX- 2 and mPGES-1 played detrimental role in CKD model of 5/6 nephrectomy [17] and AKI model of cisplatin nephropathy [18]. In diabetic kidney disease, blockade of COX-2 or PGE2 receptors of EP1 and EP4 resulted in significant protection against diabetic kidney injury [19]. In another proteinuric animal model induced by adriamycin, the overexpression of COX-2 in podocytes remarkably worsened kidney damage [20]. All of these data suggest a critical role of the COX-2/mPGES-1/PGE2 cascade in mediating kidney injury. Albumin overload-induced mitochondrial abnormality and oxidative stress was ameliorated by MnTBAP Following albumin treatment, the ROS production was significantly enhanced (Figure 8A–8B). After the administration of MnTBAP, the increments of COX-2, mPGES-1, and PGE2 were significantly reversed (Figure 9A–9E and Figure 10A–10D). However, mPGES-2, cPGES, and COX-1 were not affected by albumin or MnTBAP treatment (Figure 9B–9D). These results demonstrated that the albumin effect on the activation of COX-2/mPGES-1/PGE2 cascade was through a mitochondrial oxidative stress-mediated mechanism. Eleven days of albumin overload in mice resulted in a severe structural disruption of the mitochondria in renal tubular cells as determined by TEM (Figure 1A). Meanwhile, the oxidative stress marker of MDA in urine was elevated by albumin overload (Figure 1B). Strikingly, all these abnormalities were reversed by a mitochondrial SOD2 mimic MnTBAP (Figure 1A and 1B). DISCUSSION Proteinuria is a hallmark for the diagnosis of glomerular diseases. Growing evidence has also demonstrated that proteinuria is an independent causative factor mediating the progression of kidney diseases, possibly via the stimulation of the inflammatory response in tubular epithelial cells and the tubulointerstitial region. PGE2 is abundantly produced in the kidney and contributes to a number of physiological and pathological processes. Under pathological insults, PGE2 was mostly found to play a detrimental role in kidney diseases [12–14]. By qRT-PCR, we observed a selective upregulation of COX-2 and mPGES-1 but not COX-1, mPGES-2, and cPGES (Figure 2A–2E). By Western blotting, we further confirmed the up-regulation of COX-2 and mPGES-1 at the protein levels (Figure 3A–3C). Moreover, albumin overload significantly increased urinary PGE2 excretion (Figure 3D). Interestingly, application of MnTBAP largely normalized the stimulation of COX-2/mPGE-1/PGE2 cascade (Figure 3A–3D) without the impact on other PGE2 synthetic enzymes (Figure 2B–2D). These data suggested that albumin overload-induced oxidative stress Treating renal epithelial cells or animals with albumin has been widely used to mimic the effect of proteinuria www.impactjournals.com/oncotarget Oncotarget 9236 on kidneys. In animals, albumin overload results in the exposure of renal tubular cells to the excessive albumin filtered through the glomerular filtration barrier. Although this model cannot entirely mimic the disease status of patients with proteinuria in the clinic, it could be a suitable model for evaluating the effect of albuminuria on kidney injury because of its exclusion of non-proteinuria factors, such as diabetes, hypertension, and lipid disorders. In the present study, albumin overload for 11 days induced severe renal tubular injury accompanied by a significant increase of oxidative stress and mitochondrial morphological alteration. In line with the elevated oxidative stress, the urinary PGE2 excretion was significantly increased. After administration of a mitochondrial SOD2 mimic, the oxidative stress occurred in kidneys was robustly abolished. At the same time, the abnormality of the mitochondrial morphology in the tubular cells was strikingly improved. These results suggested that mitochondrial-derived oxidative stress strongly contributed to the albumin overload-induced mitochondrial injury and might be an important pathogenic factor in mediating proteinuria- associated kidney injury in patients. l against various insults, such as diabetes, cisplatin nephropathy, adriamycin nephropathy and renal mass reduction [17–20]. Following the administration of albumin, urinary PGE2 was significantly elevated in line with the upregulation of COX-2 and mPGES-1 in kidneys. However, COX-1, mPGES-2 and cPGES were unaffected by the albumin overload. DISCUSSION These data indicated that the activation of COX-2 and mPGES-1 might contribute to the renal PGE2 production in this model. This stimulation of COX-2/mPGES-1/PGE2 cascade was accompanied with increased oxidative stress and mitochondrial abnormality. Thus, we treated the animals with MnTBAP to evaluate the potential relationship between the activation of COX- 2/mPGES-1/PGE2 cascade and mitochondrial oxidative stress. Strikingly, MnTBAP almost entirely abolished the effect of albumin on the activation of this PGE2-generating cascade, suggesting a critical role of mitochondrial oxidative stress in this process. However, due to the complicated nature of mammalian systemic responses, we could not conclude whether these effects were directly from albumin exposure or a secondary response from kidney injury. To address this, we directly exposed mouse proximal tubular cells to the albumin and reproduced the phenomenon observed in vivo. As shown by the data, following MnTBAP treatment, the stimulation of the COX- 2/mPGES-1/PGE2 cascade was diminished in line with the PGE2 is an important inflammatory mediator that is involved in many kidney diseases [12, 13, 26, 27]. Blockade of the PGE2 synthetic enzymes [28] or antagonism of the PGE2 receptors substantially protects Figure 1: MnTBAP treatment ameliorated oxidative stress and mitochondrial abnormality induced by albumin overload in mice. (A) Mitochondrial morphology in tubular epithelial cells determined by TEM. (B) Renal TBARs levels. The values represent means ± SD (n = 8). *P < 0.01 vs. control. #P < 0.01 vs. albumin-overload mice. 1 M TBAP t t t li t d id ti t d it h d i l b lit i d d b lb i Figure 1: MnTBAP treatment ameliorated oxidative stress and mitochondrial abnormality induced by albumin overload in mice. (A) Mitochondrial morphology in tubular epithelial cells determined by TEM. (B) Renal TBARs levels. The values represent means ± SD (n = 8). *P < 0.01 vs. control. #P < 0.01 vs. albumin-overload mice. www.impactjournals.com/oncotarget Oncotarget 9237 albumin (BSA) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). MnTBAP was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit polyclonal antibody against COX-2 and rabbit polyclonal antibody against mPGES-1 were purchased from Cayman Chemical (USA). Rabbit antibody against β-actin was obtained from Cell Signaling Technology (Danvers, MA, USA). Peroxidase-conjugated AffiniPure goat anti-rabbit secondary antibody was from Zhongshan Gold Bridge Biotechnology (Beijing, China). attenuation of oxidative stress. Animal studies For the MnTBAP experiment, 8-week-old 129/ Sv male mice weighing 25–30 g were subjected to an intraperitoneal (i.p.) injection with low-endotoxin albumin (A-9430, Sigma Chemical Co., St. Louis, MO) dissolved in saline for 11 days. In brief, albumin was administered for 5 days in a stepwise incremental dose regimen, increasing from 2 mg/g body weight on the first day (D1) DISCUSSION These in vitro data clearly demonstrate a direct action of albumin on the stimulation of the COX-2/mPGES-1/PGE2 cascade via mitochondrial oxidative stress. In summary, using both animals and in vitro cells, we demonstrated that albumin enhanced mitochondrial oxidative stress to stimulate COX-2/mPGES-1/PGE2 cascade in renal proximal tubular cells. These findings suggested that the inhibition of the mitochondrial oxidative stress could protect kidneys against proteinuria- associated kidney injury, and such a protective effect might be through the inactivation of COX-2/mPGES-1/ PGE2 cascade to some extent. Reagents and antibodies DMEM medium was purchased from Gibco Corporation (Carlsbad, CA, USA). Bovine serum Figure 2: The upregulation of COX-2 and mPGES-1 was revered by MnTBAP at mRNA levels. (A) qRT-PCR analysis of mPGES-1. (B) qRT-PCR analysis of mPGES-2. (C) qRT-PCR analysis of cPGES. (D) qRT-PCR analysis of COX-1. (E) qRT-PCR analysis of COX-2. The values represent means ± SD (n = 8). *P < 0.01 vs. control. #P < 0.01 vs. albumin-overload mice. Oncotarg 9238 w impactjournals com/oncotarget Figure 2: The upregulation of COX-2 and mPGES-1 was revered by MnTBAP at mRNA levels. (A) qRT-PCR analysis mPGES-1. (B) qRT-PCR analysis of mPGES-2. (C) qRT-PCR analysis of cPGES. (D) qRT-PCR analysis of COX-1. (E) qRT-PCR analy of COX-2. The values represent means ± SD (n = 8). *P < 0.01 vs. control. #P < 0.01 vs. albumin-overload mice. Figure 2: The upregulation of COX-2 and mPGES-1 was revered by MnTBAP at mRNA levels. (A) qRT-PCR analysis of mPGES-1. (B) qRT-PCR analysis of mPGES-2. (C) qRT-PCR analysis of cPGES. (D) qRT-PCR analysis of COX-1. (E) qRT-PCR analysis of COX-2. The values represent means ± SD (n = 8). *P < 0.01 vs. control. #P < 0.01 vs. albumin-overload mice. www.impactjournals.com/oncotarget Oncotarget 9238 Cell culture studies mPTCs, an immortalized cell line, were grown in serum-free keratinocyte medium supplemented with bovine pituitary extract and epidermal growth factor (Wisent, Canada). The cells were specifically grown at 37°C with 5% CO2 and subcultured at 50–80% confluence using 0.25% trypsin-0.02% EDTA (Invitrogen). In certain experiments, the cells were preincubated with MnTBAP (100 nmol/ml) for 30 min before BSA (10 mg/ml) treatment. Quantitative real-time PCR (qRT-PCR) to the maximum dose of 10 mg/g body weight on D5, which was thereafter maintained until day 11. The control groups received a corresponding volume of saline via i.p. injection, and the albumin-treated mice received vehicle or MnTBAP (10 mg/kg/day) for 11 days. Total RNA was extracted using the TRIzol reagent (Invitrogen),. Oligonucleotides were designed using Primer3 software (available at http://frodo.wi.mit. edu/) and synthesized by Invitrogen. The sequences of the primer pairs are shown in Table 1. qRT-PCR was then used to detect the mtDNA copy number and the mRNA expression of target genes. Reverse transcription was performed using a reaction kit (Promega Reverse Transcription System) according to the manufacturer’s protocol. Real-time PCR amplification was performed using the ABI 7500 real-time PCR detection system (Foster City, CA, USA) with the SYBR Green PCR Master Mix (Applied Biosystems). The cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The mRNA levels were normalized to GAPDH as a control and calculated using the comparative cycle threshold (ΔΔCt) method. All mice were maintained on a 12 h light-dark cycle in a temperature-controlled (19–21°C) room, were fed a standard rodent diet, and were allowed free access to drinking water. At the termination of the experiments, the mice were anesthetized with an i.p. injection of a ketamine/xylazine/atropine cocktail. Plasma and kidney samples were then immediately frozen in liquid nitrogen and stored at –80°C until use. The study protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Nanjing Medical University, China. Western blotting mPTCs were lysed using a protein lysis buffer containing 50 mM Tris, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 200 mM sodium fluoride, and 4 mM sodium orthovanadate as a protease inhibitor (pH 7.5). Immunoblotting was then performed with primary Figure 3: MnTBAP treatment reversed the effects of albumin overload on the protein expressions of COX-2 and mPGES-1 and urinary PGE2 production. (A) Western blots of mPGES-1, COX-2 and β-actin. (B) Densitometric analysis of mPGES-1. (C) Densitometric analysis of COX-2. (D) Urinary PGE2 output. The values represent means ± SD (n = 8). *p < 0.01 vs. control. #p < 0.01 vs. albumin-overload mice. Figure 3: MnTBAP treatment reversed the effects of albumin overload on the protein expressions of COX-2 and mPGES-1 and urinary PGE2 production. (A) Western blots of mPGES-1, COX-2 and β-actin. (B) Densitometric analysis of mPGES-1. (C) Densitometric analysis of COX-2. (D) Urinary PGE2 output. The values represent means ± SD (n = 8). *p < 0.01 vs. control. #p < 0.01 vs. albumin-overload mice. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 9239 Measurement of thiobarbituric acid-reactive substances antibodies against COX-2 (Cayman Chemical, USA, 1:500), mPGES-1 (Cayman Chemical, USA, 1:500), and β-actin (1:1000), followed by the addition of HRP-labeled secondary antibodies. The blots were visualized using the Amersham ECL detection system (Amersham, Little Chalfont, UK). Densitometric analysis was performed using Quantity One software (Bio-Rad). The measurement of plasma thiobarbituric acid- reactive substances (TBARS) was based on the formation of malondialdehyde by using a commercially available TBARS Assay kit (10009055; Cayman Chemical), according to the manufacturer’s instructions. Transmission electron microscopy (TEM) The concentration of PGE2 in the medium was examined using a commercial EIA kit purchased from Cayman Chemical. Fresh kidney tissues were fixed in 1.25% glutaraldehyde/0.1 M phosphate buffer and post-fixed in 1% OsO4/0.1 M phosphate buffer. Ultrathin sections (60 nm) were then cut on a microtome, placed on copper grids, stained with uranyl acetate and lead citrate, and examined under an electron microscope (JEOL JEM-1010, Tokyo, Japan). Analysis of ROS production in cells ROS production in mPTCs was measured by DCFDA as described previously [29]. Oncotarget 9240 w impactjournals com/oncotarget igure 4: Albumin upregulated the mRNA expressions of COX-2 and mPGES-1 in a dose-dependent manner in mPTCs. (A) qRT-PCR analysis of mPGES-1. (B) qRT-PCR analysis of mPGES-2. (C) qRT- PCR analysis of cPGES. (D) qRT-PCR nalysis of COX-1. (E) qRT-PCR analysis of COX-2. The values represent means ± SD (n = 6). *P < 0.01 vs. control. Figure 4: Albumin upregulated the mRNA expressions of COX-2 and mPGES-1 in a dose-dependent manner in mPTCs. (A) qRT-PCR analysis of mPGES-1. (B) qRT-PCR analysis of mPGES-2. (C) qRT- PCR analysis of cPGES. (D) qRT-PCR analysis of COX-1. (E) qRT-PCR analysis of COX-2. The values represent means ± SD (n = 6). *P < 0.01 vs. control. www.impactjournals.com/oncotarget Oncotarget 9240 Figure 5: Albumin stimulated the protein expressions of COX-2 and mPGES-1 and PGE2 release in a dose-dependent manner in mPTCs. (A) Western blots of mPGES-1, COX-2, and β-actin. (B) Densitometric analysis of mPGES-1. (C) Densitometric analysis of COX-2. (D) PGE2 concentration in medium. The values represent means ± SD (n = 6). Figure 5: Albumin stimulated the protein expressions of COX-2 and mPGES-1 and PGE2 release in a dose-dependent manner in mPTCs. (A) Western blots of mPGES-1, COX-2, and β-actin. (B) Densitometric analysis of mPGES-1. (C) Densitometric analysis of COX-2. (D) PGE2 concentration in medium. The values represent means ± SD (n = 6). Figure 6: Albumin upregulated the mRNA expressions of COX-2 and mPGES-1 in a time-dependent manner in mPTCs. (A) qRT-PCR analysis of mPGES-1. (B) qRT-PCR analysis of mPGES-2. (C) qRT- PCR analysis of cPGES. (D) qRT-PCR analysis of COX-1. (E) qRT-PCR analysis of COX-2. The values represent means ± SD (n = 6). *P < 0.01 vs. control. Figure 6: Albumin upregulated the mRNA expressions of COX-2 and mPGES-1 in a time-dependent manner in mPTCs. (A) qRT-PCR analysis of mPGES-1. (B) qRT-PCR analysis of mPGES-2. (C) qRT- PCR analysis of cPGES. (D) qRT-PCR analysis of COX-1. (E) qRT-PCR analysis of COX-2. The values represent means ± SD (n = 6). *P < 0.01 vs. control. www.impactjournals.com/oncotarget Oncotarget 9241 Figure 7: Albumin stimulated the protein expressions of COX-2 and mPGES-1 and PGE2 release in a time-dependent manner in mPTCs. (A) Western blots of mPGES-1, COX-2, and β-actin. (B) Densitometric analysis of mPGES-1. (C) Densitometric analysis of COX-2. (D) PGE2 concentration in medium. Analysis of ROS production in cells The values represent means ± SD (n = 6). *P < 0.01 vs. control. Figure 7: Albumin stimulated the protein expressions of COX-2 and mPGES-1 and PGE2 release in a time-dependent manner in mPTCs. (A) Western blots of mPGES-1, COX-2, and β-actin. (B) Densitometric analysis of mPGES-1. (C) Densitometric analysis of COX-2. (D) PGE2 concentration in medium. The values represent means ± SD (n = 6). *P < 0.01 vs. control. Figure 8: MnTBAP treatment suppressed albumin-induced ROS production in mPTCs. (A) Representative image of DCF fluorescence. (B) Quantitative analysis of ROS production by DCF fluorescence. The values represent means ± SD (n = 6). *P < 0.01 vs. control. #P < 0.01 vs. albumin group. Figure 8: MnTBAP treatment suppressed albumin-induced ROS production in mPTCs. (A) Representative image of DCF fluorescence. (B) Quantitative analysis of ROS production by DCF fluorescence. The values represent means ± SD (n = 6). *P < 0.01 vs. control. #P < 0.01 vs. albumin group. www.impactjournals.com/oncotarget Oncotarget 9242 Figure 9: The upregulation of COX-2 and mPGES-1 mRNA expressions was revered by MnTBAP in mPTCs. (A) qRT- PCR analysis of mPGES-1. (B) qRT-PCR analysis of mPGES-2. (C) qRT-PCR analysis of cPGES. (D) qRT-PCR analysis of COX-1. (E) qRT-PCR analysis of COX-2. The values represent means ± SD (n = 6). *P < 0.01 vs. control. #P < 0.01 vs. albumin group. Figure 9: The upregulation of COX-2 and mPGES-1 mRNA expressions was revered by MnTBAP in mPTCs. (A) qRT- PCR analysis of mPGES-1. (B) qRT-PCR analysis of mPGES-2. (C) qRT-PCR analysis of cPGES. (D) qRT-PCR analysis of COX-1. (E) qRT-PCR analysis of COX-2. The values represent means ± SD (n = 6). *P < 0.01 vs. control. #P < 0.01 vs. albumin group. Figure 10: MnTBAP treatment reversed the effects of albumin on the protein expressions of COX-2 and mPGES-1 and PGE2 release in mPTCs. (A) Western blots of mPGES-1, COX-2 and β-actin. (B) Densitometric analysis of mPGES-1. (C) Densitometric analysis of COX-2. (D) PGE2 release in medium. The values represent means ± SD (n = 6). *P < 0.01 vs. control. #P < 0.01 vs. albumin group. Figure 10: MnTBAP treatment reversed the effects of albumin on the protein expressions of COX-2 and mPGES-1 and PGE2 release in mPTCs. (A) Western blots of mPGES-1, COX-2 and β-actin. (B) Densitometric analysis of mPGES-1. (C) Densitometric analysis of COX-2. (D) PGE2 release in medium. Statistical analysis 81570643 and 81570616), the National Key Research and Development Program (no. 2016YFC0906103), the Natural Science Foundation of Jiangsu Province (no. BK20130077), and Nanjing City Key Medical Research P j t (ZKX16057) 81570643 and 81570616), the National Key Research and Development Program (no. 2016YFC0906103), the Natural Science Foundation of Jiangsu Province (no. BK20130077), and Nanjing City Key Medical Research Project (ZKX16057) All data are presented as means ± standard deviation (SD). The statistical analysis was performed using ANOVA followed by Bonferroni’s test with SPSS 13 statistical software. P < 0.05 was considered significant. Project (ZKX16057). FUNDING 7. Lovgren AK, Kovarova M, Koller BH. cPGES/p23 is required for glucocorticoid receptor function and embryonic growth but not prostaglandin E2 synthesis. Mol Cell Biol. 2007; 27:4416–4430. This work was supported by Grants from the National Natural Science Foundation of China (nos. 81370802, 81400750, 81300591, 81670647, 81600557, www.impactjournals.com/oncotarget Author contributions SH, AZ, and ZJ designed the study and experiments. YZ, CW, CW, DD, FZ, CH, WG, GD, YZ, LC, GY, and CZ performed the research and analyzed the data. All authors interpreted and discussed the data. SH and ZJ wrote the manuscript. All authors read and approved the final manuscript. 5. Murakami M, Naraba H, Tanioka T, Semmyo N, Nakatani Y, Kojima F, Ikeda T, Fueki M, Ueno A, Oh S, Kudo I. Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2. J Biol Chem. 2000; 275:32783–32792. Abbreviations 1. Burton C, Harris KP. The role of proteinuria in the progression of chronic renal failure. Am J Kidney Dis. 1996; 27:765–775. mPTCs: mouse proximal tubule epithelial cells; CKD: chronic kidney disease; COX-1: cyclooxygenase-1; COX-2: cyclooxygenase-2; PGE2: prostaglandin E2; mPGES-1: membrane associated PGE synthase-1; mPGES-2: membrane associated PGE synthase-2; cPGES: cytosolic PGE2 synthase; BSA: bovine serum albumin; TEM: transmission electron microscopy; TBARS: thiobarbituric acid-reactive substances. 2. Eddy AA. Proteinuria and interstitial injury. Nephrol Dial Transplant. 2004; 19:277–281. 3. Zoja C, Morigi M, Remuzzi G. Proteinuria and phenotypic change of proximal tubular cells. J Am Soc Nephrol. 2003; 14:S36–41. 4. Li X, Pabla N, Wei Q, Dong G, Messing RO, Wang CY, Dong Z. PKC-delta promotes renal tubular cell apoptosis associated with proteinuria. J Am Soc Nephrol. 2010; 21:1115–1124. CONFLICTS OF INTEREST 6. Jania LA, Chandrasekharan S, Backlund MG, Foley NA, Snouwaert J, Wang IM, Clark P, Audoly LP, Koller BH. Microsomal prostaglandin E synthase-2 is not essential for in vivo prostaglandin E2 biosynthesis. Prostaglandins Other Lipid Mediat. 2009; 88:73–81. There is no conflicts of interest to disclose. Analysis of ROS production in cells The values represent means ± SD (n = 6). *P < 0.01 vs. control. #P < 0.01 vs. albumin group. www.impactjournals.com/oncotarget Oncotarget 9243 Table 1: Primer sequences for qRT-PCR Gene symbol Primer sequences GAPDH 5′- GTCTTCACTACCATGGAGAAGG - 3′ 5′- TCATGGATGACCTTGGCCAG -3′ mPGES1 5′- GGATGCGCTGAAACGTGGA - 3′ 5′- CAGGAATGAGTACACGAAGCC - 3′ mPGES2 5′- CCTCGACTTCCACTCCCTG - 3′ 5′- TGAGGGCACTAATGATGACAGAG - 3′ cPGES 5′- TGTTTGCGAAAAGGAGAATCCG - 3′ 5′- CCATGTGATCCATCATCTCAGAG - 3′ COX1 5′- ATGAGTCGAAGGAGTCTCTCG - 3′ 5′- GCACGGATAGTAACAACAGGGA - 3′ COX2 5′- AACCGTGGGGAATGTATGAG - 3′ 5′- GCAGGAAGGGGATGTTGTT - 3′ www.impactjournals.com/oncotarget Oncotarget 9244 19. Jia Z, Sun Y, Liu S, Liu Y, Yang T. COX-2 but Not mPGES-1 Contributes to Renal PGE2 Induction and Diabetic Proteinuria in Mice with Type-1 Diabetes. PLoS One. 2014; 9:e93182. 8. Stokes JB, Kokko JP. Inhibition of sodium transport by prostaglandin E2 across the isolated, perfused rabbit collecting tubule. J Clin Invest. 1977; 59:1099–1104. 9. Stokes JB. Effect of prostaglandin E2 on chloride transport across the rabbit thick ascending limb of Henle. Selective inhibitions of the medullary portion. J Clin Invest. 1979; 64:495–502. 20. Cheng H, Wang S, Jo YI, Hao CM, Zhang M, Fan X, Kennedy C, Breyer MD, Moeckel GW, Harris RC. Overexpression of cyclooxygenase-2 predisposes to podocyte injury. J Am Soc Nephrol. 2007; 18:551–559. 10. Stokes JB. Integrated actions of renal medullary prostaglandins in the control of water excretion. Am J Physiol. 1981; 240:F471–480. 21. Erkan E, Devarajan P, Schwartz GJ. Mitochondria are the major targets in albumin-induced apoptosis in proximal tubule cells. J Am Soc Nephrol. 2007; 18:1199–1208. 11. Guan Y, Zhang Y, Breyer RM, Fowler B, Davis L, Hebert RL, Breyer MD. Prostaglandin E2 inhibits renal collecting duct Na+ absorption by activating the EP1 receptor. J Clin Invest. 1998; 102:194–201. 22. Granata S, Zaza G, Simone S, Villani G, Latorre D, Pontrelli P, Carella M, Schena FP, Grandaliano G, Pertosa G. Mitochondrial dysregulation and oxidative stress in patients with chronic kidney disease. BMC Genomics. 2009; 10:388. 12. Mohamed R, Jayakumar C, Ranganathan PV, Ganapathy V, Ramesh G. Kidney proximal tubular epithelial-specific overexpression of netrin-1 suppresses inflammation and albuminuria through suppression of COX-2-mediated PGE2 production in streptozotocin-induced diabetic mice. Am J Pathol. 2012; 181:1991–2002. 23. Che R, Yuan Y, Huang S, Zhang A. Mitochondrial dysfunction in the pathophysiology of renal diseases. Am J Physiol Renal Physiol. 2014; 306:F367–378. 24. Yuan Y, Huang S, Wang W, Wang Y, Zhang P, Zhu C, Ding G, Liu B, Yang T, Zhang A. Activation of peroxisome proliferator-activated receptor-gamma coactivator 1alpha ameliorates mitochondrial dysfunction and protects podocytes from aldosterone-induced injury. Kidney Int. 2012; 82:771–789. 13. Ranganathan PV, Jayakumar C, Mohamed R, Dong Z, Ramesh G. Netrin-1 regulates the inflammatory response of neutrophils and macrophages, and suppresses ischemic acute kidney injury by inhibiting COX-2-mediated PGE2 production. Kidney Int. 2013; 83:1087–1098. 25. Wen H, Ting JP, O’Neill LA. A role for the NLRP3 inflammasome in metabolic diseases–did Warburg miss inflammation? Nat Immunol. 2012; 13:352–357. 14. www.impactjournals.com/oncotarget Ren Y, D’Ambrosio MA, Garvin JL, Wang H, Carretero OA. Prostaglandin E2 mediates connecting tubule glomerular feedback. Hypertension. 2013; 62:1123–1128. 26. Nakagawa N, Yuhki K, Kawabe J, Fujino T, Takahata O, Kabara M, Abe K, Kojima F, Kashiwagi H, Hasebe N, Kikuchi K, Sugimoto Y, Narumiya S, et al. The intrinsic prostaglandin E2-EP4 system of the renal tubular epithelium limits the development of tubulointerstitial fibrosis in mice. Kidney Int. 2012; 82:158–171. 15. Fujita T, Fuke Y, Satomura A, Hidaka M, Ohsawa I, Endo M, Komatsu K, Ohi H. PGl2 analogue mitigates the progression rate of renal dysfunction improving renal blood flow without glomerular hyperfiltration in patients with chronic renal insufficiency. Prostaglandins Leukot Ess ent Fatty Acids. 2001; 65:223–227. 27. Kvirkvelia N, McMenamin M, Chaudhary K, Bartoli M, Madaio MP. Prostaglandin E2 promotes cellular recovery from established nephrotoxic serum nephritis in mice, prosurvival, and regenerative effects on glomerular cells. Am J Physiol Renal Physiol. 2013; 304:F463–470. 16. Uriu K, Kaizu K, Hashimoto O, Komine N, Etoh S. Acute and chronic effects of thromboxane A2 inhibition on the renal hemodynamics in streptozotocin-induced diabetic rats. Kidney Int. 1994; 45:794–802. 28. Murakami M, Kudo I. Prostaglandin E synthase: a novel drug target for inflammation and cancer. Curr Pharm Des. 2006; 12:943–954. 17. Jia Z, Wang H, Yang T. Microsomal prostaglandin E synthase 1 deletion retards renal disease progression but exacerbates anemia in mice with renal mass reduction. Hypertension. 2012; 59:122–128. 29. Huang S, Zhang A, Ding G, Chen R. Aldosterone-induced mesangial cell proliferation is mediated by EGF receptor transactivation. Am J Physiol Renal Physiol. 2009; 296:F1323–1333. 18. Jia Z, Wang N, Aoyagi T, Wang H, Liu H, Yang T. Amelioration of cisplatin nephrotoxicity by genetic or pharmacologic blockade of prostaglandin synthesis. Kidney Int. 2011; 79:77–88. www.impactjournals.com/oncotarget Oncotarget 9245
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Gyroidal nanoporous carbons - Adsorption and separation properties explored using computer simulations
Condensed matter physics
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Received November 13, 2015, in final form December 17, 2015 Received November 13, 2015, in final form December 17, 2015 Adsorption and separation properties of gyroidal nanoporous carbons (GNCs) — a new class of exotic nanocar- bon materials are studied for the first time using hyper parallel tempering Monte Carlo Simulation technique. Porous structure of GNC models is evaluated by the method proposed by Bhattacharya and Gubbins. All the studied structures are strictly microporous. Next, mechanisms of Ar adsorption are described basing on the analysis of adsorption isotherms, enthalpy plots, the values of Henry’s constants, αs and adsorption poten- tial distribution plots. It is concluded that below pore diameters ca. 0.8 nm, primary micropore filling process dominates. For structures possessing larger micropores, primary and secondary micropore filling mechanism is observed. Finally, the separation properties of GNC toward CO2/CH4, CO2/N2, and CH4/N2 mixtures are dis- cussed and compared with separation properties of Virtual Porous Carbon models. GNCs may be considered as potential adsorbents for gas mixture separation, having separation efficiency similar or even higher than activated carbons with similar diameters of pores. Key words: gyroidal nanoporous carbons, adsorption, gas mixtures separation, Monte Carlo simulations ∗This paper is dedicated to Prof. Stefan Sokołowski on the occasion of his 65th birthday. Condensed Matter Physics, 2016, Vol. 19, No 1, 13003: 1–14 DOI: 10.5488/CMP.19.13003 http://www.icmp.lviv.ua/journal © S. Furmaniak, P.A. Gauden, A.P. Terzyk, P. Kowalczyk, 2016 Gyroidal nanoporous carbons — Adsorption and separation properties explored using computer simulations∗ S. Furmaniak1, P.A. Gauden1, A.P. Terzyk1, P. Kowalczyk2 1 Faculty of Chemistry, Physicochemistry of Carbon Materials Research Group, Nicolaus Copernicus University in Toruń, Gagarin St. 7, 87–100 Toruń, Poland 2 School of Engineering and Information Technology, Murdoch University, Murdoch, Western Australia 6150, Australia S. Furmaniak1, P.A. Gauden1, A.P. Terzyk1, P. Kowalczyk2 1 Faculty of Chemistry, Physicochemistry of Carbon Materials Research Group, Nicolaus Copernicus University in Toruń, Gagarin St. 7, 87–100 Toruń, Poland 2 School of Engineering and Information Technology, Murdoch University, Murdoch, Western Australia 6150, Australia 1. Introduction [16, 17, 34– 38], (iv) interaction of charged chain particles and spherical counterions in contact with charged hard wall [31], (v) analysis of the properties of two-dimensional symmetrical mixtures in an external field of square symmetry [39, 40], (vi) demixing and freezing in two-dimensional symmetrical mixtures [21], and (vii) the behaviour of mixed two component submonolayer films (Ar and Kr [41, 42] or Kr and Xe [22] on graphite). The majority of the analysed adsorbents have an ideal geometry of pores, for example a slit-like [25, 26, 32, 34, 43]. However, more complex models have also been studied, for example, pillared slit-like pores [28] and slit-like pores with walls decorated by tethered polymer brushes in the form of stripes [29]. In the last decades, novel exotic porous carbon nanostructures (such as carbon nanotubes (CNTs), single-walled carbon nanohorns, graphene and graphitic nanoribbons, ordered porous carbons, worm- like nanotubes and graphitic nanofibers, stacked-cup carbon nanofibers, cubic carbon allotropes, carbon onions, carbyne networks, and others) have been projected to be among the most useful materials for selective adsorption and separation of fluid mixtures [44–48]. However, in the theoretical studies, differ- ent carbon adsorbents are studied, such as: carbon nanotubes [13, 49–53], carbon nanohorns [13, 51, 54], 2D and 3D ordered carbon networks [55], hydrophobic virtual porous carbons (VPCs) [12, 14, 56–62], ox- idized VPCs [12, 14, 60, 62, 63], and triply periodic carbon minimal surfaces (Schwarz’s primitive and Schoen’s gyroid) [45, 59, 64–70]. Recently, scientists have paid attention to the next generation of porous carbon molecular sieves materials, i.e., crystalline exotic cubic carbon allotropes: cubic carbon poly- morphs (CCPs) [45, 71–73], diamond-like super structures of CNTs (super diamonds) [74], diamond-like frameworks [75], porous aromatic frameworks (PAFs) [76, 77], diamond-like carbon frameworks (i.e., di- amondynes, also named D-carbons) [78], tetrahedral node diamondyne [79], carbon allotropes proposed by Karfunkel and Dressler [45, 80], compressed carbon nanotubes [45, 81], sodalite-like nanostructures [45, 82], folding of graphene slit-like pore walls [52, 83], gyroidal nanoporous and mesoporous carbons (GNCs and GMCs, respectively) [84–86]. One of the most interesting and promising adsorbent from the above mentioned is GNC. In the current study, we consider nine different GNC structures having surface built in a way ensuring connection of each carbon atom with exactly three neighbours, similarly as “schwarzites”. Nicolaï et al. 1. Introduction Adsorption of gas mixtures on solid surfaces has been attracting great interest for many years [1–7]. In the last decades there has been observed an increased interest to the development of new separation and purification techniques. It is evident that adsorption using various adsorbents is still a versatile tool for these purposes. On the other hand, the basic problem appearing in experimental studies is caused by difficulties in the synthesis of nanomaterials possessing desired properties. It is still not simple to control porosity and/or the chemical nature of the surface, and the both parameters at the same time. Moreover, experimental data on mixed systems are very limited, i.e., in the case of mixtures consisting of two volatile components the problem of the surface coverage determination has not been fully solved yet. Predicting adsorption behaviour of mixtures from pure component data is very important, from both the theoretical and practical viewpoints [8–14]. It is well known that the theoretical calculations provide additional opportunities for studies to better understand the separation processes. However, despite the intensive experimental and theoretical studies, our knowledge of the properties and the structure of mixed adsorbed layers is rather sparse, especially, on new generations of nanoporous adsorbents. Computer simulation is an efficient method for resolving the above mentioned problems, since it is capable of modelling the processes of interest at the required level of detail in a controllable environ- ment, providing the necessary tools for establishing the connections between the observed phenomena © S. Furmaniak, P.A. Gauden, A.P. Terzyk, P. Kowalczyk, 2016 13003-1 S. Furmaniak et al. and their molecular-level physical background. Prof. Sokołowski’s (and co-workers) research topics of interest have also been concerned with the issue of mixtures using Monte Carlo simulations [15–22], Den- sity Functional Theory [23–28], and Dissipative Particle Dynamics [29]. Their inspiring articles discuss, for example, the following problems: (i) adsorption from mixtures of monomers [15], dimers [15], the chain molecules [30, 31], and even polymer mixtures [27], (ii) adsorption from mixtures on homogeneous [25, 32] and heterogeneous surfaces [15, 33, 34], (iii) layering transition, capillary condensation, wetting phenomena, and multilayer adsorption of binary ideal mixtures, systems exhibiting negative deviations from ideal mixing or positive one, binary mixture with partially miscible components, etc. 1. Introduction [84] confirmed that the curvature and the rigidity do not play a crucial role in the performance of GNC structures for ionic conduction. The major role, however, is played by the pore size and pore volume. Indeed, the larger the pore is, the larger is the ionic transport. Finally, the mentioned authors stated that GNC structures with tunable properties can be widely applied to water filtration or energy storage. 2. Simulation details We used the structures of nine gyroidal nanocarbons (denoted as GNC-04, GNC-07, GNC-09, GNC-11, GNC-12, GNC-13, GNC-15, GNC-18 and GNC-21) published previously by Nicolaï et al. [84] (see figure 1 in [84]). In the case of the first six systems, original boxes generated by Nicolaï et al. [84] were multiplied (eightfold) in order to obtain box size at least two times greater than the cut-off distances used during simulations described below. The porosity of all the studied carbonaceous adsorbents was characterised by a geometrical method proposed by Bhattacharya and Gubbins (BG) [87]. The implementation of the method was described in detail elsewhere [88, 89]. The BG method provided histograms of pore sizes (effective diameters — deff). These data were also used to calculate the average sizes of pores accessible for Ar atoms (deff,acc,av) [88]. In addition, the volume of pores accessible for Ar was determined using a combination of the BG method and Monte Carlo integration [88]. Argon adsorption isotherms at its boiling point (i.e., T = 87 K) on all the studied nanocarbons were 13003-2 13003-2 Gyroidal nanoporous carbons — Adsorption and separation properties simulated using the hyper parallel tempering Monte Carlo method (HPTMC) proposed by Yan and de Pablo [90]. The simulation scheme was the same as in previous work [88]. We considered 93 replicas corresponding to different relative pressure values (p/ps, where p and ps are equilibrium and saturated Ar vapour pressure, respectively) in the range 1.0 × 10−10 −1.0. Other details of the performed HPTMC simulations are described in [88]. The average numbers of Ar atoms in the simulation box were used to calculate the adsorption amount of Ar per unit of mass of the adsorbent (a) [88]. The isosteric enthalpy of adsorption (qst) was also determined from the theory of fluctuations [88, 91] to reflect the energetics of the process. In order to analyse the mechanism of Ar adsorption, we constructed high resolution αs-plots [92] based on simulated adsorption isotherms. We used Ar adsorption isotherm simulated in the ideal slit- like system composed of graphene sheets with effective pore width equal to 10 nm as the reference one. We also determined the values of Henry’s constant (KH) from the slope of the linear part of adsorption isotherms in the low-pressure range [83]. Finally, adsorption potential distribution (APD) curves [93–95] were calculated. 2. Simulation details The APD curve is the first derivative of the so-called characteristic curve, presenting adsorption amount as a function of the adsorption potential (Apot) defined as: Apot = −RT ln p ps , (2.1) (2.1) where R is the universal gas constant. The differentiation was performed numerically by the approxima- tion of the isotherms using some empirical functions and calculating their derivatives. We also simulated the adsorption and separation of three binary gas mixtures (important from prac- tical point of view): CO2/CH4, CO2/N2, and CH4/N2 on all the studied GNCs. The computations were per- formed for T = 298 K using the grand canonical Monte Carlo method (GCMC) [91, 96]. The simulation scheme was the same as in our previous works [60, 62]. Simulations were performed for the total mixture pressure ptot = 0.1 MPa (i.e., atmospheric pressure) and for the following mole fractions of components in the gaseous phase (y): 0.0, 0.01, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.975, 0.99, and Figure 1. (Color online) Comparison of (a) pore size histograms for all the studied nanocarbons deter- mined from the BG method (the subsequent histograms are shifted by 1 unit from the previous ones), (b) average sizes and (c) volume of pores accessible for Ar atoms, respectively. Figure 1. (Color online) Comparison of (a) pore size histograms for all the studied nanocarbons deter- mined from the BG method (the subsequent histograms are shifted by 1 unit from the previous ones), (b) average sizes and (c) volume of pores accessible for Ar atoms, respectively. 13003-3 S. Furmaniak et al. 1.0. For each point, the mole fractions of components in the adsorbed phase (x) were calculated from the average numbers of molecules present in the simulation box. The efficiency of the process of separation of mixtures was reflected by the value of equilibrium separation factor (the 1st component over the 2nd one): S1/2 = x1/x2 y1/y2 . (2.2) S1/2 = x1/x2 y1/y2 . (2.2) The adsorbed phase is enriched in the 1st component if S1/2 > 1. 3. Results and discussion Figure 1 (a) collects histograms of effective pore sizes, determined from the application of BG method, for all the studied GNCs. All the structures are strictly microporous, i.e., the diameters of pores do not exceed 2 nm. Generally, the size of dominant pores increases in the considered series (from the GNC- 04 up to GNC-21). However, there are two groups of nanocarbons having similar diameters of the main pores: (i) GNC-11, GNC-12 and GNC-13 — deff around 1 nm and (ii) GNC-18 and GNC-21 — deff around 1.5 nm. It should be noted that in the case of GNC-21, some amount of pores wider than in GNC-18 is also present. These regularities are reflected by the values of the average pore diameter [figure 1 (b)]. The increase in pore diameters is accomplished by the increase in pore volume from ca. 0.4 cm3/g for GNC-04 up to ca. 1.3 cm3/g for GNC-18 and GNC-21 [figure 1 (c)]. p g g Figure 2 shows the comparison of Ar adsorption isotherms simulated for all the studied GNCs. The Figure 2. (Color online) Comparison of Ar adsorption isotherms (T = 87 K) simulated for all the considered nanocarbons. Figure 2. (Color online) Comparison of Ar adsorption isotherms (T = 87 K) simulated for all the considered nanocarbons. 13003-4 Gyroidal nanoporous carbons — Adsorption and separation properties Figure 3. (Color online) Equilibrium argon configurations for selected nanocarbons and selected values of relative pressure (the frames reflect the size of simulation boxes, all the structures are in the same scale). It should be noted that this figure was created using the VMD program [97]. Figure 3. (Color online) Equilibrium argon configurations for selected nanocarbons and selected values of relative pressure (the frames reflect the size of simulation boxes, all the structures are in the same scale). It should be noted that this figure was created using the VMD program [97]. changes observed in the shape of isotherms reflect the differences in the properties of nanocarbons. Adsorption capacity varies from ca. 12 mmol/g for GNC-04 up to ca. 45 mmol/g for GNC-18 and GNC-21. These changes correspond to the differences in the pore volume [figure 1 (c)]. At the same time, the shift of the pore filling pressure toward higher values is observed. The pores of the GNC-04 structure are filled in the relative pressure range 10−8 −10−6. 3. Results and discussion (Color online) Comparison of isosteric enthalpy of Ar adsorption [the data related to figure 2; for clarity adsorption amounts are normalised by the maximum observed value (amax)]. Figure 4. (Color online) Comparison of isosteric enthalpy of Ar adsorption [the data related to figure 2; for clarity adsorption amounts are normalised by the maximum observed value (amax)]. a monolayer is formed and next the remaining free volume is filled. Figure 4 shows the plots of isosteric enthalpy of adsorption related to the isotherms shown in figure 2. For low loadings, the qst values increase as the adsorption amount rises. In this range, Ar atoms are ad- sorbed mainly on high-energetic centres. The increasing adsorption amount causes other Ar atoms to appear in the vicinity of the initially adsorbed ones. This rises the contribution of fluid-fluid interactions to the total energy of a system. Here, the only exception is GNC-21 structure. There is observed a decrease in qst for relative adsorption up to ca. 0.1. This system has probably got a heterogeneous surface. Con- sequently, the subsequent Ar atoms are adsorbed on centres having lower energy and this reduces the effects of the increase in lateral Ar-Ar interactions. In the intermediate range of adsorption, the enthalpy rises for all the structures until the entire adsorbent surface is covered. Next, the values of qst decrease since Ar is adsorbed at the places more distant from the surface and this is connected with lower solid- fluid contributions. In the case of the structures having the widest pores (especially GNC-15, GNC-18 and GNC-21), the second peak on qst is also observed. This peak is related to the total filling of pores. Comparing the enthalpy at zero coverage for all the systems, one can observe that GNC-04 has a re- markably higher value of this parameter (ca. 17.5 kJ/mol) than the other systems. This is connected with the presence of the narrowest pores having high adsorption energy. The other structures may be divided into two groups. The first one includes GNC-07, GNC-09, GNC-12, GNC-13 and GNC-21 carbons. In this case, the enthalpy at zero coverage is close to 12 kJ/mol. For the second group (GNC-11, GNC-15 and GNC-18), this enthalpy value is in the range 9–10 kJ/mol. 3. Results and discussion However, the total filling of GNC-18 and GNC-21 occurs for similar values of relative pressure (around 10−2). The middle carbons of the series (i.e., GNC-11, GNC-12 and GNC-13) are filled in the similar range of relative pressure (p/ps > 10−4). The differences in the pore filling are also clearly seen on equilibrium snapshots of Ar configurations in the simulation boxes shown in figure 3. These regularities are related to the differences in diameters of dominant pores present in the individual GNCs [figure 1 (a)]. Finally, one can observe that in the case of initial structures (up to GNC-13), the pore filling is a single-step process. However, the pores of GNC-15, GNC-18 and GNC-21 are filled in two steps. This is also caused by the rise in pore sizes. For pores wider than 1 nm, in the first step changes observed in the shape of isotherms reflect the differences in the properties of nanocarbons. Adsorption capacity varies from ca. 12 mmol/g for GNC-04 up to ca. 45 mmol/g for GNC-18 and GNC-21. These changes correspond to the differences in the pore volume [figure 1 (c)]. At the same time, the shift of the pore filling pressure toward higher values is observed. The pores of the GNC-04 structure are filled in the relative pressure range 10−8 −10−6. However, the total filling of GNC-18 and GNC-21 occurs for similar values of relative pressure (around 10−2). The middle carbons of the series (i.e., GNC-11, GNC-12 and GNC-13) are filled in the similar range of relative pressure (p/ps > 10−4). The differences in the pore filling are also clearly seen on equilibrium snapshots of Ar configurations in the simulation boxes shown in figure 3. These regularities are related to the differences in diameters of dominant pores present in the individual GNCs [figure 1 (a)]. Finally, one can observe that in the case of initial structures (up to GNC-13), the pore filling is a single-step process. However, the pores of GNC-15, GNC-18 and GNC-21 are filled in two steps. This is also caused by the rise in pore sizes. For pores wider than 1 nm, in the first step 13003-5 S. Furmaniak et al. Figure 4. (Color online) Comparison of isosteric enthalpy of Ar adsorption [the data related to figure 2; for clarity adsorption amounts are normalised by the maximum observed value (amax)]. Figure 4. 3. Results and discussion This may suggest some similarities in the surface nature of the group members (for example curvature, which is the main factor determining the energy of adsorp- tion on the surface). The above-described differences in the energy of interactions with the surface of 13003-6 Gyroidal nanoporous carbons — Adsorption and separation properties Figure 5. (Color online) Comparison of Henry’s constants related to the Ar adsorption isotherms presented on figure 2. Figure 5. (Color online) Comparison of Henry’s constants related to the Ar adsorption isotherms presented on figure 2. Figure 5. (Color online) Comparison of Henry’s constants related to the Ar adsorption isotherms presented on figure 2. Figure 6. (Color online) Comparison of αs-plots related to the Ar adsorption isotherms presented in fig- ure 2 [for clarity the adsorption amounts are normalized by the maximum observed value (amax)]. The αs is the normalized adsorption on the reference material, i.e., in the ideal slit-like system with the effective pore width equal to 10 nm. Figure 6. (Color online) Comparison of αs-plots related to the Ar adsorption isotherms presented in fig- ure 2 [for clarity the adsorption amounts are normalized by the maximum observed value (amax)]. The αs is the normalized adsorption on the reference material, i.e., in the ideal slit-like system with the effective pore width equal to 10 nm. 13003-7 S. Furmaniak et al. Figure 7. (Color online) (a) Comparison of APD curves [for clarity, all the curves are normalized by the maximum observed value (APDmax); subsequent curves are shifted by 1.35 units]. (b) Location of the main peak on the APD curves (Apot,max). (c) Correlation between the location of the main peak and the average sizes of pores accessible for Ar atoms. Figure 7. (Color online) (a) Comparison of APD curves [for clarity, all the curves are normalized by the maximum observed value (APDmax); subsequent curves are shifted by 1.35 units]. (b) Location of the main peak on the APD curves (Apot,max). (c) Correlation between the location of the main peak and the average sizes of pores accessible for Ar atoms. adsorbents fully correspond to the variation of Henry’s constants shown in figure 5. This is not surprising since solid-fluid interactions are the main factor affecting the shape of the isotherm in the low pressure range. Hence, the value of KH for GNC-04 system is at least 1000 times greater than for the other ones. 3. Results and discussion For three of the remaining structures (i.e., GNC-11, GNC-15 and GNC-18), lower values of KH (< 104 mmol/g) are recorded. The same carbons have the lowest enthalpy of adsorption at zero coverage. Figure 6 presents comparison of αs-plots related to the Ar adsorption isotherms. One can see that the adsorption process is dominated by a FS swings (GNC-04 and GNC-07) and the FS-CS swings (remaining structures) [98]. It can be noticed that with the rise in the pore diameter, the combination of primary and secondary micropore filling mechanism occurs. The boundary between those mechanisms is located for the structures with pore diameters around ca. 0.8 nm. It is also interesting that the range on αs-plots connecting FS and CS swings is not linear as it is observed for the case of slit-like carbon micropores. This can be caused by the surface curvature. Figure 7 (a) compares APD curves for all the systems studied. The presented data are complemen- tary to adsorption isotherms shown in figure 2. On all the APD curves, at least one (dominant) peak is observed. It corresponds to the pore filling. Its location [Apot,max, figure 7 (b)] is related to the pressure of the pore filling according to equation 2.1. Hence, this parameter may be correlated with the size of pores — see figure 7 (c). This figure quantitatively confirms the above-described qualitative differences in the pore filling process. The width of the main peak also provides some information on the process. The narrow peak means that condensation occurs in a narrow range of relative pressure. By contrast, a wide peak denotes a wide condensation range. For example, the pore filling in GNC-04 system occurs, as mentioned above, for 10−8 < p/ps < 10−6 and this is reflected by a wide peak with the maximum located at ca. 12.6 kJ/mol. For this system, the other two peaks are also observed (the third one with the maximum 13003-8 Gyroidal nanoporous carbons — Adsorption and separation properties Figure 8. (Color online) Comparison of equilibrium separation factors [S1/2, equation (2.2)] for the ad- sorption of all the mixtures (T = 298 K, ptot = 0.1 MPa) on all the nanocarbons studied. The data plotted as the function of the 1st component mol fraction in the gaseous phase (y1, the 1st component is CO2 for CO2/CH4 and CO2/N2 mixtures and CH4 for CH4/N2 mixture). Figure 8. 3. Results and discussion (Color online) Comparison of equilibrium separation factors [S1/2, equation (2.2)] for the ad- sorption of all the mixtures (T = 298 K, ptot = 0.1 MPa) on all the nanocarbons studied. The data plotted as the function of the 1st component mol fraction in the gaseous phase (y1, the 1st component is CO2 for CO2/CH4 and CO2/N2 mixtures and CH4 for CH4/N2 mixture). Figure 8. (Color online) Comparison of equilibrium separation factors [S1/2, equation (2.2)] for the ad- sorption of all the mixtures (T = 298 K, ptot = 0.1 MPa) on all the nanocarbons studied. The data plotted as the function of the 1st component mol fraction in the gaseous phase (y1, the 1st component is CO2 for CO2/CH4 and CO2/N2 mixtures and CH4 for CH4/N2 mixture) Figure 8. (Color online) Comparison of equilibrium separation factors [S1/2, equation (2.2)] for the ad- sorption of all the mixtures (T = 298 K, ptot = 0.1 MPa) on all the nanocarbons studied. The data plotted as the function of the 1st component mol fraction in the gaseous phase (y1, the 1st component is CO2 for CO2/CH4 and CO2/N2 mixtures and CH4 for CH4/N2 mixture). at ca. 45 kJ/mol is very broad). These peaks reflect the other sub-steps of the Ar density rise in pores of this structure. Similar interpretation also concerns the additional peaks observed for GNC-11 and GNC-12. In the case of GNC-18 and GNC-21 structures, the observed second peak is related to the above mentioned monolayer formation. A slightly different scenario occurs for GNC-15 carbon. Here, the dominant peak is connected with the monolayer formation and the second low (also wide) peak reflects the filling of the remaining pore volume. This fact explains why the location of the main peak for this structure deviates from the distinct trend visible for all the other GNCs in correlation shown in figure 7 (c). Figure 8 presents a comparison of equilibrium separation factors for adsorption of all three studied mixtures for different compositions of gaseous phase. In addition, figure 9 directly compares the effi- ciency of separation of equimolar mixtures for all the studied systems. The separation is a consequence of differences in the adsorption of mixture components. Since the critical temperature for the studied gases decreases significantly in the sequence CO2 > CH4 > N2, the adsorption affinity decreases in the 13003-9 S. Furmaniak et al. Figure 9. 3. Results and discussion (Color online) Comparison of equilibrium separation factors for adsorption of equimolar (y = 0.5) mixtures: (a) CO2/CH4, (b) CO2/N2 and (c) CH4/N2 (ptot = 0.1 MPa, T = 298 K) — see figure 8. Figure 9. (Color online) Comparison of equilibrium separation factors for adsorption of equimolar (y = 0.5) mixtures: (a) CO2/CH4, (b) CO2/N2 and (c) CH4/N2 (ptot = 0.1 MPa, T = 298 K) — see figure 8. same sequence (for the subsequent gases, the process occurs for increasing value of reduced tempera- ture). Consequently, for the given GNC, the highest equilibrium separation factor is observed for CO2/N2 and the smallest one for CH4/N2 mixture. The qualitative differences in separation efficiency between the structures studied are the same for all the mixtures studied. The highest values of equilibrium separation factor (remarkably higher than for the other systems) are observed for GNC-04 carbon. This is connected Figure 10. (Color online) Comparison of equilibrium separation factors [(a) CO2/CH4, (b) CO2/N2 and (c) CH4/N2] for mixture adsorption (ptot = 0.1 MPa, T = 298 K) on GNC-04 and GNC-21 nanocrabons and on three virtual porous carbons (VPCs): d0.5, d0.9 and d1.3 [88] (the data for VPCs taken from our previous paper [60]). In addition, panel (d) presents the pore size histograms for the presented VPCs [88] (the subsequent histograms are shifted by 0.15 units from the previous ones). Figure 10. (Color online) Comparison of equilibrium separation factors [(a) CO2/CH4, (b) CO2/N2 and (c) CH4/N2] for mixture adsorption (ptot = 0.1 MPa, T = 298 K) on GNC-04 and GNC-21 nanocrabons and on three virtual porous carbons (VPCs): d0.5, d0.9 and d1.3 [88] (the data for VPCs taken from our previous paper [60]). In addition, panel (d) presents the pore size histograms for the presented VPCs [88] (the subsequent histograms are shifted by 0.15 units from the previous ones). 13003-10 Gyroidal nanoporous carbons — Adsorption and separation properties with the presence of the narrowest pores. Among the other nanocarbons, GNC-07 and GNC-09 also ex- hibit higher values of S1/2. However, they are lower than in the case of GNC-04 since these structures have wider pores. The efficiency of separation for the next GNCs is similar. Nevertheless, some small dif- ferences are also noticeable (see insets in figure 9). The GNC-11, GNC-15 and GNC-18 structures are less efficient in comparison with the adjacent carbons in the series. 3. Results and discussion These regularities are analogous to that observed for Henry’s constants shown in figure 5 and discussed above. These facts suggest that in the case of GNCs with pores wider than ca. 1 nm, the main factor affecting the efficiency of the separation process is the energetics of fluid interaction with the curved surface of the nanocarbons studied. p g Finally, figure 10 compares the equilibrium separation factors (all three mixtures studied) for GNC-04 and GNC-21 structures and for three virtual porous carbons (VPCs) described in detail previously [88] and having different porosity — see figure 10 (d). As one can see, the GNC-21 structure exhibits a sep- aration efficiency similar to the d0.5 carbon. The main pores of both adsorbents have a similar width. However, this VPC has also some narrower micropores which presence probably positively affects the separation efficiency. Such micropores are absent in the case of GNC-21 and, nonetheless, this nanocar- bon exhibits similar values of S1/2. This is the consequence of the adsorption energetics on a curved surface of this structure. On the contrary, the GNC-04 nanocarbon exhibits the efficiency of the CO2/CH4 mixture separation similar to the d1.3 carbon. This VPC has micropores distributed in the range up to ca. 1 nm [figure 10 (d)]. A large part of them has diameters similar to or lower than the GNC-04 struc- ture. In the case of CO2/N2 and CH4/N2 mixtures, the values of equilibrium separation factors for GNC-04 are higher than for d1.3 carbon. This comparison (especially for CO2/CH4 mixture — similar efficiency for both adsorbents and for CH4/N2 mixture — higher efficiency for GNC-04) may suggest that a regu- larly curved surface of gyroidal carbons exhibits higher affinity to CH4 molecules than a heterogeneous surface of activated carbons. Summing up, the GNCs studied may be considered as potential adsorbents for gas mixture separation. The efficiency of this process is similar to or higher than for activated carbons with similar diameters of pores. The GNC-04 or similar structures seem to be quite promising materials for this purpose since this nanocarbon contains narrow and quite uniform pores (ca. 0.5 nm). 4. Conclusions Adsorption and separation properties of GNCs — a new class of exotic nanocarbon materials, are stud- ied for the first time using computer simulation technique. All the structures studied are strictly micro- porous. The mechanisms of Ar adsorption are described basing on the analysis of adsorption isotherms, enthalpy plots, the values of Henry’s constants, αs and adsorption potential distribution plots. Below the pore diameters ca. 0.8 nm, primary micropore filling process dominates. For structures possessing larger micropores, primary and secondary micropore filling mechanisms are observed. GNCs may be consid- ered as potential adsorbents for gas mixture separation, having separation efficiency similar to or higher than this for activated carbons with similar diameters of pores. 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Graphics, 1996, 14, 33; doi:10.1016/0263-7855(96)00018-5 98. Kaneko K., Ishii C., Kanoh H., Hanzawa Y., Setoyama N., Suzuki T., Adv. Colloid Interface Sci., 1998, 77, 295; doi:10.1016/S0001-8686(98)00050-5. 89. Furmaniak S., Terzyk A.P., Gauden P.A., Kowalczyk P., Harris P.J.F., Koter S., J. Phys.: Condens. Matter, 2013, 25, 015004; doi:10.1088/0953-8984/25/1/015004. 90. Yan Q., de Pablo J.J., J. Chem. Phys., 1999, 111, 9509; doi:10.1063/1.480282. 91. Frenkel D., Smit B., Understanding Molecular Simulation, Academic Press, San Diego, 1996. g g 92. Setoyama N., Suzuki T., Kaneko K., Carbon, 1998, 36, 1459; doi:10.1016/S0008-6223(98)00138-9. Гiроїднi нанопористi вуглецi — адсорбцiя i особливостi роздiлення, дослiдженi з використанням комп’ютерного моделювання С. Фурманяк1, П.А. Гауден1, А.П. Тержик1, П. Ковальчик2 1 Хiмiчний факультет, Дослiдницька група фiзикохiмiї вуглецевих матерiалiв, Унiверситет Нiколауса Копернiкуса в Торунi, Торунь, Польща 2 Школа iнженерiї та iнформацiйних технологiй, Унiверситет Мердока, Мердок, Захiдна Австралiя, 6150, Австралiя С. Фурманяк1, П.А. Гауден1, А.П. Тержик1, П. Ковальчик2 1 Хiмiчний факультет, Дослiдницька група фiзикохiмiї вуглецевих матерiалiв, Унiверситет Нiколауса Копернiкуса в Торунi, Торунь, Польща 2 Школа iнженерiї та iнформацiйних технологiй, Унiверситет Мердока, Мердок, Захiдна Австралiя, 6150, Австралiя Адсорбцiя i особливостi роздiлення у гiроїдних нанопористих вуглецях (GNC), новому класi екзотичних нановуглецевих матерiалiв, вперше дослiджено, використовуючи метод моделювання Монте Карло з гi- перпаралельним темперуванням. Пориста структура GNC моделей оцiнюється методом, запропонова- ним Бхатачарiя i Губiнсом. Всi дослiдженi структури є строго мiкропористi. Крiм того, механiзми адсорбцiї Ar описуються на основi аналiзу iзотерм адсорбцiї, кривих ентальпiї, значень сталих Генрi, αs та кри- вих розподiлу потенцiалу адсорбцiї. Зроблено висновок, що при дiаметрах пор близьких або менших за 0.8 nm домiнує процес заповнення первинних мiкропор. Для структур, що мають бiльшi мiкропори, спостерiгається механiзм заповнення первинних та вторинних мiкропор. Нарештi, описано властивостi роздiлення CO2/CH4, CO2/N2 i CH4/N2 сумiшей у GNC i виконано порiвняння з властивостями роздiлення моделей Вiртуального Пористого Вуглецю. Гiроїднi пористi вуглецi можна розглядати як потенцiйнi ад- сорбенти для роздiлення газових сумiшей, якi володiють аналогiчною або навiть вищою ефективнiстю роздiлення, нiж активований вуглець з подiбним дiаметром пор. Ключовi слова: гiроїднi нанопористi вуглецi, адсорбцiя, роздiлення газових сумiшей, моделювання Монте Карло Ключовi слова: гiроїднi нанопористi вуглецi, адсорбцiя, роздiлення газових сумiшей, моделювання Монте Карло 13003-14
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https://www.nature.com/articles/srep17132.pdf
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High nutrient-use efficiency during early seedling growth in diverse Grevillea species (Proteaceae)
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High nutrient-use efficiency during early seedling growth in diverse Grevillea species (Proteaceae) PEN received: 26 February 2015 accepted: 09 September 2015 Published: 26 November 2015 Tianhua He, William M. Fowler & Casey L. Causley Several hypotheses have been proposed to explain the rich floristic diversity in regions characterised by nutrient-impoverished soils; however, none of these hypotheses have been able to explain the rapid diversification over a relatively short evolutionary time period of Grevillea, an Australian plant genus with 452 recognised species/subspecies and only 11 million years of evolutionary history. Here, we hypothesise that the apparent evolutionary success of Grevillea might have been triggered by the highly efficient use of key nutrients. The nutrient content in the seeds and nutrient-use efficiency during early seedling growth of 12 species of Grevillea were compared with those of 24 species of Hakea, a closely related genus. Compared with Hakea, the Grevillea species achieved similar growth rates (root and shoot length) during the early stages of seedling growth but contained only approximately half of the seed nutrient content. We conclude that the high nutrient-use efficiency observed in Grevillea might have provided a selective advantage in nutrient-poor ecosystems during evolution and that this property likely contributed to the evolutionary success in Grevillea. Mediterranean climate regions, such as those in southwest Western Australia (SWA), the South African Cape region, and the Mediterranean Basin, have particularly diverse species and endemic rich flora and are considered globally significant1,2. Numerous hypotheses have been proposed to explain the floristic diversity of these biodiversity hotspots3. For example, Hopper proposed that landscape age contributed to the high diversity in SWA4, and Lengyel et al. reported higher global diversification rates in myrmeco- chorous lineages relative to their sister groups and speculated that myrmecochory contributed to the high diversification of SWA flora5. However, none of the current hypotheses can explain the extreme diversity of many species-rich genera in Australia, such as Grevillea in the family Proteaceae. y p g y Grevillea is a predominantly Australian genus containing 362 currently recognised species or 452 taxa, which includes subspecies, with five species present in New Guinea, New Caledonia, and Sulawesi6. The evolutionary time period for species accumulation has emerged as a significant explanatory variable for biodiversity3,4, and this explanation is supported by fossil evidence7. Grevillea is one of the youngest genera, with only c. Department of Environment and Agriculture, Curtin University, PO Box U1987, Perth, WA 6845, Australia. Correspondence and requests for materials should be addressed to T.H. (Tianhua.He@curtin.edu.au) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | 5:17132 | DOI: 10.1038/srep17132 High nutrient-use efficiency during early seedling growth in diverse Grevillea species (Proteaceae) PEN 11.2 million years of evolutionary history8, and it is the most species-rich genus in the Proteaceae family; thus, the greater-time-for-speciation theory does not explain the high numbers of species in this genus. However, Hopper proposed that taxa in old and climate-buffered landscapes, including Grevillea and other genera, such as the closely related genus Hakea, typically have a low extinc- tion rate4. Grevillea species are generally dispersed by ants, which has been hypothesised to be a key evo- lutionary novelty that may account for the high diversification rate in this genus5. However, many other plant lineages in SWA, typically species that co-occur with Grevillea and are also dispersed by ants, have markedly fewer species within their genera. For example, ant-dispersed Daviesia (Fabaceae) diversified into 127 species over c. 26 million years, whereas ant-dispersed Bossiaea (Fabaceae) diversified into 80 species within c. 10 million years of evolutionary history3. Therefore, myrmecochory alone is unlikely to contribute to the high diversification rate in Grevillea. With a relatively short evolutionary history of 11.2 million years and as the third largest plant genus in Australia after Acacia and Eucalyptus, Grevillea probably has one of the highest net speciation rates of all large Australian plant clades. Scientific Reports | 5:17132 | DOI: 10.1038/srep17132 1 www.nature.com/scientificreports/ Hakea Grevillea t-test, P value Number of species 149a 362b – Age (million years)c 15.8 11 – Seed mass (mg)d 30.4 (2.6–501.0) 34.9 (2.8–244.0) – Seed mass (mg)e 33.3 ±  29.2 39.5 ±  15.5 t =  − 0.40, P =  0.703 Seed N (%) 7.61 ±  1.36 3.53 ±  0.79 t =  − 1.09, P <  0.001 Seed P (%) 1.36 ±  0.38 0.63 ±  0.23 t =  − 1.48, P <  0.001 Germination rate 0.82 ±  0.12 0.32 ±  0.15 t =  − 5.07, P <  0.001 Days to first true leaf 32 ±  6 31 ±  5 t =  0.02, P =  0.942 Number of leaves 16.7 ±  5.4 8.3 ±  1.2 t =  − 3.57, P =  0.002 Root/shoot biomass 1.67 ±  0.71 1.34 ±  0.55 t =  − 1.27, P =  0.218 Total dry biomass (g) 0.54 ±  0.39 0.28 ±  0.13 t =  − 1.09, P =  0.093 Root length (mm) 468 ±  198 580 ±  181 t =  − 1.48, P =  0.152 Table 1. Seed mass and nutrient concentration (N and P), seed generation, and seedling growth in Hakea and Grevillea. High nutrient-use efficiency during early seedling growth in diverse Grevillea species (Proteaceae) PEN a6; b6; c8; d28; e:species examined in this study. Table 1. Seed mass and nutrient concentration (N and P), seed generation, and seedling growth in Hakea and Grevillea. a6; b6; c8; d28; e:species examined in this study. Species in the family Proteaceae are highly adapted to and became diversified on the most nutrient-impoverished soils observed worldwide. Highly infertile soils have long been demonstrated to influence biological and physiological responses and speciation patterns4,9. Species have developed diverse morphological and physiological adaptations to limited nutrient availability in order to success- fully establish in nutrient-limited soils10–13. These species typically exhibit highly efficient photosynthetic phosphorus use14. p p In southwestern Australia, seed germination and successful seedling establishment predominantly occurs following winter rains after summer fires. Seed-stored nutrients, particularly nitrogen (N) and phosphorus (P), are crucial for the emergence and early growth of seedlings, and the efficient use of these nutrients is critical for the successful establishment of seedlings in severely nutrient-poor and water-limited environments. Nutrient limitation has recently been proposed as a potential driver for variation in species diversity15. Lambers et al. suggested that high plant species diversity on infertile soils, particularly in species-rich biomes, is associated with functional diversity for nutritional strategies, including nutrient-use efficiency16. Despite extensive research on plant species richness in relation to nutrient availability, few studies have attempted to explore the relationship between nutrient-use effi- ciency and speciation patterns. Here, we compare the nutrient concentration and content in seeds and nutrient-use efficiency during early seedling growth of Grevillea with those of the closely related genus Hakea, another Australian genus with 149 species that efficiently use seed nutrients during early seedling growth16. Our study aimed to provide unique insights into the potential mechanisms underlying the evolutionary success (i.e., high diversification rate) of Grevillea. Scientific Reports | 5:17132 | DOI: 10.1038/srep17132 Results d Seed mass varies markedly in both Grevillea (2.8–244 mg, 116 species/subspecies surveyed) and Hakea (2.6–501 mg, 153 species/subspecies surveyed), and Grevillea has a slightly higher median seed mass than Hakea (24.2 vs. 18.7 mg). The average seed masses of the 30 Grevillea species and 29 Hakea species used in the present study were statistically similar (Table 1). Nutrient assays on the 30 Grevillea species and 29 Hakea species revealed significantly higher concentrations of N and P in the Hakea seeds than in the Grevillea seeds (Table 1; Table S1). The total N and P contents per seed in Hakea were also much higher than in Grevillea (N: 2.66 vs. 0.56 mg, P =  0.002; P: 0.47 vs. 0.17 mg, P =  0.008; Fig. 1).h g g g The seeds of the 25 Hakea species that were tested readily germinated without requiring pre-treatment, and they had an overall germination rate of 82%. Grevillea seeds (29 species) pre-treated with smoke water exhibited a significantly lower germination rate of 32% (Table 1). However, for the 24 Hakea and 12 Grevillea species that yielded a sufficient number of germinants for growth comparisons, the time period to expand the first true leaves was similar, although Grevillea plants had significantly fewer leaves after growing for 100 days. Both Hakea and Grevillea diverted more resources to the roots than to the shoots, with a statistically similar root:shoot ratio. Grevillea and Hakea species accumulated similar amounts of dry biomass and had similar root depths in the soil after 100 days of growth (Table 1). Scientific Reports | 5:17132 | DOI: 10.1038/srep17132 2 www.nature.com/scientificreports/ 0 2 4 6 8 Hakea Grevillea Seed N (mg) 0.0 0.5 1.0 1.5 Grevillea Hakea Seed P (mg) Figure 1. Comparison of nitrogen (N) and phosphorus (P) content per seed of the test species of Hakea and Grevillea. The middle lines in the box represent the median values, and the bottom and top of the box are the first and third quartiles. The ends of the whiskers are 1.5 interquartile range (IQR) above the third quartile and 1.5 IQR below the first quartile. 0.0 0.5 1.0 1.5 Grevillea Hakea Seed P (mg) 0 2 4 6 8 Hakea Grevillea Seed N (mg) Figure 1. Comparison of nitrogen (N) and phosphorus (P) content per seed of the test species of Hakea and Grevillea. Results d The middle lines in the box represent the median values, and the bottom and top of the box are the first and third quartiles. The ends of the whiskers are 1.5 interquartile range (IQR) above the third quartile and 1.5 IQR below the first quartile. 0 0.2 0.4 0.6 0.8 Hakea Grevillea Biomass/N (mg/mg) 0 1 2 3 4 Grevillea Hakea Biomass/P (mg/mg) 0 0.4 0.8 1.2 1.6 Hakea Grevillea Root length/N (m/mg) 0 2 4 6 8 Grevillea Hakea Root length/P (m/mg) Figure 2. The nutrient-use efficiency (N and P) of the test species of Hakea and Grevillea. The middle lines in the box represent the median values, and the bottom and top of the box are the first and third quartiles. The ends of the whiskers are 1.5 interquartile range (IQR) above the third quartile and 1.5 IQR below the first quartile. Figure 2. The nutrient-use efficiency (N and P) of the test species of Hakea and Grevillea. The middle lines in the box represent the median values, and the bottom and top of the box are the first and third quartiles. The ends of the whiskers are 1.5 interquartile range (IQR) above the third quartile and 1.5 IQR below the first quartile. Further analysis of the seed nutrient content and seedling growth rate revealed that Grevillea species had significantly higher nutrient-use efficiencies (Table 1; Fig. 2). Compared with Hakea, for each unit of P in the seed, Grevillea accumulated 1.6-fold more dry biomass and had roots that descended 2.9 times deeper into the soil, whereas for each unit of N in the seed, Grevillea accumulated 1.4-fold more dry biomass and had taproots that descended 3.1 times deeper into the soil (Table 1, Fig. 2). Discussionh The seed nutrient concentration in Grevillea reported in the present study, particularly with respect to P, was only half of that reported in previous scattered studies. For example, Hocking reported an average of 11.4 ±  2.6 mg.g−1 P for ten Grevillea species17. The results of the present study on 30 species reported Scientific Reports | 5:17132 | DOI: 10.1038/srep17132 3 www.nature.com/scientificreports/ an average of 6.3 ±  2.3 mg.g−1. This discrepancy is unlikely to be a consequence of methodological errors in our analysis because the 29 Hakea species were assayed in the same batch in the present study, and they yielded an average P content of 13.6 ±  3.8 mg.g−1, which is similar to the 15.1 ±  4.2 mg.g−1 P concentration reported for 13 species from ten previous studies (except Hakea pycnoneura, which was reported to have an unusually high P concentration of 36 mg.g−1)18. Interestingly, although the N and P concentrations in the seeds were lower in Grevillea than in Hakea, the N:P ratios in both taxa groups were similar. Notably, the lower seed N and P concentrations in Grevillea relative to those in Hakea did not reflect dilution by seed mass because the tested species in the two groups had similar seed masses. l y p g p With only half the seed nutrient content compared with species from the closely related genus Hakea, the species in Grevillea achieved similar levels of seedling growth during the early stages. Indeed, both groups of plants expanded their first true leaves within a similar time, accumulated similar amounts of dry biomass, and diverted similar proportions of resources to the shoots and roots. Consequently, the results of this analysis suggest that compared with Hakea, Grevillea species use nutrients signifi- cantly more efficiently than do Hakea. This efficiency represents a clear ecological advantage over Hakea because Grevillea species can accumulate twice as much dry biomass and descend their roots three times deeper into the soil for every unit of nutrient in their seeds. Seed-stored nutrients (N and P) are crucial for the successful establishment of seedlings in nutrient-impoverished landscapes. The rapid descent of the taproots to reach more reliable soil moisture reserves prior to the onset of summer droughts is critical for the survival of seedlings, particularly in Mediterranean climates with dry summer conditions19. Discussionh Using fewer nutrients, Grevillea species can grow roots equal in length to those of Hakea and survive dry Mediterranean-type summers equally as well, and these characteristics provide a selective advantage to Grevillea compared with other organisms that might require additional nutrients in nutrient-impoverished soils. g q p Seed production is inversely proportional to the per-seed reserve of nutrients, particularly P, in nutrient-impoverished soils20. In the fire-prone environments of SWA, which are generally characterised by poor soil, resprouters (populations that predominantly regenerate through sprouts from the trunk or underground organs) typically produce fewer seeds than nonsprouters (populations that predominantly regenerate through seeds)21. However, no significant differences were observed in the P content between nonsprouting and resprouting species growing on P-impoverished soil in a survey of 41 species in SWA18. The lower nutrient requirements for seedling growth in Grevillea relative to Hakea, which has much higher nutrient (N and P) concentrations, might allow Grevillea species to produce more seeds regard- less of the post-fire regeneration mode. Because of challenges in gauging the size of the soil seedbank, systematic surveys of Grevillea seed production are rare, although Pickup et al. reported a large seedbank for Grevillea rivularis at 193 ±  73 seeds m–2 22. Because of its capacity to grow to 2.5 m tall and 3 m wide, this plant species has the potential to produce up to 2000 seeds per plant, which is much higher than that of most Hakea species23. Levin24 proposed that propagule-rich lineages are likely to have high speciation rates because the increased number of seeds increases the opportunities for ecological and geographical speciation, and this hypothesis could also be true for Grevillea. Interestingly, high propagule pressure has also been proposed as one of the mechanisms facilitating successful invasion25. g Species in the Proteaceae family grow well in the world’s most nutrient-impoverished landscapes, particularly P-impoverished soils. Apart from diverse and efficient nutrient-acquisition strategies26, high nutrient-use efficiency is likely another key adaptation. Recent studies have observed high photosyn- thetic P-use efficiency in SWA Proteaceae species10. Lambers et al. reported that Proteaceae species such as Hakea species (Grevillea species were not included) from severely P-impoverished soils extensively replace phospholipids with galactolipids and sulpholipids during leaf development to achieve a high photosynthetic P-use efficiency, and proposed the “sulphur-for-phosphorus” hypothesis16. Sulpice et al.27 observed that Proteaceae species grow with very low levels of ribosomes, particularly plastidic ribosomes, at early stages of leaf development. Discussionh Many of the above studies involved Hakea species, although they rarely included Grevillea species; thus, additional research is required to test whether Grevillea have developed peak nutrient-use efficiency. p pfi y It can be speculated that once Grevillea overcome the barriers imposed by the two most limiting factors to its development, low soil nutrient availability and periodic water availability, these plants can invade most habitats. Indeed, species of Grevillea have been observed throughout Australia. High nutrient-use efficiency has interacted with multiple factors, including myrmecochory, a predominantly sexual reproduction strategy, and a high seed output, to allow the Grevillea genus to diversify into a group with 452 species/subspecies within only 11 million years. Scientific Reports | 5:17132 | DOI: 10.1038/srep17132 Methodsh The seeds of Grevillea and Hakea used for the nutrient analyses and germination and seedling growth comparisons were acquired from Nindethana Seed Service (Albany, Western Australia, Australia). Genus-wide seed mass information was extracted from the Seed Information Database28. The nutrient analysis was conducted on seeds from 59 species (29 Hakea and 30 Grevillea, Table S1). These spe- cies cover the major intra-generic taxonomy sections and provenances; therefore, they represent a wide diversity of the respective genera. For each species, up to 20 g of dry seeds were analysed for N and P concentrations at the ChemCentre (Perth, Western Australia, Australia) using the combustion method for N and inductively coupled plasma spectrometry for P. Scientific Reports | 5:17132 | DOI: 10.1038/srep17132 4 www.nature.com/scientificreports/ A total of 52 species (25 Hakea and 27 Grevillea species, Table S1) were used in the germination trials. Each species had three replicates, and there were up to 30 seeds per replicate (depending on seed size). Each replicate was contained within a 120-mm sterile Petri dish with 2 layers of sterilized Whatman Grade 1 filter paper and sealed with Parafilm. Petri dishes with Hakea seeds were filled with 5 mL autoclaved deionised water, whereas those with Grevillea seeds (because of inherent dormancy) were filled with 5 mL 10% smoke water solution (Regen 2000 Smokemaster); previous studies have sug- gested that Grevillea seed germination is enhanced by smoke stimulation29. To reduce the risk of fungal growth throughout the germination experiment, the smoke water was passed through a 0.20-μ m filter. Germination was conducted in an environmental chamber at a constant 15 °C with a 12 hour light/dark cycle. The germinants (seeds with radicle emergence ≥ 1 mm) were counted and recorded every two days for a period of up to 60 days. Seeds that did not germinate were assessed for viability using a cut test to visually assess the health of the embryo. The Petri dishes were randomised within the environmental chamber in each experiment. p For each of the species germinated in the above-described germination phase (12 Grevillea and 24 Hakea species, with no or minimal germination recorded in some species), ten germinants (or fewer when available) were individually planted in PVC tubes (100 cm in length and 5 cm in diameter) with a substrate of washed white sand (with no nutrients) and placed in a hoop house. References 1. Cowling, R. M., Rundel, P. W., Lamont, B. B., Arroyo, M. K. & Arianoutsou, M. Plant diversity in mediterranean-climate regions. Trends Ecol. Evol. 11, 362–366 (1996). 2. Myers, N., Mittermeier, R. A., Mittermeier, C. G., da Fonseca, G. A. B. & Kent, J. Biodiversity hotspots for conservation priorities Nature 403, 853–858 (2000).h 3. Cook, L. G., Hardy, N. B. & Crisp, M. D. Three explanations for biodiversity hotspots: small range size, geographical overlap and time for species accumulation. An Australian case study. New Phytol. doi: 10.1111/nph.13199 (2014). p y y p 4. Hopper, S. D. OCBIL theory: towards an integrated understanding of the evolution, ecology and conservation of biodiversity on old, climatically buffered, infertile landscapes. Plant Soil 322, 49–86 (2009).il climatically buffered, infertile landscapes. Plant Soil 322, 49–86 (2 f 5. Lengyel, S., Gove, A. D., Latimer, A. M., Majer, J. D. & Dunn, R. R. Ants sow the seeds of global diversification in flowering plants. PLoS ONE 4, 5 (2009). p 6. Makinson, R.O. In Flora of Australia, Vol. 17A (ed Makinson, O.) Ch. 1, 1–20 (CSIRO Publishing, 2000).l . Makinson, R.O. In Flora of Australia, Vol. 17A (ed Makinson, O d d l l d 7. Sniderman, J. M. K., Jordan, G. J. & Cowling, R. M. Fossil evidence for a hyperdiverse sclerophyll flora under a non-Mediterr type climate. Proc. Natl. Acad. Sci. USA 110, 3423–3428 (2013).i 8. Sauquet, H. et al. Contrasted patterns of hyperdiversification in mediterranean hotspots. Proc. Natl. Acad. Sci. USA 106, 221–225 (2009).fi 9. Orians, G. H. & Milewski, A. V. Ecology of Australia: the effects of nutrient-poor soils and intense fires. Biol. Rev. 82, 393–423 (2007). 10. Denton, M. D., Veneklaas, E. J., Freimoser, F. M. & Lambers, H. Banksia species (Proteaceae) from severely phosphorus- impoverished soils exhibit extreme efficiency in the use and re-mobilization of phosphorus. Plant Cell Environ. 30, 1557–1565 (2007). 11. Lambers, H. & Shane, M. W. Role of root clusters in phosphorus acquisition and increasing biological diversity in agriculture in Scale and Complexity in Plant Systems Research: Gene-Plant-Crop Relations (eds. Spiertz, J. H. J., Struik, P. C., Laar, H. H.) 237–250 (Springer, 2007). p g 12. Lambers, H., Raven, J. A., Shaver, G. R. & Smith, S. E. Plant nutrient-acquisition strategies change with soil age. Trends Evol. 23, 95–103 (2008). 13. Sander, J. & Wardell-Johnson, G. Methodsh The seedlings were watered (20 mL) every 2 to 3 days and grown for 100 days (from late winter to spring 2014) prior to harvesting. The measurements taken at harvest were leaf number, shoot length, root length and fresh and dry shoot and root mass. The dry biomass of the shoot and root was recorded after the samples were oven dried at 70 °C for 48 hours.hfii The nutrient-use efficiency (N and P) was defined as the amount of biomass produced or root length gained per unit of nutrient (mg) in the seeds. The nutrient-use efficiencies of Hakea and Grevillea were compared. Comparisons of the parameter values between Grevillea and Hakea were analysed with a t-test and implemented in PAST30. P <  0.05 was considered to represent statistical significance. Acknowledgementsh g This work was supported by Australian Research Council (DP120103389). TH acknowledges the support from a Curtin Research Fellowship. We thank Barry Price (ChemCentre, Western Australia) for assistance in seed nutrient assay, and Sophie Fox, Sh-Hoob El-Ahmirand, Thien Tran Duc for assistance in glasshouse and germination components. g This work was supported by Australian Research Council (DP120103389). TH acknowledges the support from a Curtin Research Fellowship. We thank Barry Price (ChemCentre, Western Australia) for assistance in seed nutrient assay, and Sophie Fox, Sh-Hoob El-Ahmirand, Thien Tran Duc for assistance in glasshouse and germination components. References 10, 307–315 (UWA Publishing, 2014).fi 26. Lambers, H. in Plant life on the sandplains in Southwest Australia, a global biodiversity hotspot (ed Lambers, H.) Ch. 10, 307–315 (UWA Publishing, 2014). 27 Sulpice R et al Low levels of ribosomal RNA partly account for the very high photosynthetic phosphorus use efficiency of (UWA Publishing, 2014). 27. Sulpice, R. et al. Low levels of ribosomal RNA partly account for the very high photosynthetic phosphorus-use efficiency of g 7. Sulpice, R. et al. Low levels of ribosomal RNA partly account for the very high photosynthetic phosphorus-use efficiency o Proteaceae species. Plant Cell Environ. 37, 1276–1298 (2014). p 8. Royal Botanic Gardens Kew. Seed Information Database (SID). Version 7.1. (2014) Available at: http://data.kew.org/sid/ Date o access: 10/12/2014. 9. Morris, E. C. Germination response of seven east Australian Grevillea species (Proteaceae) to smoke, heat exposure and scarification. Aust. J. Bot. 48, 179–189 (2000).t i 0. Hammer, Ø., Harper, D. A. T. & Ryan, P. D. 2001. PAST: Paleontological statistics software package for education and data analysis. Palaeontol. Electron. 4, 1–9 (2001). Author Contributions T.H. and C.C. designed the experiment, W.F. conducted the germination and growth experiment, T.H. and W.F. analysed the data; C.C. prepared materials for nutrient analysis. T.H. and W.F. wrote manuscript; All authors contributed to revision and reviewed the manuscript. References Fine-scale patterns of species and phylogenetic turnover in a global biodiversity hotspot: implications for climate change vulnerability. J. Veg. Sci. 22, 766–780 (2011). 14. Lambers, H. et al. Proteaceae from severely phosphorus-impoverished soils extensively replace phospholipids with galactolipids and sulfolipids during leaf development to achieve a high photosynthetic phosphorus-use efficiency. New Phytol. 196, 1098–1108 (2012). 15. Venterink, O. H. Does phosphorus limitation promote species-rich plant communities? Plant and Soil 345, 1–9 (2011). 16. Lambers, H., Brundrett, M. C., Raven, J. A. & Hopper, S. D. Plant mineral nutrition in ancient landscapes: high plant diversity on infertile soils is linked to functional diversity for nutritional strategies. Plant and Soil 334, 11–31 (2010) 17. Hocking, P. J. Mineral nutrient composition of leaves and fruit of selected species of Grevillea from south-western Australi special reference to Grevillea leucopteris Meissn. Aust. J. Bot. 34, 155–164 (1986). p p 18. Groom, P. K. & Lamont, B. B. Phosphorus accumulation in Proteaceae seeds: a synthesis. Plant Soil 334, 61–72 (2010). 19. Enright, N. J. & Lamont, B. B. Survival, growth and water relations of Banksia seedlings in a sand mine rehabilitation sit adjacent scrub-heath sites. J. Appl. Ecol. 29, 663–671 (1992). j pp 0. Henery, M. L. & Westoby, M. Seed mass and seed nutrient content as predictors of seed output variation between species. Oiko 92, 479–490 (2001).ti 21. Lamont, B. B. & Wiens, D. Are seed set and speciation always low among species that resprout after fire, and why? Evol. Ecol. 17, 277–292 (2003).l 22. Pickup, M., McDougall, K. L. & Whelan, R. J. Fire and flood: soil-stored seed bank and germination ecology in the endangered Carrington Falls Grevillea (Grevillea rivularis, Proteaceae). Austral Ecol. 28, 128–136 (2003). g 3. Groom, P. K. & Lamont, B. B. In Gondwanan heritage. Past, present and future of the Western Australian biota (eds Hopper, S D. et al.) 39–24 (Surrey Beatty and Sons, 1996). Scientific Reports | 5:17132 | DOI: 10.1038/srep17132 5 5 www.nature.com/scientificreports/ 4. Levin, D. A. Ancient dispersals, propagule pressure, and species selection in flowering Plants. Syst. Bot. 31, 443–448 (2006).h 25. Lockwood, J. L., Cassey, P. & Blackburn, T. M. The more you introduce the more you get: the role of colonization pressure and propagule pressure in invasion ecology. Divers. Distrib. 15, 904–910 (2009). 6. Lambers, H. in Plant life on the sandplains in Southwest Australia, a global biodiversity hotspot (ed Lambers, H.) Ch. Scientific Reports | 5:17132 | DOI: 10.1038/srep17132 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: He, T. et al. High nutrient-use efficiency during early seedling growth in iverse Grevillea species (Proteaceae). Sci. Rep. 5, 17132; doi: 10.1038/srep17132 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. 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Reactive Oxygen Species, Antioxidant Agents, and DNA Damage in Developing Maize Mitochondria and Plastids
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ORIGINAL RESEARCH published: 19 May 2020 doi: 10.3389/fpls.2020.00596 Edited by: Edited by: Francisco J. Corpas, Consejo Superior de Investigaciones Científicas (CSIC), Spain Reviewed by: Carlos Guillermo Bartoli, Instituto de Fisiología Vegetal (INFIVE), Argentina Daniel Gallie, University of California, Riverside, United States *Correspondence: Arnold J. Bendich bendich@uw.edu Specialty section: This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science Received: 28 November 2019 Accepted: 20 April 2020 Published: 19 May 2020 Citation: Tripathi D, Nam A, Oldenburg DJ and Bendich AJ (2020) Reactive Oxygen Species, Antioxidant Agents, and DNA Damage in Developing Maize Mitochondria and Plastids. Front. Plant Sci. 11:596. doi: 10.3389/fpls.2020.00596 Specialty section: This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science Reactive Oxygen Species, Antioxidant Agents, and DNA Damage in Developing Maize Mitochondria and Plastids Diwaker Tripathi, Andy Nam, Delene J. Oldenburg and Arnold J. Bendich* Department of Biology, University of Washington, Seattle, WA, United States Maize shoot development progresses from non-pigmented meristematic cells at the base of the leaf to expanded and non-dividing green cells of the leaf blade. This transition is accompanied by the conversion of promitochondria and proplastids to their mature forms and massive fragmentation of both mitochondrial DNA (mtDNA) and plastid DNA (ptDNA), collectively termed organellar DNA (orgDNA). We measured developmental changes in reactive oxygen species (ROS), which at high concentrations can lead to oxidative stress and DNA damage, as well as antioxidant agents and oxidative damage in orgDNA. Our plants were grown under normal, non-stressful conditions. Nonetheless, we found more oxidative damage in orgDNA from leaf than stalk tissues and higher levels of hydrogen peroxide, superoxide, and superoxide dismutase in leaf than stalk tissues and in light-grown compared to dark-grown leaves. In both mitochondria and plastids, activities of the antioxidant enzyme peroxidase were higher in stalk than in leaves and in dark-grown than light-grown leaves. In protoplasts, the amount of the small-molecule antioxidants, glutathione and ascorbic acid, and catalase activity were also higher in the stalk than in leaf tissue. The data suggest that the degree of oxidative stress in the organelles is lower in stalk than leaf and lower in dark than light growth conditions. We speculate that the damaged/fragmented orgDNA in leaves (but not the basal meristem) results from ROS signaling to the nucleus to stop delivering DNA repair proteins to mature organelles producing large amounts of ROS. Keywords: maize, ROS, plastids, mitochondria, protoplasts, DNA damage INTRODUCTION Received: 28 November 2019 Accepted: 20 April 2020 Published: 19 May 2020 Reactive oxygen species (ROS) can have both detrimental and beneficial effects on plants. At high concentrations, ROS can lead to oxidative stress by causing damage to various biomolecules. ROS are produced as unavoidable byproducts of electron transport reactions in both respiration and photosynthesis, and damage-defense measures are employed to ameliorate oxidative stress. But ROS also function in modifying the cell wall during development, as signaling molecules to maintain cellular and organismal homeostasis, and to regulate plant development (Mittler, 2017; Smirnoffand Arnaud, 2019). For example, ROS can affect the distribution of chloroplasts within a cell and the ability to resist pathogen attack (Park et al., 2018; Ding et al., 2019), as well as the fate of stem cells (Zeng et al., 2017; Yang et al., 2018). Citation: Tripathi D, Nam A, Oldenburg DJ and Bendich AJ (2020) Reactive Oxygen Species, Antioxidant Agents, and DNA Damage in Developing Maize Mitochondria and Plastids. Front. Plant Sci. 11:596. doi: 10.3389/fpls.2020.00596 May 2020 | Volume 11 | Article 596 1 Frontiers in Plant Science | www.frontiersin.org Organellar ROS and DNA Damage in Maize Tripathi et al. is increased by environmental change (extreme temperature, intense light, high salinity, water or nutrient deprivation) or deleterious mutations (Halliwell, 2006; Noctor and Foyer, 2016; Brunkard and Burch-Smith, 2018; Foyer, 2018; Bokhari and Sharma, 2019; Zandalinas et al., 2019). Our approach has been to monitor changes in orgDNA during the normal development of the wild-type plant, without the imposition of genotoxic agents or extreme environments. In particular, we wish to investigate potential ROS signaling that leads to the demise of orgDNA in differentiated somatic cells but not in germline cells. Reactive oxygen species can be produced in chloroplasts, mitochondria, and several other plant cell compartments (Janku et al., 2019). Since ROS are produced during the partial reduction of molecular oxygen, one way to avoid the potential damaging effects of ROS is to maintain certain cells under hypoxic conditions. In mammals, a hypoxic niche is maintained during early development in cells that will develop into gametes (embryonic stem cells) and later in the stem cells that provide the differentiated cells of adult tissues (Mohyeldin et al., 2010). Similarly, the non-green cells of the shoot apical meristem in Arabidopsis are also maintained in a hypoxic niche for 5 weeks during the development of the inflorescence meristem of the adult plant (Weits et al., 2019). Thus, in both animals and plants, the DNA that will be transmitted to the next generation is protected from potential oxidative stress associated with respiration and photosynthesis (Mohyeldin et al., 2010; Considine et al., 2017). is increased by environmental change (extreme temperature, intense light, high salinity, water or nutrient deprivation) or deleterious mutations (Halliwell, 2006; Noctor and Foyer, 2016; Brunkard and Burch-Smith, 2018; Foyer, 2018; Bokhari and Sharma, 2019; Zandalinas et al., 2019). Our approach has been to monitor changes in orgDNA during the normal development of the wild-type plant, without the imposition of genotoxic agents or extreme environments. In particular, we wish to investigate potential ROS signaling that leads to the demise of orgDNA in differentiated somatic cells but not in germline cells. Citation: Here, we report on the types and levels of ROS in mitochondria, plastids, and whole cells during maize seedling development in light and dark growth conditions so as to assess the correlation between ROS and orgDNA degradation. We also report on antioxidant agents and oxidative damage to orgDNA as assessed by levels of 8-hydroxydeoxyguanasine (8-OHdG) and 8-oxoguanine (8-oxoG). , ) Although the nucleus is not a major site of ROS production, ROS signaling molecules generated elsewhere in the cell can be moved to the nucleus to modulate expression of nuclear genes (Noctor and Foyer, 2016). The highest concentration of ROS should be found in the parts of the cell that produce most of the ROS, such as chloroplasts and mitochondria, but most research on DNA damage has focused on nuclear DNA. The most severe type of DNA damage is a double-strand break, and both homologous recombination (HR) and non-homologous end-joining (NHEJ) are used to repair this break in the nuclear DNA of yeasts and mammals (Runge and Li, 2018; Scully et al., 2019). Both HR and NHEJ are also found in the plant nucleus (Spampinato, 2017), but thus far only HR has been identified in either mitochondria or plastids of plants (Boesch et al., 2011; Christensen, 2018). Other DNA damage repair systems found in the nucleus may also be found in mitochondria and plastids. For example, Arabidopsis organellar DNA (orgDNA) polymerases can perform microhomology-mediated end-joining (MMEJ) in vitro (Garcia-Medel et al., 2019), proteins associated with base excision repair (BER) have been found in Arabidopsis plastids and mitochondria (Gutman and Niyogi, 2009; Boesch et al., 2011), and the BER system is the major pathway for repair of oxidatively damaged DNA (Markkanen, 2017). Frontiers in Plant Science | www.frontiersin.org Plant Tissue Zea mays (inbred line B73) seeds were imbibed overnight and sown in Sunshine soil Mix #4 and vermiculite (1:1 ratio). The seedlings were grown for 12 days with a 16 h light/8 h dark photoperiod (light-grown) or in continuous dark for 12 days (dark-grown). The light intensity was ∼500 µmol s−1 m−2. Seedlings were washed with 0.5% sarkosyl for ∼3 min and then rinsed with distilled water. For each assay, tissue was harvested from 20 to 25 plants as follows: Stalk lower (base of stalk 5 mm above the node); Stalk upper (top of stalk 5 mm below the ligule of the first leaf), leaf blades (L1 or L1 + L2 + L3). Stalk tissue was composed of several concentric rings of leaves, the outermost being the first leaf sheath. L1 was the fully expanded blade, whereas L2 and L3 were still developing. The coleoptile was removed before the extraction of plastids, mitochondria, and protoplasts. Isolation of Plastids and Mitochondria For plastids, 30% and 70% Percoll solutions adjusted to the equivalent osmolarity of 1x CDB were prepared (for example: for 30%, 12 mL Percoll + 8 mL 5x Chlp Gradient Buffer + 20 mL dH2O and for 70%, 28 mL Percoll + 8 mL 5x Chlp Gradient Buffer + 4 mL dH2O; 5x Chlp Gradient Buffer is 1.65 M D-sorbitol, 40 mM Hepes pH 7.6, 4 mM EDTA, 2 mM MgCl2, 0.2% BSA). For two-step gradients, 15 mL 30% Percoll was layered onto 15 mL of 70% Percoll in a 40-mL centrifuge tube. Then 2–4 mL of plastid solution was gently layered on top, followed by centrifuged for 30 min at 1,500 × g using a JA-20 fixed-angle rotor. Plastids were removed from the 30/70 Percoll interface, transferred to a centrifuge tube and washed 2– 3 times with CDB (using 10x the volume of recovered plastid solution), followed by centrifugation of 3,000 × g for 8 min to pellet plastids. The purified plastids were then resuspended in a small volume of CDB. A similar process was used for purification of mitochondria, except for the following minor changes. A two- step 28% and 45% Percoll gradient, with solutions adjusted to the equivalent osmolarity of 1x MDB, was used (for example: for 28%, 11.2 mL Percoll + 20 mL 2x Mito Gradient Buffer + 8.8 mL dH2O and for 45%, 18 mL Percoll + 20 mL 2x Mito Gradient Buffer + 2 mL dH2O; 2x Mito Gradient Buffer is 0.8 M D- sorbitol, 40 mM Hepes pH 7.6, 4 mM EDTA, 2 mM MgCl2, 0.2% BSA). Centrifugation was done for 20 min at 20,000 × g using a JA-20 fixed-angle rotor, mitochondria were recovered from the 28/45 Percoll interface, washed 2–3 times with MDB, pelleted by centrifugation for 15 min at 20,000 × g, and resuspended in small volume of MDB. Finally, plastids and mitochondria were stored in CDB or MDB. Freshly isolated plastids and mitochondria were used in the ROS assays. 0.5% Sarkosyl (5 min), 0.6% sodium hypochlorite (10 min), and 70% ethanol (10 s) and rinsed with sterile water. 0.5 mm strips were cut from the middle part of four or five leaves and stalks. Isolation of Plastids and Mitochondria Tissues were digested in the enzyme solution (1.5% cellulase R10 and 0.3% macerozyme (Yakult Honsha) in 0.6 M mannitol, 10 mM MES pH 5.7, 1 mM CaCl2, 5mM β-mercaptoethanol, 0.1% BSA for 30 min in a vacuum and 2 h at room temperature with agitation at 80 rpm. Protoplasts were stored overnight at 4◦C. The suspension containing protoplasts was filtered through a 35 µm nylon mesh. Protoplasts were pelleted by centrifuging at 150 × g, and the pellet was washed twice and then stored in W1 buffer (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES pH 5.7). Protoplasts were counted using a counting chamber slide. Freshly isolated protoplasts were used for the assays. Isolation of DNA From Organelles Isolation of DNA From Organelles Plastid and mtDNA were extracted using cetyltrimethylammonium bromide (CTAB) as described by Rogers and Bendich (1985) with minor modifications. An equal volume of 2x CTAB buffer [2% CTAB (w/v), 100 mM Tris/HCl (pH 8.0), 20 mM EDTA, 1.4 M NaCl, 1% polyvinylpyrrolidone (M 40000; w/v); preheated to 65◦C] and Proteinase K (20 µg/ml) were added to the resuspended plastids or mitochondria and incubated at 65◦C for 1 h. Then 0.1 M phenylmethylsulfonyl fluoride was added, followed by incubation at room temperature for 1 h. Then RNase A was added to 100 µg/mL, and the samples were kept at 60◦C for 15 min. Next, potassium acetate was added to 400 mM, and the mixtures were kept on ice for 15 min before centrifugation at 12,000 × g for 10 min at 4◦C. Equal volumes of chloroform:isoamyl alcohol (24:1) were added, the tubes were shaken, and then centrifuged at 12,000 × g for 1 min. After isopropanol precipitation, the DNA pellet was suspended in 10 mM Tris (pH 8), 1 mM EDTA (TE), and precipitated with two volumes of 100% ethanol overnight at −20◦C before pelleting. DNA pellets were washed three times with 70% ethanol, dried, and then resuspended in TE. Quantitation was performed using the Quant-IT DNA quantitation kit (Thermo Fisher Scientific). For superoxide detection, MitoSOX Red (Thermo Fisher Scientific) was used as a mitochondrial superoxide indicator. Oxidation of MitoSOX Red indicator (or dihydroethidium) by superoxide results in the formation of 2-hydroxyethidium that exhibits fluorescence at 510/580 nm. Amplex Red Assays for H2O2 and Peroxidase Activity For measuring H2O2 and peroxidase levels, we used the Amplex red dye. Amplex red reagent (10-acetyl-3,7- dihydroxyphenoxazine from Thermo Fisher Scientific) reacts with H2O2 in a 1:1 stoichiometry to produce highly fluorescent resorufin that can be measured by absorbance at 560 nm. The manufacturer’s protocol was followed to measure H2O2 and peroxidase. Briefly, H2O2 and peroxidase standards were prepared by serial dilution. Equal volumes (as above) of plastids/mitochondria/protoplasts and H2O2/peroxidase standard solutions were added to the Amplex red reagent and incubated at room temperature for 30 min. The absorbance was measured, and H2O2 and peroxidase levels were calculated using standard curves. Isolation of Plastids and Mitochondria Isolation of Plastids and Mitochondria Plastids and mitochondria were isolated using high-salt buffer (HSB; 1.25 M NaCl, 40 mM HEPES pH 7.6, 2 mM EDTA pH 8, 0.1% BSA) (Oldenburg et al., 2006, 2013). 0.1% β-mercaptoethanol (Sigma-Aldrich) was added to the buffer before grinding the tissue samples. Briefly, leaf and stalk tissues were homogenized in HSB using a blender, and the homogenate was filtered through 1–3 layers of Miracloth (EMD Millipore). The homogenate was differentially centrifuged first at low speed (500 × g for 5 min) to remove nuclei. Then the supernatant was centrifuged (3,000 × g for 10 min) to pellet plastids. The resulting supernatant was centrifuged at 20,000 × g for 15 min) to pellet mitochondria. The plastid and mitochondria pellets were washed three times with chloroplast dilution buffer (CDB; 0.33 M D- sorbitol, 20 mM HEPES pH 7.6, 2 mM EDTA, 1 mM MgCl2, 0.1% BSA) and mitochondria dilution buffer (MDB; 0.4 M D- sorbitol, 0.1 M HEPES pH 7.6, 2 mM EDTA, 1 mM MgCl2, 0.1% BSA), respectively. The plastids and mitochondria were further purified using discontinuous (step) Percoll gradients as As maize plants develop from the meristem at the base of the shoot (the basal meristem) to the leaves, the size of orgDNA [referring to both plastid DNA (ptDNA) and mitochondrial DNA (mtDNA)] decreases from molecules equal to or greater than the size of the genome (570 kb for mtDNA and 140 kb for ptDNA) in the meristem to much smaller fragments in the leaf (Oldenburg and Bendich, 2004, 2015; Kumar et al., 2014). In dark-grown plants, high-integrity ptDNA (large complex- branched molecules) is retained in the leaves, and a rapid decline in ptDNA copy number is observed after transfer from dark to light growth conditions (Zheng et al., 2011). We interpreted this decline in molecular integrity to ROS-induced DNA damage that was not repaired, followed by degradation of the unrepaired orgDNA molecules (Oldenburg and Bendich, 2015). Through the decades, research has focused on the damage caused by ROS. More recently, however, the positive aspects of ROS signaling have been appreciated when oxidative stress May 2020 | Volume 11 | Article 596 2 Organellar ROS and DNA Damage in Maize Tripathi et al. follows. Assays of ROS and Antioxidant Agents ROS Marker Dyes Fluorescein and rhodamine dyes are chemically reduced to colorless, non-fluorescent dyes. These “dihydro” derivatives are readily oxidized back to the parent dye by ROS and thus can serve as fluorogenic probes for detecting oxidative activity in cells and tissues. The fluorogenic probes and CellROX Green reagent (Thermo Fisher Scientific) were used to measure ROS in plastids, and the rhodamine dye DHR123 (Thermo Fisher Scientific) was used to measure ROS in mitochondria and protoplasts. Equal volumes of centrifuged pellets of isolated plastids, mitochondria, and protoplasts from leaf/stalk were used for each comparative measurement. Resuspended plastids/mitochondria/protoplasts were incubated with 5 µM CellROX or DHR123 for 30 min at 37◦C before the fluorescence units were measured using a Victor plate reader (Perkin Elmer) at 485/520 nm (for CellROX) and 507/529 nm (for DHR123). Ascorbic Acid Assays The levels of ascorbic acid (AsA) in protoplasts were quantified using the Cell Biolabs’ OxiSelectTM Ascorbic Acid Assay kit according to the manufacturer’s protocol (Cell Biolabs). The assay was based on the Ferric Reducing/Antioxidant Ascorbic Acid (FRASC) chemistry driven by the electron-donating reducing power of antioxidants. The assay employs ascorbate oxidase, which allows the user to differentiate the AsA content from other antioxidants present within the samples. AsA levels in a sample are determined by measuring the difference in optical density between two sample wells, one with and one without the enzyme. In samples, the ferrous iron was chelated to a colorimetric probe to form a product that was measured at 540. AsA levels (nM) were determined using the standard curve. For plastids, immunofluorescence imaging was done using a Nikon Microphot Epifluorescence microscope and images acquired with a QImaging Retiga 1300 10-bit digital camera using OpenLab image capture and analysis software. Imaging of 8-oxoG/Alexa 488 plastids was done using a 470/40ex and 525/50em filter set and autofluorescence imaging of plastids with a 470/20ex and 514em filter set. For individual plastids, the 8-oxoG/Alexa 488 mean fluorescence intensity (FI) was measured as pixel values (0 to 1023) (background mean FI was also measured and subtracted from plastid mean FI). As a control, plastids incubated with the Alexa secondary Ab and without the 8-oxoG primary antibody were also imaged. Immunofluorescence analysis of 8-oxoG/Alexa was also performed with plastids from light- and dark-grown entire seedling shoots (stalk and leaves), and the fluorescence intensity was evaluated visually (“by eye”) using a scale of undetectable, weak, or high fluorescence. 8-OxoG immunofluorescence assay Plastids and mitochondria from light-grown stalk lower, stalk upper and L1 tissues were fixed in 4% formaldehyde/Dulbecco’s phosphate buffered saline (PBS; Gibco) in 1 mM EDTA for 10 min, pelleted, and washed twice in PBS/EDTA. Then organelles were permeabilized in 0.1% Triton X- 100/PBS/EDTA for 5 min, followed by washing twice. Fixed and permeabilized plastids were incubated in blocking solution (2% BSA/PBS/EDTA) for 30 min. Organelles were incubated with primary antibody anti-8-oxoguanine mouse monoclonal (1:1000 dilution; 1 µL of 0.5 mg/mL anti-8-oxoG in 1 mL 1% BSA/PBS/EDTA blocking solution) (Millipore-Sigma MAB3560- C) for 1 h at room temperature, then washed three times. Next, organelles were incubated with secondary antibody goat anti- mouse IgM conjugated with Alexa Fluor 488 (1:1000 dilution; 1 µL of 2 mg/mL Alexa in 1 mL 1% BSA/PBS/EDTA blocking solution) (Invitrogen A21042) for 1 h at room temperature, then washed three times. Catalase Assay y Catalase activity in protoplasts was quantified using the OxiSelectTM catalase activity assay according to the manufacturer’s protocol (Cell Biolabs) that involves decomposition of H2O2 into water and oxygen, which is proportional to the concentration of catalase. After the reaction, the catalase is quenched with sodium azide, and the remaining H2O2 facilitates the coupling reaction of 4-aminophenazone (4-aminoantipyrene, AAP) and 3,5-dichloro-2-hydroxy- benzenesulfonic acid (DHBS) in the presence of horseradish peroxidase (HRP) catalyst. The product, quinoneimine dye, was measured at 520 nm using a 96-well microtiter plate. Hydrogen peroxide ‘working solution’ was added to each well. Incubation was at room temperature for 40–60 min with vigorous mixing. The absorbance was read at 520 nm. The activity in the samples was determined by interpolation of the catalase standard curve. Glutathione Assays Total glutathione (GSH) levels in protoplasts were determined by the OxiSelectTM Total Glutathione Assay Kit as per the manufacturer’s protocol (Cell Biolabs). In this assay, GSH reductase reduces oxidized glutathione (GSSG) to reduced glutathione (GSH) in the presence of NADPH. Subsequently, the chromogen reacts with the thiol group of GSH to produce a colored compound that absorbs at 405 nm. The total glutathione content (µM) was determined by comparison with the predetermined GSH standard curve. The mitochondria were stained with MitoTracker Red CMXRos (Invitrogen/Molecular Probes) (incubation in 1x PBS/EDTA/200 nM CMXRos for 1 h) prior to fixation. Imaging of mitochondria with the MitoTracker dye and 8-oxoG/Alexa 488 was done using an Olympus IX81 microscope and images were acquired with a Hamamatsu Orca Flash 2.8 CMOS 12- bit digital camera. A TRITC filter set (556/20 ex and 614/30 Protoplast Isolation Superoxide dismutase (SOD) activity was measured using a SOD colorimetric activity kit and the manufacturer’s protocol (Thermo Fisher Scientific). This assay measures all types of Maize protoplasts were isolated from the leaf and stalk tissues, as described by Sheen (1995). Briefly, seedlings were washed with May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 3 Organellar ROS and DNA Damage in Maize Tripathi et al. Biolabs) following the manufacturer’s protocol, which includes digestion of DNA to nucleosides. Equal amounts of the digested ptDNA and mtDNA and 8-OHdG standards were added to wells of 8-OHdG/BSA-conjugate preabsorbed microwell strips and incubated at room temperature for 10 min on an orbital shaker. Then anti-8-OHdG antibody was added to each well, and the plate was incubated at room temperature for 1 h. The strips were washed with Wash Buffer three times, and the diluted secondary antibody-enzyme conjugate was added for incubation at room temperature for 1 h. After washing, a substrate solution was added, and incubation was at room temperature for 2 min. The absorbance of each microwell was measured using 450 nm, and the 8-OHdG level was measured using a standard curve. SOD activity, including Cu/Zn, Mn, and FeSOD types. Samples (plastids/mitochondria/protoplasts) were diluted in colored sample diluent and added to the wells of a 96-well plate. The substrate was added followed by Xanthine Oxidase Reagent and incubation at room temperature for 20 min. Superoxide is generated by the xanthine oxidase that converts a colorless substrate to a yellow-colored product, which was quantified at 450 nm by an absorbance assay. A SOD standard curve was used for all samples. DNA Damage Assays ELISA 8-OHdG assay The quantitative measurement of 8-OHdG was determined by the OxiSelectTM Oxidative DNA Damage ELISA kit (Cell May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 4 Organellar ROS and DNA Damage in Maize Tripathi et al. RESULTS We previously reported changes in the structure of orgDNA molecules during maize development and proposed that light triggers orgDNA degradation probably due to ROS-induced damage without subsequent repair (Oldenburg and Bendich, 2004; Oldenburg et al., 2006, 2013; Zheng et al., 2011; Kumar et al., 2014). There is a gradient in cell and organellar development from the base of the stalk to the tip of the maize leaf (Sylvester et al., 1990; Stern et al., 2004). Here, we measured the levels of ROS, antioxidant agents, and orgDNA damage at three stages of maize development: stalk lower (the base of the stalk), stalk upper (top of the stalk), and the blades from the first three leaves (Table 1 and see “Materials and Methods” section). L1 refers to the first and oldest leaf. L2 and L3 refers to the second and third leaves, respectively. The tissue with the lowest value in each set of assays was used as the baseline for comparison with other tissues and is set at 1 (see “Materials and Methods” section and see Supplementary Material for statistical analyses). Since DHR123 and CellROX report ROS in a general sense, we used other dyes to focus on specific types of ROS molecules. Hydrogen peroxide is a major non-radical oxygen species that is generated during photosynthetic and respiratory electron transport chain reactions, as well as in peroxisomes (Mhamdi et al., 2010). We used the Amplex red assay for H2O2 in organelles isolated from leaf and stalk tissues. The lowest H2O2 level was detected in stalk lower (plastids and mitochondria) and stalk (for protoplasts) tissues, with the highest level in L1 and leaf (Figures 1D–F and Supplementary Table S3). Superoxide, a free-radical oxygen species, is also present in plant organelles. Using the mitochondrial-specific superoxide dye, MitoSOX red, we measured the superoxide level in mitochondria and protoplasts of leaf and stalk tissues (Figures 1G,H and Supplementary Table S5). We found that MitoSOX red, which was developed for mitochondria, did not work with isolated chloroplasts. The level of superoxide in mitochondria was higher by 2.3-fold for L1 than in stalk lower and 3-fold for leaf tissue than in stalk, respectively (Figures 1G,H). To summarize, both H2O2 and O2•−were lower in stalk than leaves, so that the level of ROS clearly increases as the seedlings develop from stalk to leaf blade tissue. RESULTS In order to compare properties of orgDNA molecules during maize development, we previously used equal volumes of isolated packed organelles, and here we use the same standard to assess changes in ROS and orgDNA damage. Organelle number per cell, organelle size, and protein amount and composition per organelle all change greatly during the transition from promitochondria and proplastids to mature organelles. Therefore, neither an equal number of organelles nor an equal amount of protein is a good standard for comparison. How changes in ROS assessed using isolated organelles might reflect changes in the organelles within the plant will be considered later (see “Discussion” section). ROS Levels Increase During Development em) was used for the MitoTracker dye and a FITC filter set (485/20 ex and 516/11 em) for 8-oxoG/Alexa 488. For individual mitochondria, the 8-oxoG/Alexa 488 mean fluorescence intensity (FI) was measured as pixel values (0 to 4096) (background mean FI was also measured and subtracted from mitochondria mean FI). As a control, mitochondria incubated with the Alexa secondary Ab and without the 8-oxoG primary antibody were also imaged. Differences in fluorescence intensity, higher for mitochondria (Figure 6B) than plastids (Figure 6A), can be attributed to the use of two different systems for image acquisition, the Hamamatsu 12-bit and the QImaging 10- bit, respectively. To measure the ROS level, we used ROS-indicator dyes that quantify general ROS components or that are specific for either superoxide anion (O2•−) or hydrogen peroxide (H2O2). We used these dyes (see “Materials and Methods” section) with isolated plastids and mitochondria and with whole-cell protoplasts. The ROS-indicator dyes are oxidized to fluorescent products that were quantified using a microplate reader. Figure 1 shows relative ROS levels using the dyes DHR123 for mitochondria and protoplasts and CellROX for plastids (Supplementary Table S1). Figure 1A shows that the ROS level is lowest in the plastids isolated from the stalk lower tissue. As the developmental gradient proceeds from stalk lower to stalk upper to the blade of L1, the ROS level increases to 2.6 in L1 compared to stalk lower. Unless accompanied by a corresponding increase in antioxidant defense, we would expect greater ROS damage to molecules of ptDNA in the green leaf blade than in the stalk (see “Discussion” section). A similar developmental increase of 2.7- fold was found for ROS in mitochondria isolated from the same tissues (Figure 1B), as well as 2.6-fold for protoplasts obtained from the total leaf (L1 + L2 + L3) blades and total stalk (stalk lower + stalk upper) tissues (Figure 1C). These data indicate that the increase in ROS during the development from stalk lower to leaf blade can be attributed to the maturation of plastids and mitochondria (and probably to the ROS byproducts of electron transport chains used in both photosynthesis and respiration), rather than other parts of the cell where ROS can be produced. Statistical Analysis All assays were performed at least three times with similar results. For Figures 1–5, the values in each bar graph are shown as mean relative values ± SE from three independent assays (biological replicates). Statistically significant differences between tissues were assessed by the Student’s t-test and/or by the ANOVA, and Tukey honest significant difference test and are shown as asterisks, where ∗P-value ≤0.05, ∗∗P-value ≤0.01, ∗∗∗P- value ≤0.001, and P-values > 0.05 are indicated on respective graphs (see Supplementary Material). Frontiers in Plant Science | www.frontiersin.org ROS Levels Are Higher in Light-Grown Than Dark-Grown Plants We previously reported that orgDNA maintenance is influenced by responses to light signals: light that led to the greening of seedling leaves also triggered the demise of both ptDNA May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 5 Organellar ROS and DNA Damage in Maize Tripathi et al. E 1 | ROS levels during maize development. Plastids and mitochondria were isolated from light-grown maize seedling tissues (Stalk lower, basal 1/3 of stalk; pper, upper 2/3 of stalk; L1, leaf 1). Protoplasts were isolated from Leaf (combined leaves L1, L2, and L3) and the entire Stalk (stalk lower and upper). For t of assays, equal volumes of plastids, mitochondria, or protoplasts were used. Measurements are given relative to the tissue with the lowest value (stalk r stalk), which is set at one (see the section “Materials and Methods”). (A–C) The level of reactive oxygen species (ROS; superoxide anion (O2•−), hydroxyl HO•), and H2O2) was measured using the oxidative stress marker fluorescence dyes DHR123 for mitochondria and protoplasts and CellROX green for . (D–F) H2O2 in was measured using the Amplex red assay. The hydrogen peroxide concentrations in µM were measured. (G,H) Superoxide anion was ed using the mitochondrial-specific superoxide fluorescence dye, MitoSOX. All assays were performed at least three times. Statistically significant differences easured using ANOVA statistic test with post hoc analysis using Tukey’s HSD and are shown as asterisks, where *P-value ≤0.05, **P-value ≤0.01, ue ≤0.001. P-values > 0.05 are indicated on respective graphs. FIGURE 1 | ROS levels during maize development. Plastids and mitochondria were isolated from light-grown maize seedling tissues (Stalk lower, basal 1/3 of stalk; Stalk upper, upper 2/3 of stalk; L1, leaf 1). Protoplasts were isolated from Leaf (combined leaves L1, L2, and L3) and the entire Stalk (stalk lower and upper). For each set of assays, equal volumes of plastids, mitochondria, or protoplasts were used. Measurements are given relative to the tissue with the lowest value (stalk lower or stalk), which is set at one (see the section “Materials and Methods”). (A–C) The level of reactive oxygen species (ROS; superoxide anion (O2•−), hydroxyl radical (HO•), and H2O2) was measured using the oxidative stress marker fluorescence dyes DHR123 for mitochondria and protoplasts and CellROX green for plastids. (D–F) H2O2 in was measured using the Amplex red assay. The hydrogen peroxide concentrations in µM were measured. ROS Levels Are Higher in Light-Grown Than Dark-Grown Plants (G,H) Superoxide anion was measured using the mitochondrial-specific superoxide fluorescence dye, MitoSOX. All assays were performed at least three times. Statistically significant differences were measured using ANOVA statistic test with post hoc analysis using Tukey’s HSD and are shown as asterisks, where *P-value ≤0.05, **P-value ≤0.01, ***P-value ≤0.001. P-values > 0.05 are indicated on respective graphs. Frontiers in Plant Science | www.frontiersin.org May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org Frontiers in Plant Science | www.frontiersin.org 6 Organellar ROS and DNA Damage in Maize Tripathi et al. FIGURE 2 | ROS levels in light and dark conditions. Plastids, mitochondria, and protoplasts were isolated from light-grown (light) and dark-grown (dark) maize seedling leaves (L1 + L2 + L3). Equal volumes of plastids, mitochondria or protoplasts were used for each set of assays. The assay measurements are given relative to the tissue with the lowest value (dark-grown samples) which is set at one. (A–C) The levels of ROS (DHR123 for plastids and mitochondria and CellROX for protoplasts) were assayed as in Figure 1. (D–F) The level of H2O2 was measured using the Amplex red as in Figure 1. (G,H) The levels of superoxide anion (O2•−) were measured using MitoSOX as in Figure 1. All assays were performed at least three times. Statistically significant differences were measured using ANOVA statistic test with post hoc analysis using Tukey’s HSD and are shown as asterisks, where *P-value ≤0.05, **P-value ≤0.01, ***P-value ≤0.001. GURE 2 | ROS levels in light and dark conditions. Plastids, mitochondria, and protoplasts were isolated from light-grown (light) and dark-grown (dark) maize dling leaves (L1 + L2 + L3). Equal volumes of plastids, mitochondria or protoplasts were used for each set of assays. The assay measurements are given relative he tissue with the lowest value (dark-grown samples) which is set at one. (A–C) The levels of ROS (DHR123 for plastids and mitochondria and CellROX for toplasts) were assayed as in Figure 1. (D–F) The level of H2O2 was measured using the Amplex red as in Figure 1. (G,H) The levels of superoxide anion (O2•−) FIGURE 2 | ROS levels in light and dark conditions. Plastids, mitochondria, and protoplasts were isolated from light-grown (light) and dark-grown (dark) maize seedling leaves (L1 + L2 + L3). Equal volumes of plastids, mitochondria or protoplasts were used for each set of assays. ROS Levels Are Higher in Light-Grown Than Dark-Grown Plants The assay measurements are given relative to the tissue with the lowest value (dark-grown samples) which is set at one. (A–C) The levels of ROS (DHR123 for plastids and mitochondria and CellROX for protoplasts) were assayed as in Figure 1. (D–F) The level of H2O2 was measured using the Amplex red as in Figure 1. (G,H) The levels of superoxide anion (O2•−) were measured using MitoSOX as in Figure 1. All assays were performed at least three times. Statistically significant differences were measured using ANOVA statistic test with post hoc analysis using Tukey’s HSD and are shown as asterisks, where *P-value ≤0.05, **P-value ≤0.01, ***P-value ≤0.001. and mtDNA in maize (Oldenburg et al., 2006; Zheng et al., 2011; Kumar et al., 2014). Here, we test the hypothesis that the increased level of ROS in light-grown maize correlates with increased damage to orgDNA. We quantified ROS in plastids, mitochondria, and protoplasts from light- and dark-grown total leaf blade (L1 + L2 + L3) tissues, using the tissue with the May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 7 et al. Organellar ROS and DNA Damage in Maize RE 3 | Antioxidant agents during maize development. Organelles and protoplasts were isolated, as in Figure 1. The assay measurements are given relative to ssue with the lowest value which is set at one. (A–C) The level of superoxide dismutase (SOD) enzyme activity was measured as U/mL using an immunoassay. ) The Amplex red assay was used for the peroxidase (PRX) enzyme activity and measured as mU/mL. (G) A colorimetric assay was used for determining ase (CAT) enzyme activity and measured as U/mL. (H) Total glutathione (GSH) and (I) ascorbic acid (AsA) levels (µM and nM, respectively) in protoplasts were mined by colorimetric assays. All assays were performed at least three times. Statistically significant differences were measured using ANOVA statistic test with hoc analysis using Tukey’s HSD and are shown as asterisks, where *P-value ≤0.05, **P-value ≤0.01, ***P-value ≤0.001. P-values > 0.05 are indicated in the ective graphs. Tripathi et al. Organellar ROS and DNA Damage in Maize FIGURE 3 | Antioxidant agents during maize development. Organelles and protoplasts were isolated, as in Figure 1. The assay measurements are given relative to the tissue with the lowest value which is set at one. ROS Levels Are Higher in Light-Grown Than Dark-Grown Plants (A–C) The level of superoxide dismutase (SOD) enzyme activity was measured as U/mL using an immunoassay. (D–F) The Amplex red assay was used for the peroxidase (PRX) enzyme activity and measured as mU/mL. (G) A colorimetric assay was used for determining catalase (CAT) enzyme activity and measured as U/mL. (H) Total glutathione (GSH) and (I) ascorbic acid (AsA) levels (µM and nM, respectively) in protoplasts were determined by colorimetric assays. All assays were performed at least three times. Statistically significant differences were measured using ANOVA statistic test with post hoc analysis using Tukey’s HSD and are shown as asterisks, where *P-value ≤0.05, **P-value ≤0.01, ***P-value ≤0.001. P-values > 0.05 are indicated in the respective graphs. lower amount of ROS as the baseline for comparison. The light/dark ratios were similar, ranging from 2.5 to 3.5, for general ROS (Figures 2A–C and Supplementary Table S2), H2O2 (Figures 2D–F and Supplementary Table S4), and O2•− (Figures 2G,H and Supplementary Table S6). Although it might be expected that these light/dark ratios would be similar for plastids and protoplasts, it is notable that the light/dark ratios are also in the range of 2.5 to 3 for isolated, non-green May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 8 Organellar ROS and DNA Damage in Maize Tripathi et al. RE 4 | Antioxidant agents from maize seedlings grown under light or dark conditions. Organelles and protoplasts were isolated, as in Figure 2. The assay rements are given relative to the tissue with the lowest value, which is set at one. (A–C) The level of superoxide dismutase (SOD) enzyme activity was red as U/mL using an immunoassay. (D–F) The level of peroxidase (PRX) enzyme activity was measured as mU/mL using the Amplex red assay. (G) A metric assay was used for determining catalase (CAT) enzyme activity and measured as U/mL. (H) Total glutathione (GSH) and (I) ascorbic acid (AsA) leve d nM, respectively) in protoplasts were determined by colorimetric assays. All assays were performed at least three times. Statistically significant differen measured using ANOVA statistic test with post hoc analysis using Tukey’s HSD and are shown as asterisks, where *P-value ≤0.05, **P-value ≤0.01, lue ≤0.001. FIGURE 4 | Antioxidant agents from maize seedlings grown under light or dark conditions. Organelles and protoplasts were isolated, as in Figure 2. ROS Levels Are Higher in Light-Grown Than Dark-Grown Plants The assay measurements are given relative to the tissue with the lowest value, which is set at one. (A–C) The level of superoxide dismutase (SOD) enzyme activity was measured as U/mL using an immunoassay. (D–F) The level of peroxidase (PRX) enzyme activity was measured as mU/mL using the Amplex red assay. (G) A colorimetric assay was used for determining catalase (CAT) enzyme activity and measured as U/mL. (H) Total glutathione (GSH) and (I) ascorbic acid (AsA) levels (µM and nM, respectively) in protoplasts were determined by colorimetric assays. All assays were performed at least three times. Statistically significant differences were measured using ANOVA statistic test with post hoc analysis using Tukey’s HSD and are shown as asterisks, where *P-value ≤0.05, **P-value ≤0.01, ***P-value ≤0.001. May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 9 Organellar ROS and DNA Damage in Maize Tripathi et al. E 5 | 8-hydroxydeoxyguanosine (8-OHdG) representing orgDNA damage during maize development. Equal amounts of DNA extracted from isolated p ochondria were used to measure the 8-OHdG levels using a competitive ELISA assay. The levels of 8-OHdG in ptDNA and mtDNA were determined wn relative to stalk lower for (A,B) and relative to dark-grown leaves for (C,D). All assays were performed at least three times. Statistically significant ces were measured using ANOVA statistic test with post hoc analysis using Tukey’s HSD and are shown as asterisks, where *P-value ≤0.05, e ≤0.01, ***P-value ≤0.001. P-values > 0.05 are indicated on respective graphs. FIGURE 5 | 8-hydroxydeoxyguanosine (8-OHdG) representing orgDNA damage during maize development. Equal amounts of DNA extracted from isolated plastids and mitochondria were used to measure the 8-OHdG levels using a competitive ELISA assay. The levels of 8-OHdG in ptDNA and mtDNA were determined (ng/mL) and shown relative to stalk lower for (A,B) and relative to dark-grown leaves for (C,D). All assays were performed at least three times. Statistically significant differences were measured using ANOVA statistic test with post hoc analysis using Tukey’s HSD and are shown as asterisks, where *P-value ≤0.05, **P-value ≤0.01, ***P-value ≤0.001. P-values > 0.05 are indicated on respective graphs. May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 10 Organellar ROS and DNA Damage in Maize Tripathi et al. TABLE 1 | Assays for ROS and antioxidant agents. Antioxidant Enzyme Activity in Light and Dark Conditions The ROS level in the organelles isolated from leaf tissue of maize was higher for light-grown than dark-grown plants (Figure 2), and this difference may be attributed to lower antioxidant enzyme activity in the light. To test this idea, we measured the antioxidant activities of SOD, peroxidase, and catalase in leaves (L1 + L2 + L3) grown in light and dark conditions, as described above. We found 2–3.4 times higher SOD activity in the organelles and protoplasts of light-grown than dark- grown plants (Figures 4A–C and Supplementary Table S8). In contrast, the activity of peroxidase was 2–2.7 times lower for light-grown than dark-grown plants (Figures 4D–F and Supplementary Table S10). The catalase activity was 2.4 times lower in protoplasts from light compared to dark (Figure 4G and Supplementary Table S12). mitochondria because there are no known photoreceptors in mitochondria. ROS Levels Are Higher in Light-Grown Than Dark-Grown Plants Assay Method Mechanism Detection General ROS Dihydrorhodamine 123 and CellROX dyes Dyes fluoresce green upon oxidation Fluorescence Hydrogen peroxide Amplex red assay (Amplex red reagent + horseradish peroxidase) Generation of fluorescent resorufin Absorbance/ fluorescence Superoxide MitoSOX dye Dye oxidized by superoxide in mitochondria Fluorescence Superoxide dismutase Colorimetric immunoassay Generation of a yellow product Absorbance Peroxidase Amplex red assay (Amplex red reagent + hydrogen peroxide) Generation of fluorescent resorufin Absorbance/ fluorescence Catalase Colorimetric assay Coupling of quinoneimine dye with H2O2 Absorbance Glutathione Colorimetric assay Generation of a colored compound by a reaction of chromogen with glutathione Absorbance Ascorbic acid Colorimetric assay Ferric reducing/antioxidant ascorbic acid (FRASC) chemistry Absorbance TABLE 1 | Assays for ROS and antioxidant agents. localized to the cytosol, plastids, mitochondria, and peroxisomes in plants (Liebthal et al., 2018). Our peroxidase assay measured all types of peroxidase activities. We found more peroxidase activity in organelles isolated from stalk lower compared to organelles from L1 and more in protoplasts from stalk than from leaf (Figures 3D–F and Supplementary Table S9). Catalase has high specificity for H2O2 and acts by the dismutation of two molecules of H2O2 to water and O2 (Mhamdi et al., 2010). Although the CAT-3 isoform of catalase was reported in maize mitochondria (Scandalios et al., 1980), and some catalase was reported in other cellular compartments including chloroplasts (Mhamdi et al., 2010), the peroxisome has been considered as the main location of catalase within plant cells, with mitochondrial/chloroplast catalase as possible contamination from broken peroxisomes (Corpas et al., 2017). Here, we report total cellular catalase activity in maize protoplasts prepared from stalk and leaf tissue. In our assays, catalase activity was higher in protoplasts from stalk than from leaf (Figure 3G and Supplementary Table S11). In all antioxidant enzyme assays, the tissue with the lowest enzyme activity was used as the baseline for comparison with other tissues. Our assays suggest that high peroxidase and catalase activities result in the relatively low H2O2 level in the stalk, whereas low peroxidase and catalase activities result in high H2O2 levels in the leaf. Frontiers in Plant Science | www.frontiersin.org Change in Antioxidant Enzyme Activity During Development Antioxidant enzymes can modulate the levels of ROS and reduce oxidative stress (Asada, 2006; Soares et al., 2019). The SOD enzymes remove superoxide by catalyzing its dismutation and reducing it to H2O2. Plants have MnSODs in the mitochondria and peroxisomes, Cu/ZnSODs in the chloroplast, peroxisomes, and cytosol, and FeSODs in the chloroplast (Alscher et al., 2002; Pilon et al., 2011). Our SOD assay measured all types of SOD activities. We measured SOD activity in organelles and protoplasts isolated from leaf and stalk tissues. As shown in Figures 3A–C and Supplementary Table S7, SOD activity was highest in organelles isolated from L1 and higher in protoplasts from leaf than from stalk. Levels of Small-Molecule Antioxidants Levels of Small-Molecule Antioxidants The non-enzymatic antioxidant system in plants includes low- mass metabolites like GSH, AsA, phenolic compounds, and proline. These antioxidants manage the ROS homeostasis by removing, transforming, or neutralizing the oxidant pool (Diaz- Vivancos et al., 2015; Smirnoff, 2018; Soares et al., 2019). To determine if small antioxidants are involved in maintaining low levels of ROS in maize, we quantified the levels of GSH and AsA in protoplasts isolated from leaf and stalk tissues. We found that the level of GSH was 2.2 times higher in stalk compared to leaf (Figure 3H and Supplementary Table S11). And we found that the level of AsA was 1.2 times higher in stalk than in leaf (Figure 3I and Supplementary Table S11). The stability and accumulation of H2O2 are mainly influenced by the activity of the antioxidative system. In plants, several antioxidant enzymes metabolize H2O2. Ascorbate peroxidases (APX), peroxiredoxins, glutathione/thioredoxin peroxidases, glutathione S-transferases, and catalases are such enzymes (Cerny et al., 2018; Foyer, 2018). APXs have high specificity for H2O2, are present in chloroplast and mitochondria, and perform the final step of conversion of free radicals to water and oxygen (Maruta et al., 2016). Other peroxidases, such as peroxiredoxins, are May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 11 Organellar ROS and DNA Damage in Maize Tripathi et al. Supplementary Table S13). The level was 1.4–1.6 times higher in orgDNAs isolated from L1 compared to orgDNAs isolated from stalk lower. We also found 1.4 times higher 8-OHdG in the orgDNA of leaves from seedlings grown in the light than in the dark (Figures 5C,D and Supplementary Table S14). The higher 8-OHdG levels strongly suggest greater oxidative damage in the organelles from leaf than stalk. We also performed the GSH and AsA assays in protoplasts isolated from the light-and dark-grown leaves. As shown in Figures 4H,I and Supplementary Table S12, both GSH and AsA levels were higher in the protoplasts isolated from the dark-grown plants. In protoplasts from the dark-grown leaves, the GSH level was 2.8 times higher, and the AsA level was 1.4 times higher than protoplasts from light-grown plants. In summary (Table 2), we found higher levels of ROS (H2O2 and superoxide) and higher SOD activity in leaf than stalk tissues, but lower activities for peroxidase and catalase in leaf than stalk tissues. Levels of Small-Molecule Antioxidants Light-grown leaves had higher levels of H2O2, superoxide, and SOD but lower activities of peroxidase and catalase than dark-grown leaves. Levels of GSH and AsA also changed, lower in light-grown leaf than in stalk tissue. Immunofluorescence microscopy was performed using isolated plastids and mitochondria from light-grown maize tissues (stalk lower, stalk upper, and L1) with a primary antibody to 8-oxoG and a secondary antibody containing the fluorescent dye Alexa 488. For both plastids and mitochondria, the fluorescence intensity per organelle was higher for the leaf than the stalk tissues (Figures 6A,B). 8-OxoG immunofluorescence was also evaluated for plastids from light- and dark-grown entire seedling shoots (stalk and leaves). In this assay, the fluorescence intensity was assessed visually and scored as undetectable, weak, or high. For light, 75 out of 107 plastids were scored as “high”; for dark, 0 of 48 plastids were scored as “high.” Each of these results indicate that there are more 8-oxoG lesions in ptDNA from photosynthetically active leaf chloroplasts than non-photosynthetic stalk plastids. Levels of orgDNA Oxidative Damage Change During Development and in Light/Dark Conditions Reactive oxygen species generate various modified DNA bases, and 7,8-dihydro-8-oxoguanine (8-oxoguanine, 8-oxoG) is the most common modified base found in DNA from bacteria, the eukaryotic nucleus, and mitochondria of animals, plants, and yeast (Imlay, 2013; Wallace, 2013; Bokhari and Sharma, 2019). During repair via the BER pathway, the damaged nucleoside, 8-hydroxydeoxyguanasine (8-OHdG), is released and has been used as a biomarker for oxidative stress (Shen et al., 2007). 8- oxoG lesions in DNA have also been assessed in rat liver cells by immunofluorescence (Kemeleva et al., 2006). DISCUSSION Most of the changes we report for ROS and antioxidant agents during maize development might have been anticipated as a mitigating response to damage resulting from oxidative stress. In some cases, however, the anticipated damage-defense relationship was not observed suggesting a beneficial role for ROS unrelated to damage. We now consider how ROS and antioxidant agents may influence the maintenance or degradation of orgDNA during the transition from stem cell to leaf. We used antibodies to 8-OHdG and a competitive ELISA method to measure the relative amounts of oxidative damage in orgDNA from maize tissues. In our ELISA assay, we found a higher level of 8-OHdG in ptDNA and mtDNA isolated from the blade of L1 compared to stalk lower tissue (Figures 5A,B and ROS and Antioxidant Agents in Plant Cells and Organelles: An Overview (A) The number of plastids measured and the average mean fluorescence intensity ± standard error was 22 and 0.8 ± 0.2 for Stalk lower, 18 and 1.2 ± 0.3 for Stalk upper, and 20 and 10.6 ± 1.4 for L1. (B) The number of mitochondria measured and the average mean fluorescence intensity ± standard error was 19 and 45 ± 7 for Stalk lower, 15 and 56 ± 10 for Stalk upper, and 33 and 136 ± 9 for L1. For both plastids and mitochondria, Stalk lower and Stalk upper are significantly different compared to L1 with P-value < 0.0001 using ANOVA with post hoc Tukey HSD, and there is no significant difference between Stalk lower and Stalk upper. Differences in the Fluorescence intensity may be attributed to different systems used to acquire images for plastids and mitochondria (see “Materials and Methods” section). increases from stalk to green leaf (Figure 1G), but we do not know the molar concentration in either tissue. And superoxide levels were determined only for mitochondria, not for plastids, even in the protoplast assays. The assay for H2O2 does report concentration, although this is for a given volume of pooled organelles or protoplasts. For plastids from the stalk lower, the concentration ranged from 2.5–5.3 µM H2O2 and increased to 8.5–19.6 in the green leaf. Similar values were found for mitochondria, 3.3–4.3 in stalk lower and 8.2–11.7 in leaf. Not surprisingly, the cellular H2O2 concentration was higher, 18.7– 24.5 µM for protoplasts from light-grown leaves. The molarity of H2O2 extracted from various plants has been reported, but these values enormously exceed the values for animal cells, probably do the extremely high levels in the apoplastic parts of plant tissues (cell walls and intercellular spaces) (Foyer and Noctor, 2016; Noctor et al., 2018). Previous estimates of the absolute concentrations of ROS molecules within plant cells are problematic, in part due to technical difficulties, and even newer methods employing genetically engineered ROS-sensor proteins (HyPer) only report relative differences in ROS levels between samples (such as control and high light exposure) or along a cellular gradient (Exposito-Rodriguez et al., 2017; Smirnoffand Arnaud, 2019). Our data were obtained with isolated organelles and protoplasts, including many wash steps, so that apoplastic sources do not affect our ROS and antioxidant data. Maize is a C4 plant containing both mesophyll and bundle sheath cells. Isolated Organelles and Organelles in the Plant Any biochemical property may be altered by removing molecules or organelles from intact tissue before analysis, and the nature of ROS can make measurements especially challenging (Noctor et al., 2016). In order to mitigate effects of inadvertent oxidation on subsequent assays of ROS, antioxidant enzymes, and orgDNA damage, we employed low temperature and reducing conditions during the isolation of organelles. Furthermore, our data are reported as relative values among tissues handled and analyzed in parallel, so that potential isolation artifacts were controlled. The overall conclusions, summarized in Figure 7, present a relationship between redox status and DNA damage that would not be anticipated from artifactual data. germline meristem and highly fragmented/damaged orgDNA in developing somatic cells. ROS and Antioxidant Agents in Plant Cells and Organelles: An Overview (A) The number of plastids measured and the average mean fluorescence intensity ± standard error was 22 and 0.8 ± 0.2 for Stalk lower, 18 and 1.2 ± 0.3 for Stalk upper, and 20 and 10.6 ± 1.4 for L1. (B) The number of mitochondria measured and the average mean fluorescence intensity ± standard error was 19 and 45 ± 7 for Stalk lower, 15 and 56 ± 10 for Stalk upper, and 33 and 136 ± 9 for L1. For both plastids and mitochondria, Stalk lower and Stalk upper are significantly different compared to L1 with P-value < 0.0001 using ANOVA with post hoc Tukey HSD, and there is no significant difference between Stalk lower and Stalk upper. Differences in the Fluorescence intensity may be attributed to different systems used to acquire images for plastids and mitochondria (see “Materials and Methods” section). ROS and Antioxidant Agents in Plant Cells and Organelles: An Overview TABLE 2 | Results summary for plastids, mitochondria, and protoplasts. Assay Results General ROS Leaf > stalk Light > dark Hydrogen peroxide Leaf > stalk Light > dark Superoxide* Leaf > stalk Light > dark Superoxide dismutase Leaf > stalk Light > dark Peroxidase Leaf < stalk Light < dark Catalase** Leaf < stalk Light < dark Glutathione** Leaf < stalk Light < dark Ascorbic acid** Leaf < stalk Light < dark *Superoxide assay was not performed for plastids. **Assays were performed only for protoplasts. TABLE 2 | Results summary for plastids, mitochondria, and protoplasts. g Although chloroplasts and mitochondria are the main sites of ROS production, ROS profoundly influence the chemistry in peroxisomes, cytosol, and vacuoles (Asada, 2006; Noctor and Foyer, 2016; Kohli et al., 2019). Cells also contain many protein and small-molecule antioxidant agents that both counteract oxidative stress and facilitate signaling the redox status of the cell to the nucleus, probably in the form of H2O2 (Cerny et al., 2018; Locato et al., 2018; Smirnoffand Arnaud, 2019; Soares et al., 2019). These include various SOD enzymes that convert the highly reactive O2•−to the mobile but less reactive H2O2 (Alscher et al., 2002; Pilon et al., 2011), catalases that remove H2O2 (Zamocky et al., 2008; Corpas et al., 2017), and APXs and glutathione peroxidases (GTXs) (Maruta et al., 2016; Liebthal et al., 2018) that also remove H2O2, as well as GSH, AsA, and other small-molecule antioxidants (Noctor et al., 2012; Diaz- Vivancos et al., 2015; Smirnoff, 2018). The redox “objective” indicated by levels of these antioxidant agents, as well as oxygen content, seems to be directed at suppressing oxidative damage in the meristem while tolerating some oxidative damage in the green leaf. One consequence is seen as pristine orgDNA in the May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 12 Organellar ROS and DNA Damage in Maize Tripathi et al. FIGURE 6 | 8-oxoguanine (8-oxoG) in maize plastids and mitochondria. Plastids (A) and mitochondria (B) were isolated from light-grown maize seedling tissues (Stalk lower, basal 1/3 of stalk; Stalk upper, upper 2/3 of stalk; L1, leaf 1). Fluorescence intensity was measured for individual plastids and mitochondria that were imaged by immunofluorescence microscopy using a primary antibody to 8-oxoG coupled with a secondary fluorescence antibody. All assays were performed at least three times. ROS and Antioxidant Agents in Plant Cells and Organelles: An Overview The isolated organelles and protoplasts prepared using our methods are mostly derived from mesophyll cells (Kumar et al., 2015), so that our ROS and antioxidant data represent a specific subset of differentiated maize cells. Whereas accurate measurements of the concentrations of ROS molecules and antioxidants in plant cell compartments are needed to better understand the influence of ROS in oxidative stress and signaling, comparisons of the relative levels in isolated organelles can provide insights, as described below. FIGURE 6 | 8-oxoguanine (8-oxoG) in maize plastids and mitochondria. Plastids (A) and mitochondria (B) were isolated from light-grown maize seedling tissues (Stalk lower, basal 1/3 of stalk; Stalk upper, upper 2/3 of stalk; L1, leaf 1). Fluorescence intensity was measured for individual plastids and mitochondria that were imaged by immunofluorescence microscopy using a primary antibody to 8-oxoG coupled with a secondary fluorescence antibody. All assays were performed at least three times. (A) The number of plastids measured and the average mean fluorescence intensity ± standard error was 22 and 0.8 ± 0.2 for Stalk lower, 18 and 1.2 ± 0.3 for Stalk upper, and 20 and 10.6 ± 1.4 for L1. (B) The number of mitochondria measured and the average mean fluorescence intensity ± standard error was 19 and 45 ± 7 for Stalk lower, 15 and 56 ± 10 for Stalk upper, and 33 and 136 ± 9 for L1. For both plastids and mitochondria, Stalk lower and Stalk upper are significantly different compared to L1 with P-value < 0.0001 using ANOVA with post hoc Tukey HSD, and there is no significant difference between Stalk lower and Stalk upper. Differences in the Fluorescence intensity may be attributed to different systems used to acquire images for plastids and mitochondria (see “Materials and Methods” section). FIGURE 6 | 8-oxoguanine (8-oxoG) in maize plastids and mitochondria. Plastids (A) and mitochondria (B) were isolated from light-grown maize seedling tissues (Stalk lower, basal 1/3 of stalk; Stalk upper, upper 2/3 of stalk; L1, leaf 1). Fluorescence intensity was measured for individual plastids and mitochondria that were imaged by immunofluorescence microscopy using a primary antibody to 8-oxoG coupled with a secondary fluorescence antibody. All assays were performed at least three times. The Transition From Stem Cells to Leaf Cells Our data are shown as the relative levels of several types of ROS and antioxidant agents, using the tissue with the lowest level as the reference point. Most assays report fluorescence units from reactive dyes or enzyme activity, neither of which provide concentration level. For example, we find that superoxide During maize leaf development, new cells arise from the basal meristem, begin expansion/elongation in the stalk region, and become fully differentiated in the leaf blade. The developing May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 13 Tripathi et al. Organellar ROS and DNA Damage in Maize FIGURE 7 | Changes in ROS and antioxidant agents during maize chloroplast and mitochondrial development. There is a gradient in cellular and organellar development from the basal meristem (Stalk lower) to the fully expanded leaf blade in maize. In the undeveloped proplastids and promitochondria, the activity of peroxidase was high, facilitating the maintenance of minimal ROS levels and protecting orgDNA from oxidative damage. In chloroplasts and mitochondria, there were high levels of ROS, the byproducts of photosynthesis and respiration. Superoxide dismutase (SOD) converted some superoxide (O2•−) to H2O2, but the activity of peroxidase was low. Thus, the orgDNA was subjected to extensive oxidative damage and became fragmented. Levels of glutathione (GSH), ascorbic acid (AsA), and catalase activity were obtained from protoplasts from the entire stalk and leaf blades (L1–L3), whereas both protoplasts and isolated organelles were used to assay ROS and enzyme activities for SOD and peroxidase. FIGURE 7 | Changes in ROS and antioxidant agents during maize chloroplast and mitochondrial development. There is a gradient in cellular and organellar development from the basal meristem (Stalk lower) to the fully expanded leaf blade in maize. In the undeveloped proplastids and promitochondria, the activity of peroxidase was high, facilitating the maintenance of minimal ROS levels and protecting orgDNA from oxidative damage. In chloroplasts and mitochondria, there were high levels of ROS, the byproducts of photosynthesis and respiration. Superoxide dismutase (SOD) converted some superoxide (O2•−) to H2O2, but the activity of peroxidase was low. Thus, the orgDNA was subjected to extensive oxidative damage and became fragmented. Levels of glutathione (GSH), ascorbic acid (AsA), and catalase activity were obtained from protoplasts from the entire stalk and leaf blades (L1–L3), whereas both protoplasts and isolated organelles were used to assay ROS and enzyme activities for SOD and peroxidase. The Transition From Stem Cells to Leaf Cells reason is that mutations that either over-express or reduce levels of some SODs reveal that these proteins have only minor roles in photoprotection or protection from oxidative damage (Alscher et al., 2002; Pilon et al., 2011). However, severe developmental defects were observed in mutants deficient for two plastid FeSODs, and H2O2 was proposed to coordinate chloroplast-nuclear gene expression (Myouga et al., 2008). Therefore, a major function of SOD may be to convert an immobile ROS species (superoxide) to a mobile species (H2O2), which is the likely signaling molecule that communicates the redox status of the cell to the nucleus (Brunkard and Burch-Smith, 2018; Mullineaux et al., 2018; Janku et al., 2019). In the lower stalk, however, the low level of SOD combined with the high activities of catalase and peroxidase and higher levels of GSH and AsA (Figure 3) serves to maintain a relatively high ratio of superoxide to H2O2 that is thought to be required for “stemness” in the stem cells of the shoot apical meristem of Arabidopsis (Zeng et al., 2017; Yang et al., 2018). cells in the stalk are in an etiolated state, shielded from light first by the coleoptile and later by the outer leaf sheath. Only after the leaf blade tip emerges are the cells exposed to light and begin the final differentiation process to photosynthetic capability. It is this final step where a sudden increase in ROS due to photosynthesis may result in oxidative stress or may merely be part of the normal cellular signaling process. Antioxidant agents are usually considered as defensive agents that relieve oxidative stress and damage caused by high levels of ROS. We find that superoxide, SOD, and 8-oxoG all increase during leaf development (Table 2 and Figure 7), a result consistent with a damage-response function for the antioxidant enzyme SOD. But perhaps SOD does not act only to reduce oxidative stress when it converts superoxide to H2O2 for two reasons. Since H2O2 is more stable than superoxide, a consequence of SOD activity is the replacement of one type of ROS with a more persistent type. The neutralization of superoxide as a damaging agent involves a second step in which H2O2 is removed by catalase and peroxidases. But as the leaf develops from the lower stalk, the increase in cellular H2O2 is accompanied by a decrease in cellular catalase and peroxidase activities and GSH and AsA levels. Frontiers in Plant Science | www.frontiersin.org age and Repair of Organellar DNA Damage and Repair of Organellar DNA In maize, the DNA molecules in mitochondria and plastids isolated from the stalk lower tissue are pristine, as expected for stem cells, but these orgDNAs are highly degraded in green leaves (Oldenburg and Bendich, 2004, 2015; Oldenburg et al., 2013). Our present data show that during leaf development there is an increase in ROS and 8-oxoG (representing orgDNA damage). We infer that the demise of orgDNA begins with increased ROS production and that damaged-but-not-repaired orgDNA is degraded by default as occurs in Escherichia coli (Skarstad and Boye, 1993). Our data show that ROS, H2O2, superoxide, and SOD levels are lower in dark-grown leaves than in those grown in the light. Previously, we reported that the amount and molecular integrity of ptDNA rapidly decline upon transfer of maize seedlings from dark to light growth conditions (Zheng et al., 2011). We speculate that in the dark, the ROS byproducts of respiration lead to mtDNA damage and that a small amount of H2O2 signals the nucleus to express and deliver DNA repair proteins to the mitochondria and plastids. In the light, the green cells of the maize leaf blade produce sufficient ATP by photophosphorylation and no longer require transcription from either mtDNA to support respiration or ptDNA to support photosynthesis. A larger amount of H2O2 from the chloroplasts now signals the nucleus to cut offthe supply of DNA repair proteins (such as RecA) and the damaged DNA in both organelles disintegrates. [The theoretical problem of continued photosynthesis in single-season grasses, such as maize, using long-lived mRNAs without the support of functional ptDNA has been considered elsewhere (Oldenburg and Bendich, 2015)]. p g y g p Pristine DNA in the gametes is required for maintaining the lineage of a sexually reproducing organism. High-quality DNA is maintained by damage repair in a eukaryote with a single cell type, such as yeast or Chlamydomonas, regardless of the metabolic cost of DNA repair. But for a species with embryonic development (like maize), the high cost of DNA repair can be reduced by powering germline cells with “quiet” metabolism (neither respiration nor photosynthesis) and the somatic cells with “active” metabolism (both respiration and photosynthesis) (Bendich, 2010). Of course, full repair of DNA damage is required in the meristem. age and Repair of Organellar DNA But low oxygen, low H2O2, and high peroxidase and catalase reduce the potential for oxidative DNA damage, lowering the cost to repair both orgDNA and nuclear DNA. Thus, the local repair of a low level of damage to orgDNA would suffice, and repair pathways including BER, HR, and perhaps MMEJ operate in both plastids and mitochondria in some plants (Boesch et al., 2011; Garcia-Medel et al., 2019). Maize was not among those investigated, although its plastid proteome does contain DNA repair proteins, and most of these (including RecA) were found in greater abundance in the proplastids at the leaf base than in chloroplasts at the leaf tip (Majeran et al., 2012). Although wild type Arabidopsis ptDNA contains some ssDNA, a large increase in ssDNA regions was found in a cprecA mutant (Rowan et al., 2010). In maize leaf, if RecA expression is turned offfollowing ROS signaling, ssDNA should accumulate in orgDNA leading to its demise. For somatic cells in the leaf, the plant can “afford” to not repair all of the high oxidatively damaged copies of their organellar genomes, thereby reducing the high cost of orgDNA repair, although repair of nuclear DNA is required for cellular homeostasis and checkpoint control of cell division. ( g ) The changes in ROS and antioxidant enzyme levels (SOD and peroxidase) during maize seedling development and under light and dark growth conditions show the same trends in both plastids and mitochondria. However, since mitochondria have no known light receptors, it is unclear why the mtDNA suffers the same light-induced demise as does ptDNA. One possible explanation comes from cultured human retinal pigment epithelial cells: the electron transport chain generates ROS when cells are exposed to blue light (King et al., 2004). In maize, we reported lower amounts of DNA per plastid in blue light than in white light and concluded that blue light suppresses ptDNA replication/repair and induces degradation (Oldenburg et al., 2006). One way for the nucleus to coordinate organellar activities is expression of dual-targeted proteins (those delivered to both plastids and mitochondria), including the replication/repair protein RecA (Oldenburg and Bendich, 2015). Thus, regardless of the organellar source of H2O2, considered the main signaling molecule, the nucleus perceives the redox status of the cell and dictates the fate of the orgDNA. The Transition From Stem Cells to Leaf Cells The second To summarize, our data support a signaling role for ROS in the lower stalk to maintain the stem cells (that later lead to the gametes) and in leaf cells developing photosynthetic capacity. 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This research was funded by the Junat Fund (a private charitable fund). Mitochondrial immunofluorescence data were acquired using an Olympus IX81 microscope located in the Biology Imaging Facility at the University of Washington. CONCLUSION Studies on ROS in plants typically consider the effects following biotic and abiotic stress. Here, we focus on changes in ROS and antioxidant agents under normal, non-stressful growth conditions and show that the ROS levels increase in whole cells and in plastids and mitochondria during maize leaf development. Although we report changes in the relative levels of ROS and antioxidant agents, a deeper understanding of ROS in oxidative stress and signaling may be gained when new methods are developed to measure absolute concentrations. Previously, we showed differences in the maintenance and degradation of ptDNA between maize and other plants, including tobacco and Arabidopsis (Shaver et al., 2006; Rowan and Bendich, 2009). These differences could be due to variations in response to ROS signaling. We propose that orgDNA degradation in maize leaf is a result of an increase in oxidative damage to orgDNA and ROS-signaling that leads to a decrease in orgDNA repair systems. Although mechanisms for orgDNA repair in plants have been addressed recently (Baruch-Torres and Brieba, 2017; Garcia- Medel et al., 2019), additional insight may be found elsewhere. The “SOS response” in E. coli is initiated by accumulation of single-stranded DNA (ssDNA) during replication of DNA containing lesions (Maslowska et al., 2019). For both bacteria and May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 15 Organellar ROS and DNA Damage in Maize Tripathi et al. SUPPLEMENTARY MATERIAL DT and AN performed the experiments. DT, AN, and DO analyzed the data. DT, DO, and AB wrote the manuscript. All authors have read and approved the final version of the manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2020.00596/ full#supplementary-material REFERENCES D., and Zhao, H. (2007). 8-Hydroxy-2’- deoxyguanosine (8-OH-dG) as a potential survival biomarker in patients with nonsmall-cell lung cancer. Cancer109, 574–580. doi: 10.1002/cncr.22417 Mullineaux, P. M., Exposito-Rodriguez, M., Laissue, P. P., and Smirnoff, N. (2018). ROS-dependent signalling pathways in plants and algae exposed to high light: comparisons with other eukaryotes. Free Radic. Biol. Med. 122, 52–64. doi: 10.1016/j.freeradbiomed.2018.01.033 Skarstad, K., and Boye, E. (1993). Degradation of individual chromosomes in recA mutants of Escherichia coli. J. Bacteriol. 175, 5505–5509. Smirnoff, N. (2018). Ascorbic acid metabolism and functions: a comparison of plants and mammals. Free Radic. Biol. 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Biophys. Acta 1807, 989–998. REFERENCES doi: 10.1016/j.bbabio.2010.11.002 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. j Rogers, S. O., and Bendich, A. J. (1985). Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol. Biol. 5, 69–76. doi: 10.1007/BF00020088 Rowan, B. A., and Bendich, A. J. (2009). The loss of DNA from chloroplasts as leaves mature: fact or artefact? J. Exp. Bot. 60, 3005–3010. doi: 10.1093/jxb/ erp158 Copyright © 2020 Tripathi, Nam, Oldenburg and Bendich. 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. Rowan, B. A., Oldenburg, D. J., and Bendich, A. J. (2010). RecA maintains the integrity of chloroplast DNA molecules in Arabidopsis. J. Exp. Bot. 61, 2575–2588. doi: 10.1093/jxb/erq088 May 2020 | Volume 11 | Article 596 Frontiers in Plant Science | www.frontiersin.org 17
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Evaluation Of Online Development Examinations Of Infectious Diseases And Clinical Microbiology (IDCM) Expertise Students
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Evaluation Of Online Development Examinations Of Infectious Diseases And Clinical Microbiology (IDCM) Expertise Students Sakarya Universitesi Tip Fakultesi Research article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/11 Page 1/11 Background Education is defined as the process of creating desired behavioral change in the individual's behavior through his own life. The extent to which the knowledge provided by the training is achieved and the extent to which the development is achieved can only be learned by measurement and evaluation. The first aim of measurement and evaluation is to see what the learners gain in knowledge and competence levels and to monitor their individual development. The second aim is to monitor the effectiveness of the educational content and to organize the educational content according to the results, and to guide the students about their knowledge and competencies. (1) During medical education, formative assessment and summative assessment are recommended. Formative evaluation is an intermittent assessment and evaluation while training is in progress. The aim is to determine the learning deficiencies of the learner (2-5). Online formative assessments in medical education have been increasing in recent years with the aim of measuring the knowledge of research assistants according to a national norm, determining minimum national standards in the specialization training program, and measuring the quality of teaching under the training program. The appreciation of the online exams is due to its encouragement of self-learning in medical education. Self-learning students can assess their work themselves, identify their strengths and weaknesses. Students who participate in the formative examinations are more successful in the grades-based exams. (6-8) Throughout the country, different disciplines such as Turkish Orthopedics and Traumatology Training Council (TOTEK), Turkish Chest Diseases Competency Board, Turkish Medical Oncology Association, and EMEK are doing assistant development exams. EMEK and Turkish Chest Disease Competency Board hold development exams online (9-11). Infectious Diseases Society of America (IDSA) conducts development examinations online (12). Formative evaluations made online in the literature are reported to increase internal motivation for learning, to improve knowledge and to contribute to determining individual learning needs. Online exams often use multiple choice exam methods (6,13). In this study, it is aimed to evaluate the learning objectives and examinations, and to report the experience obtained in the research assistant exams of EMEK. Abstract Background: Formative examinations are required to monitor the development of research assistants in the field of Infectious Diseases and Clinical Microbiology. However, the preparation, conduct and evaluation of these exams also require especial features and experience. In this study, it is aimed to review the experiences obtained in the evaluation exams of research assistants conducted by the education commission (EMEK) in the field of IDCM specialization. Methods: There are two online exams per year for research assistants in the IDGM field. In this study, the examinations between 2014-2018 were evaluated. The validity and reliability analyzes for each exam were performed and the difficulty and discrimination indexes of the questions were calculated. The topics with the least correct answers were determined. Results: Over the years, an increase in the number of research assistants was observed. The internal consistency coefficient of the exams was the lowest of the cronbach alpha value of 0,69 and the highest of 0,92. The mean score of the exam was over 70. Some of the questions needed to be reviewed and corrected. Although clinically rare diseases and basic microbiology questions have been answered less frequently, the rate of responding to diseases that are frequently seen in the clinic was higher. Conclusion: Online examinations facilitate broad-based measurement and evaluation at national level. With the increase of our online exam experience, we found that there are still parts to be improved. Keywords: Online examination, Expertise students, Development exams, Measurement and evaluation Methods Page 2/11 Page 2/11 Exam Method The date and time of the exam will be announced on the internet in the platforms where every research assistant can be informed and the applications are taken online. Research assistants taking IDCM education in Turkey are participating in this voluntary examination. The research assistants who are applying for the test attend the exam by logging into the system with their own passwords in the clinic where they receive their education. The examiners are appointed by the responsible institutions of education. Assistant examinations are held twice a year with six-month intervals. The scope and distribution of the questions are determined according to TUKMOS curriculum subjects and the questions are prepared by EMEK executive board in the form of multiple choice test format (5 choices). All questions are reviewed by EMEK executive board and questions are validated. The exams which are made online from a central system and consist of 50 questions, have a 1 hour duration. Each question is 2 points, maximum exam score is 100 points. As this is an exam with formative purpose development, no passing grade has been determined. The exams are held at lunchtime. This timeframe appears to be a reasonable period that will not only disrupt the clinical work but also make the organization difficult. After Exam Feedback Feedback is given to each center after the exam. The result of the examination is not given to any person other than the research assistant himself and the education supervisor of the institution he / she is studying. Through the examinations, it is aimed to evaluate the professional knowledge development of the expertise students according to years and to evaluate the educational institutions' deficiencies according to these results. Exam Statistics The numerical data of the exams were obtained by reviewing retrospective data records. The data obtained were tabulated. Data were compiled as MS excel data. Reliability and Validity of Exams Cronbach's alpha reliability coefficient was calculated statistically. Student's T test was used to compare the means for countable values, chi-square and Fisher's corrected chi-square tests were used to compare the qualitative values. P <0.05 was considered significant. Discussion Medical specialization students are both educated in the clinic and serve in the provision of services. For this reason, it is very difficult for them to go to another center to take their exams. This process brings both time consuming and economic burdens. These requirements have been a starting point for online surveys and assessments (10). Because the students are located in different provinces, the problem of time and space is solved easily with the central online examination. It is ensured that the trainers evaluate this process by the central examination of the students receiving education at different faculties and different geographic regions at the same time. As a matter of fact, it is possible for the students of different educational institutions to evaluate themselves. In addition, it is ensured that research assistants in all educational institutions can see the shortcomings of students and their programs according to their questions. If technology can be used well, more communication, interaction and cooperation can be achieved with online exams. There are studies showing that the use of online evaluation has contributed positively to the learning and teaching process. There are publications reporting that students feel more comfortable in their online surveys and evaluations, their negative attitudes towards learning and measurement-evaluation have disappeared and their academic achievement has increased (14,15). When the seniority of the students taking the exam is examined, it was observed that the first year research assistants participated in the exam in the most intense way and it was noticed that the number of research assistants who took the exam in the following years decreased according to seniority. This may be due to different reasons. This may be due to the fact that the first year assistants are more willing to test themselves and are in the clinic. Last-year assistants in the period of rotation or thesis preparation may be less motivated. The online evaluation systems have many positive contributions to the education process. When appropriate infrastructure is provided to the web environment, it is accessible and applicable from anywhere. Students' achievements in exams can be seen instantly in the system (2). Online exams allow the creation of a question bank. The students who prepare the exam keep their knowledge up-to-date and follow the subjects they will prepare for the exam carefully. Exams are done through the network, thus paper and publication expenditures are also eliminated (2,16). Results It was found that the number of research assistants who wanted to test themselves in EMEK exams increased gradually (Figure 1). In particular, the participation rate of the 1st year research assistants was found to be quite high (Figure 2). The distribution of the research assistants, the mean grade point of the exams and the cronbach's alpha values are presented in Table 1. The cronbach alpha value of the exams was found to be lowest at 0,69 and highest at 0,92 (Table 1). In each exam period as a result of the exam analysis the most and least correctly answered questions were found and presented in Table 2. The discriminant rate of the exam questions was 16% in 400 questions. 84% of the questions had to be corrected before being added to the question bank (Table 3). The mean difficulty index of the exams was found to be at least 69 and at most 84.7, therefore it is determined to be in the easy question class. Table 3 presents the discrimination and mean difficulty index ratios of the questions according to the exams. Table 4 presents the distribution of subjects with low correct answer rate (difficulty index: difficult question) in 400 questions in online exams in 2014-2018. Page 3/11 Discussion It takes a long time for classical examinations to be conducted and evaluated by academics. Thanks to online exams, trainers also save time. In addition, faculty members can look at the response rates of the questions and focus on subjects in that students' knowledge is insufficient, so that these subjects can be better understood. Individualization of the exam, instant feedback, quick scoring, and no limitations in terms of time and place are the advantages of the online exam (2,17). As a matter of fact, EMEK exams were conducted with one hour duration and many research assistants were evaluated online. An exam is expected to be reliable, valid, useful and contributing to education. The fact that the margin of error in the exam is low means that it is a very reliable test (5). The measurement method should be valid and reliable (18,19). Cronbach's alpha reliability coefficient is mostly used to measure the reliability of a test. The higher the coefficient, the higher the internal consistency. For reliable tests, this coefficient is expected to be 0.80 or higher. It has also been reported that 0.70 or more can be accepted for formative exams (20). When EMEK examinations are considered, it is seen that the best value in reliability is taken in the first exam and the most negative measurement is taken in May 2017 exam. The high margin of error in this period is attributed to both the reduction of motivation in the examiners and the technical disadvantages in the preparation of the questions and in the implementation of the online exam. However, the reliability coefficient of the EMEK research assistant development examinations, which were made for formative purposes, was found to be quite satisfactory (Table 1). The discrimination index is an index used to separate those who do not have the characteristic to be measured by the test. Questions with high levels of discriminative features increase the reliability of the test. The discriminative feature of a question with a discriminant index of 0.40 or higher is defined as very high. The discriminativity of a question with index 0.30-0.39 is not complete, so it needs to be reviewed. The question with a 0.20-0.29 index should be corrected and developed, and should not be reused without any changes. Discussion 0.19 index of a question is low, the question must be removed Page 4/11 Page 4/11 from the test and not transferred to the question bank (20). In an exam made for the evaluation of research assistants, the expected feature of the questions is to distinguish the student who does not know the subject matter (20). When we look at the most of the EMEK examinations, although the discrimination rate is sufficient in some questions, it is necessary to review many questions (Table 2). In the examinations, the question rate which can distinguish the student who does not know the subject with the relevant subject is found to be 16%. However, the percentage of questions that should not be used after reviewing is 43% in total. 41% of the exam questions should be reviewed and included in the question bank after making the necessary corrections. These data suggest that it is necessary to receive specific training for measurement and evaluation during question preparation. In the analysis of exams, the investigation and interpretation of why the difficulty of the questions may have been caused is a gain of the evaluation (2,17,20). Table 4 presents the subjects in which the students have difficulty in answering correctly and have lack of knowledge. The remarkable result in this table is that less common or rare infections and basic medical science questions are answered less accurately. This will be the educational gain of the units with particular attention to these topics in the training process. In the online applications, problems such as the emergence of unexpected technical problems, infrastructure and hardware dependence, security problems, students' use of different computers and especially requirement of having a computer are the limitations of online exams. No matter how good the network is, there may be some problems such as power failure or voltage change, lack of features of the computer being used. In addition, although online exams are strictly assigned to supervisors, cheating is much easier than classical exams. For this reason, the security of the system, (the user codes must not be created on the systems) communication security, difficulties in identification of the user and cheating affect the reliability of the online examination negatively (2,17). It is the limitation of the EMEK examinations that the number of questions in categorized subject titles are not determined by taking into consideration the learning objectives of the IDCM. Conclusions As a result, the EMEK research assistants examinations conducted twice a year contribute to the evaluation of the training of the research assistants, but the examinations have aspects to be developed. Online exams make it easy to measure. With the online exams to be made, it will be easier to monitor the learning progress of the research assistant by monitoring the cognitive development of each assistant and reviewing the training programs. List Of Abbreviations IDCM: Infectious Diseases and Clinical Microbiology Society of Turkey EMEK: IDCM’s Education Commission TOTEK: Turkish Orthopedics and Traumatology Training Council TOTEK: Turkish Orthopedics and Traumatology Training Council IDSA: Infectious Diseases Society of America TUKMOS: Medical Expertise Board Curriculum Creation and Standard Setting System o Authors' contributions: d item analysis of data. Contributed significantly to the design and writing of the article. TÖK, conducted and interpreted item analysis of data. Contributed significantly to the design and writing of the article. Read and endorsed the last article. OK, designed the article and revised the study. Read and endorsed the last article. Acknowledgements: We would like to express our gratitude to the EMEK study group for their contributions to the examination and the preparation of the questions. Acknowledgements: We would like to express our gratitude to the EMEK study group for their contributions to the examination and the preparation of the questions. EMEK study group; Aliye BAŞTUĞ, Ayhan AKBULUT, Ayşe BATIREL, Bilgin ARDA, Bircan ÜNAL KAYAASLAN, Canan AĞALAR, Cemal BULUT, Çiğdem KADER, Dilek Yıldız SEVGİ, Derya ÖZTÜRK ENGİN, Esragül AKINCI, Haluk VAHABOĞLU, Hürrem BODUR, İlker İnanç BALKAN, İftihar KÖKSAL, İrfan ŞENCAN,   İsmail Yaşar AVCI, Meltem TAŞBAKAN, Meltem Arzu YETKİN, Nail ÖZGÜNEŞ, Nazif ELALDI, Nurettin ERBEN, Pınar ÖNGÜRÜ,  Salih CESUR, Sibel  GÜNDEŞ, Seniha ŞENBAYRAK, Yasemin ÇAĞ EMEK study group; Aliye BAŞTUĞ, Ayhan AKBULUT, Ayşe BATIREL, Bilgin ARDA, Bircan ÜNAL KAYAASLAN, Canan AĞALAR, Cemal BULUT, Çiğdem KADER, Dilek Yıldız SEVGİ, Derya ÖZTÜRK ENGİN, Esragül AKINCI, Haluk VAHABOĞLU, Hürrem BODUR, İlker İnanç BALKAN, İftihar KÖKSAL, İrfan ŞENCAN,   İsmail Yaşar AVCI, Meltem TAŞBAKAN, Meltem Arzu YETKİN, Nail ÖZGÜNEŞ, Nazif ELALDI, Nurettin ERBEN, Pınar ÖNGÜRÜ,  Salih CESUR, Sibel  GÜNDEŞ, Seniha ŞENBAYRAK, Yasemin ÇAĞ EMEK study group; Aliye BAŞTUĞ, Ayhan AKBULUT, Ayşe BATIREL, Bilgin ARDA, Bircan ÜNAL KAYAASLAN, Canan AĞALAR, Cemal BULUT, Çiğdem KADER, Dilek Yıldız SEVGİ, Derya ÖZTÜRK ENGİN, Esragül AKINCI, Haluk VAHABOĞLU, Hürrem BODUR, İlker İnanç BALKAN, İftihar KÖKSAL, İrfan ŞENCAN,   İsmail Yaşar AVCI, Meltem TAŞBAKAN, Meltem Arzu YETKİN, Nail ÖZGÜNEŞ, Nazif ELALDI, Nurettin ERBEN, Pınar ÖNGÜRÜ,  Salih CESUR, Sibel  GÜNDEŞ, Seniha ŞENBAYRAK, Yasemin ÇAĞ Declarations Ethics approval and consent to participate: Ethical approval was not obtained because our study did not include human and animal experiments. Required permission has been obtained from IDCM. Page 5/11 Consent for publication: ‘’Not applicable’’ Availability of data and materials: I can confirm I have included a statement regarding data and material availability in the declaration section of my manuscript. Competing interests: No competing financial interests exist. Funding: Our study does not have any funding. Funding: Our study does not have any funding. Funding: Our study does not have any funding. References 1. Amine Z, Khoo HE. Basics in Medical Education. Ed. Amin Z. Basics in medical education. World Scientific. 2009. 2nd ed. Singapore: 372 p 2. Dennick R, Wilkinson S, Purcell N. Online eAssessment: AMEE Guide No. 39. Med Tea 3. Rolfe I, McPherson J. Formative assessment: how am I doing? Lancet. 1995; 345: 83 3. Rolfe I, McPherson J. Formative assessment: how am I doing? Lancet. 1995; 345: 837-839. 4. Prober CG, Khan S. Medical education reimagined: A call to action. Acad Med. 2013; 88: 1407-1410. 4. Prober CG, Khan S. Medical education reimagined: A call to action. Acad Med. 2013; 8 5. Tavakol M, Dennick R. The foundations of measurement and assessment in medical education. Med Teach. 2017; 39: 1010-1015. 5. Tavakol M, Dennick R. The foundations of measurement and assessment in medical education. Med Teach. 2017; 39: 1010-1015. 6. Nagandla K, Sulaiha S, Nalliah S. Online formative assessments: exploring their educational value. J Adv Med Educ Prof. 2018;6: 51-57. 6. Nagandla K, Sulaiha S, Nalliah S. Online formative assessments: exploring their educational value. J Adv Med Educ Prof. 2018;6: 51-57. 7. Carrillo-de-la-Peña MT, Baillès E, Caseras X, Martínez A, Ortet G, Pérez J. Formative assessment and academic achievement in pre-graduate students of health sciences. Adv Health Sci EducTheory Pract. 2009; 14: 61-67. 7. Carrillo-de-la-Peña MT, Baillès E, Caseras X, Martínez A, Ortet G, Pérez J. Formative assessment and academic achievement in pre-graduate students of health sciences. Adv Health Sci EducTheory Pract. 2009; 14: 61-67. 8. Institute of Medicine (US) Committee on Planning a Continuing Health Professional Education Institute. Redesigning Continuing Education in the Health Professions. Washington (DC): National Academies Press (US); 2010. 8. Institute of Medicine (US) Committee on Planning a Continuing Health Professional Education Institute. Redesigning Continuing Education in the Health Professions. Washington (DC): National Academies Press (US); 2010. 9. http://www.totbid.org.tr 10. http://www.tghyk.org/RAPOR2018.pdf 11. https://kanser.org/saglik/userfiles/file/TTOD_2017 12. https://www.idsociety.org/ 13. Gikandi JW, Morrow D, Davis NE. Online formative assessment in higher education: A review of the literature. Comput Educ. 2011; 57: 2333-2351. 13. Gikandi JW, Morrow D, Davis NE. Online formative assessment in higher education: A review of the literature. Comput Educ. 2011; 57: 2333-2351. 13. Gikandi JW, Morrow D, Davis NE. Online formative assessment in higher education: A review of the literature. Comput Educ. 2011; 57: 2333-2351. Page 6/11 Page 6/11 14. Black P, Wiliam D. Inside the Black Box: Raising Standards through Classroom Assessment. Phi Delta Kappan. 2010; 92: 81-90. 15. Olsen JB, Maynes DD, Slawson D, Ho K. Comparisons of Paper-Administered, Computer-Administered and Computerized Adaptive Achievement Tests. JECR. 1989; 5: 311-326. 16. Ataoğlu S. Principles of Medical Education, Educational Objectives and Evaluation Strategy. Duzce Med J. 2018; 20: 57-58. 17. Tasdemir S, Balcı M, Cabi A, Altin M, Cabi O. The Design and Application of Online Exam System Supported by Database. IJAMEC. 2015;3: 204. 18. Morrison J. Evaluation. In: Cantillon P, Wood D (Ed). ABC of Learning and Teaching in Medicine. 2th ed. Singapore: wiley-Blackwell: 2010 19. RL Linn - Educational Accountability Systems. In: Ryan KE, Shepard LA. (Eds). The future of test-based educational accountability. 1.th ed. New York: Roultledge, 2008: 3-24 20. Atılgan H, Kan A, Aydın B. Measurement and evaluation in education. Hakan Atılgan (ed.) Anı publishing 10th Edition Ankara.2017: 3-440 20. Atılgan H, Kan A, Aydın B. Measurement and evaluation in education. Hakan Atılgan (ed.) Anı publishing 10th Editio Ankara.2017: 3-440 Tables consistency values The year of Education 2014 June 2015 June 2015 December 2016 June 2016 December 2017 May 2017 December 2018 September Total* 1.year 1 3 15 26 60 61 84 71 321 2.year 9 16 24 16 27 29 25 23 169 3.year 23 16 24 19 37 28 35 17 199 4.year 12 11 19 22 19 19 15 3 120 5.year 19 22 23 18 19 8 4 3 116 Unspecified 16 -                            -                             -                         1 - 1 60 78 Total 80 68 105 101 163 145 164 177   Grade point mean 77 74 69 77 71 79 74 72.15   Cronbach's Alpha coefficient 0,92 0,78 0.72 0,87 0,82 0,69 0,79 0,71 Page 7/11 Page 7/11 Table 2. The most and least correctly answered questions in EMEK exam according to years Year/Month Most Correctly Answered Question Title Least Correctly Answered Question Title 2014/June   Antibiotic diarrhea   Staphylococcal Microbiology 2015/June   Description of pandemic   Hemorrhagic fever   2015/December   AIDS patient monitoring   Antibiotic susceptibility testing   2016/June   EBV infection   CMV retiniti   2016/December   Saprophyticus microbiology   Cat Scratch Disease Clinic   2017/May Epidemiology of Leptospira   Group A streptococcal microbiology 2017/December Asymptomatic bacteriuria   The question of hepatitis D   2018/September Management of candidemia Live vaccines Typhoid vaccine   Table 3. Discrimination and mean difficulty index statistics of the questions according to the exams Year/Month Most Correctly Answered Question Title Least Correctly Answered Question Title 2014/June   Antibiotic diarrhea   Staphylococcal Microbiology 2015/June   Description of pandemic   Hemorrhagic fever   2015/December   AIDS patient monitoring   Antibiotic susceptibility testing   2016/June   EBV infection   CMV retiniti   2016/December   Saprophyticus microbiology   Cat Scratch Disease Clinic   2017/May Epidemiology of Leptospira   Group A streptococcal microbiology 2017/December Asymptomatic bacteriuria   The question of hepatitis D   2018/September Management of candidemia Live vaccines Typhoid vaccine   crimination and mean difficulty index statistics of the questions according to the exams Table 3. Tables Discrimination and mean difficulty index statistics of the questions according to the exams Page 8/11 QUESTION FEATURES 2014   n=50 (%) 2015 June n=50 (%) 2015 December n=50  (%) 2016 June n=50 (%) 2016 December n=50 (%) 2017 June n=50 (%) 2017 December n=50 (%) 2018 September n=50 (%) Total   n=400 (%)   Discriminative   7 (14%)   10 (20%)   6 (12%)   19 (38%)   8 (16%)   4 (8%)   6 (12%)   5 (10%)   65 (16%) Not fully discriminative Must be reviewed   4 (8%)   7 (14%)   10 (20%)   10 (20%)   13 (26%)   10 (20%)   11 (22%)   15 (30%)   80 (20%) Unable to discriminate, should be corrected and developed   9 (18%)   9 (18%)   8 (16%)   10 (20%)   13 (26%)   8 (16%)   16 (32%)   11 (22%)   84 (21%) Unable to discriminate should be removed from the test   30 (60%)   24 (48%)   26 (32%)   11 (22%)   16 (32%)   28 (56%)   17 (34%)   19 (38%)   171 (43%) MEAN DIFFICULTY INDEX   84.73   73.73   69.47   72   71.53   78.59   74.50   72.46   Table 4. Scope of the questions that are difficult to answer Table 4. Scope of the questions that are difficult to answer Page 9/11 Page 9/11 SUBJECT-QUESTION CONTENT SCOPE Acute pancreatitis Clinic Antibiotic resistance mechanisms Clinical Microbiology-Pharmacology General knowledge of antibiotics Microbiology-Pharmacology Arthropod-induced infections Clinic Special microbiological properties of bacteria Bacteriology Treatment of botulism Clinic Clinic of viral rash diseases Clinic Immunological response to infection Immunology Infective endocarditis Clinic Febrile neutropenia, organ transplantation Clinic Isolation methods specific to diseases Hospital infections Etiology of hospital-acquired pneumonia Hospital infections Drug fever properties General infectious diseases Implant surgical infections Clinic Cat scratch disease Clinic Diagnosis and treatment of chronic hepatitis Clinic Travel infections Clinic Typhoid vaccine Clinic Tuberculin skin test Clinic Laboratory findings of viral infections Virology Virus infrastructure Virology Figures Page 10/11 Figures Figure 1 Number of students taking EMEK exams over the years Figure 1 Figure 1 Number of students taking EMEK exams over the years Number of students taking EMEK exams over the years Figure 2 The number of students taking exams according to their years being as an assistant Figure 2 Figure 2 The number of students taking exams according to their years being as an assistant The number of students taking exams according to their years being as an assistant The number of students taking exams according to their years being as an assistant The number of students taking exams according to their years being as an assistant Page 11/11
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Heterochromatin in the fungal plant pathogen, Zymoseptoria tritici: Control of transposable elements, genome plasticity and virulence
Frontiers in genetics
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TYPE Review 21 November 2022 10.3389/fgene.2022.1058741 PUBLISHED DOI OPEN ACCESS EDITED BY Anna Muszewska, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland REVIEWED BY Thierry C. Marcel, INRA Centre Versailles-Grignon, France Hector M. Mora-Montes, Universidad de Guanajuato, Mexico *CORRESPONDENCE Simon K. Whitehall, simon.whitehall@ncl.ac.uk Heterochromatin in the fungal plant pathogen, Zymoseptoria tritici: Control of transposable elements, genome plasticity and virulence Callum J. Fraser and Simon K. Whitehall* Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom SPECIALTY SECTION This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Genetics RECEIVED 30 September 2022 04 November 2022 PUBLISHED 21 November 2022 ACCEPTED CITATION Fraser CJ and Whitehall SK (2022), Heterochromatin in the fungal plant pathogen, Zymoseptoria tritici: Control of transposable elements, genome plasticity and virulence. Front. Genet. 13:1058741. doi: 10.3389/fgene.2022.1058741 COPYRIGHT © 2022 Fraser and Whitehall. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Genetics Heterochromatin is a repressive chromatin state that plays key roles in the functional organisation of eukaryotic genomes. In fungal plant pathogens, effector genes that are required for host colonization tend to be associated with heterochromatic regions of the genome that are enriched with transposable elements. It has been proposed that the heterochromatin environment silences effector genes in the absence of host and dynamic chromatin remodelling facilitates their expression during infection. Here we discuss this model in the context of the key wheat pathogen, Zymoseptoria tritici. We cover progress in understanding the deposition and recognition of heterochromatic histone post translational modifications in Z. tritici and the role that heterochromatin plays in control of genome plasticity and virulence. KEYWORDS heterochromatin, Zymoseptoria tritici, histone H3 lys 27 trimethylation, histone H3 lys 9 methylation, transposable element, fungal plant pathogen, genome plasticity Introduction The wheat pathogen, Zymoseptoria tritici Zymoseptoria tritici is responsible for Septoria tritici blotch (STB), a world-wide foliar disease of wheat that is characterised by the appearance of necrotic lesions and in some cases the death of the plant. Yields in an infected crop may be reduced by 30–50% if the disease is not properly controlled via the application of fungicides (Fones and Gurr, 2015). However, the increasing incidence of fungicide resistance means that Z. tritici is a major threat to wheat production (Torriani et al., 2015). Infection is initiated when Z. tritici spores on the leaf surface germinate and hyphae invade plant tissue through stomata. This is followed by a slow, asymptomatic colonisation of the apoplastic space of the surrounding mesophyll. After approximately 10 days (depending on temperature), there is an abrupt switch from “stealth” infection to necrotrophic growth during which plant cells undergo programmed cell death. The resulting release of nutrients from the dying plant tissue facilitates a rapid 01 frontiersin.org Fraser and Whitehall 10.3389/fgene.2022.1058741 Croll et al., 2013; Möller et al., 2019). Core chromosomes from different isolates also exhibit significant structural polymorphism resulting from the insertion or deletion of clusters of TEs (Plissonneau et al., 2016; Plissonneau et al., 2018). The TEassociated chromosomal regions that are present or absent in a strain specific manner (which have been termed accessory, orphan or dispensable regions), are enriched for putative effector genes and account for substantial variations in gene content. Indeed, comparative analysis of the genomes of five Z. tritici isolates identified a core set of 9149 genes and a further 6600 strainspecific accessory genes (Plissonneau et al., 2018). increase in fungal biomass and the formation of pycnidia which contain pycnidiospores that are released to infect the leaves of neighbouring plants (Steinberg, 2015). Effector genes, repetitive elements and compartmentalised genomes Plant colonizing fungi produce a repertoire of secreted effector proteins that modulate plant immunity and host cell physiology (Uhse and Djamei, 2018). The expression of effectors is tightly regulated so that they are produced at the appropriate stage and level during infection (Uhse and Djamei, 2018). Accordingly, Z. tritici undergoes several major gene expression reprogramming events during the infection of wheat leaves (Kellner et al., 2014; Rudd et al., 2015). These events include a large up-regulation of secreted proteins approximately 9 days after infection which likely underpins the shift to necrotrophic growth. After approximately 14 days there is a general reduction in the expression of secreted proteins in favour of cell wall and carbohydrate degrading enzymes (Rudd et al., 2015). However, the mechanisms by which these large scale transcriptional reprogramming events are regulated remains to be determined. The distribution of effector genes in the genomes of filamentous plant pathogens is not random. Instead effector genes, and genes involved in the production of secondary metabolites (SM), tend to be located in clusters within, or close to, regions of the genome that are enriched in repetitive sequences such as transposable elements (TEs) (Dong et al., 2015). An association with TEs is significant because it is increasingly clear that these elements can be utilised by cells as sources of genetic variability that facilitate genome evolution (Colonna Romano and Fanti, 2022). Mobilization of TEs to new sites in the genome may influence the expression of adjacent genes and also their repetitive nature makes them potential substrates for recombination and thus drivers of genomic rearrangements. That TE/repeat-enriched genomic regions are rapidly evolving has given rise to the “two speed genome hypothesis” where specific compartments provide sources of genetic plasticity that drive adaptation to the host species (Dong et al., 2015; Seidl and Thomma, 2017; Fouche et al., 2018). TEs and heterochromatin domains Host cells employ a variety of defence mechanisms that restrict the activity of TEs to tolerable levels (Goodier, 2016). TEs are often embedded in heterochromatin, a condensed form of chromatin that is typically refractory to transcription and other DNA-dependent processes (Allshire and Madhani, 2018; Marsano and Dimitri, 2022). A key property of heterochromatin is the potential to spread from nucleation sites to cover extended domains (Allshire and Madhani, 2018) and as a result, genes in the vicinity of TEs can be subject to heterochromatic silencing (Choi and Lee, 2020). Indeed, the expression of effector and SM genes is suppressed by heterochromatin in a variety of plantcolonizing fungi (Connolly et al., 2013; Chujo and Scott, 2014; Soyer et al., 2014; Studt et al., 2016). As discussed further below, heterochromatin has also been linked to the pathogenicity of Z. tritici; putative effector genes that are upregulated during the switch to necrotrophic growth are enriched in heterochromatic regions of the genome (Meile et al., 2018; Soyer et al., 2019) and loss of key heterochromatin regulators interferes with the ability of Z. tritici to infect wheat leaves (Möller et al., 2019) (Table 1). Heterochromatin histone posttranslational modifications Heterochromatin can be subdivided into two basic forms: constitutive and facultative (Liu et al., 2020). Constitutive heterochromatin is typically associated with highly repetitive regions such as telomeres and centromeres and often plays an important architectural role in chromosomes. Facultative heterochromatin is considered to be a more fluid subclass of heterochromatin which can be remodelled in response to appropriate internal or external signals. For example, in metazoans it is associated with developmentally regulated genes and similarly, facultative heterochromatin regulates the expression of SM genes in some filamentous fungi (Liu et al., 2020; Ridenour et al., 2020). Heterochromatic regions are characterised by the presence of specific histone post-translational modifications (PTMs). TEs and genome plasticity in Z. tritici There is growing evidence indicating that TEs have shaped the compartmentalisation of the Z. tritici genome. Z. tritici has 13 essential core chromosomes and up to 8 conditionally dispensable accessory chromosomes that are gene poor and especially enriched with TEs (Goodwin et al., 2011). Accessory chromosomes are frequently lost during vegetative growth and are prone to rearrangements in meiosis with the breakpoints frequently mapping close to TEs (Wittenberg et al., 2009; Goodwin et al., 2011; Frontiers in Genetics 02 frontiersin.org Fraser and Whitehall 10.3389/fgene.2022.1058741 TABLE 1 Summary of the virulence phenotypes of Z. tritici heterochromatin mutants. Mutant Protein/Function Virulence phenotypea References(s) Δkmt1 Histone H3 lysine 9 methyltransferase Severely impaired/abolished. No necrosis or pycnidia during screening period Möller et al., 2019; Fraser et al., 2022 Δkmt6 Histone H3 lysine 27 methyltransferase Mildly impaired. Some reduction in necrosis and pycnidia Möller et al., 2019 Δkmt1 Δkmt6 Histone H3 lysine 9 & 27 methyltransferases Severely impaired/abolished No necrosis or pycnidia during screening period Möller et al., 2019 Δcbx1 Heterochromatin Protein 1 (HP1) Moderately impaired. Reduced pycnidia Fraser et al., 2022 Δcbx2 H3K9me-binding chromodomain protein No detectable impairment Fraser et al., 2022 a As determined by wheat infection assays. H3K27me3-enriched accessory chromosomes are highly unstable and are frequently lost during cell division. Deletion of kmt6 decreases the frequency with which these chromosomes are lost indicating that H3K27me3 promotes their instability (Möller et al., 2019). Increased H3K27me3 is also correlated with an elevated frequency of accessory chromosome loss in Fusarium fujikuroi (Janevska et al., 2018). Analysis of kmt6 mutants has also demonstrated that H3K27me3 promotes the rate of spontaneous mutations (Habig et al., 2021). In contrast, H3K9me2/3 functions to suppress spontaneous mutation rates and genomic instability. Deletion of kmt1 is associated with increased accessory chromosome loss, de-repression of TEs and an elevated incidence of chromosomal rearrangements with break points that often map close to TE clusters (Möller et al., 2019; Habig et al., 2021). The high level of chromosomal instability observed in kmt1 mutants is driven by the widespread re-localisation of H3K27me3 such that it invades regions previously occupied by H3K9me2/3. Accordingly, deletion of kmt6 in a Δkmt1 background suppresses genomic instability (Möller et al., 2019). In addition to TEs, a large set of protein encoding genes is upregulated in the absence of kmt1, which is consistent with H3K9me2/3 playing an important role in gene silencing. However, the majority of these genes are not directly associated with H3K9me2/3 and the changes in their expression may result, at least in part, from the re-distribution of H3K27me3 that occurs in a kmt1 mutant background (Möller et al., 2019). The majority of H3K27me3-marked genes and TEs located on core chromosomes are not activated by its removal and in contrast to some other filamentous fungi, heterochromatic PTMs do not play a major role in the control of Z. tritici SM genes (Möller et al., 2019; Hassani et al., 2022). However, heterochromatin is implicated in the regulation of effector gene expression and thus the interaction of Z. tritici with its host. Analysis of putative effector genes revealed a significant association with genomic loci that are marked with H3K27me3 or overlapping H3K27me2 and H3K9me3 under axenic growth conditions (Soyer et al., 2019). The regulation of four of these effectors (AvStb6, Avr3D1, QTL_5, and Mycgr3G76589) has been analysed using fluorescent reporter genes (Meile et al., 2020). These loci are silenced under axenic Di- and tri-methylation of histone H3 on lysine 9 (H3K9me2/ 3) are hallmarks of constitutive heterochromatin while facultative heterochromatin in metazoans is classically marked by histone H3 that is trimethylated on lysine 27 (H3K27me3) (Liu et al., 2020). The genomic locations of heterochromatic PTMs in a Z. tritici reference strain have been mapped using ChIP-seq (Schotanus et al., 2015). H3K9me3 was found to cover around 22% of the genome but only a very small number of protein coding genes were found to be associated with this PTM. Instead H3K9me3 predominantly maps to TEs and to subtelomeric regions. H3K27me3 is also found associated with subtelomeric regions and TEs but, in comparison to H3K9me3, has a broader genomic distribution that encompasses specific genic regions. Furthermore, accessory chromosomes are particularly enriched for H3K27me3. In contrast to some other organisms, pericentomeric regions of Z. tritici chromosomes are not marked with H3K9me3. Indeed, Z. tritici centromeres are quite unusual, being relatively small and lacking extensive AT–rich tracts (Schotanus et al., 2015). The Z. tritici enzymes responsible for catalysing these modifications are conserved SET [Su (var), Enhancer of zeste, Trithorax] domain lysine methyltransferases (KMTases) (Qian and Zhou, 2006). H3K9me2/3 is mediated by Kmt1 which is a homolog of human Suv39H1/2 (Schizosaccharomyces pombe Clr4, Neurospora crassa DIM5) and H3K27me3 by Kmt6 which is a homolog of Drosophila E(Z) and human EZH2/1 (Möller et al., 2019). The functions of H3K9me2/ 3 and H3K27me3 in Z. tritici have been investigated through the analysis of mutant strains lacking these KMTases. Deletion of kmt1 results in a severe reduction of fitness in vitro and sensitivity to a range of abiotic stresses (osmotic, oxidative and genotoxic). Furthermore, Δkmt1 strains have severely reduced virulence in wheat infections assays indicating that H3K9me2/3 is important for growth in planta. In contrast, loss of kmt6 (and thus H3K27me3) does not reduce growth in vitro and is associated with only modest reduction in virulence (Möller et al., 2019) (Table 1). H3K27me3 and H3K9me2/3 modifications play opposing roles in the regulation of genome stability. As outlined above, the Frontiers in Genetics 03 frontiersin.org Fraser and Whitehall 10.3389/fgene.2022.1058741 FIGURE 1 The deposition and recognition of heterochromatic histone PTMs in Z. tritici. The top panel shows the predicted composition of Kmt1 (histone H3K9) and Kmt6 (histone H3K27) KMTase complexes. The bottom panel summarises proteins known to be, or predicted to be, involved in the recognition of H3K9me2/3 and H3K27me3. Predicted proteins, activities and interactions are shown in grey. The Ensembl Fungi ID of genes encoding putative proteins is indicated. knockdown of CUL4 reduces H3K9me levels (Higa et al., 2006). As such, a functional link between CUL4 mediated ubiquitylation and methylation of H3K9 may be broadly conserved. conditions but expression is induced during host colonization. Furthermore, the increase in expression at the switch to necrotrophic growth coincides with a decrease in the associated levels of H3K27me3 at all four genes and all but one also had a reduction in H3K9me3 levels. This is consistent with the active remodelling of heterochromatic domains during infection. Moving these effector genes to an ectopic location results in loss of silencing under axenic conditions and aberrant in planta expression indicating that genomic environment is an important facet of their regulation (Meile et al., 2020). H3K9me2/3 readers in Z. tritici Histone PTMs exert their function by acting as docking sites for proteins (termed “readers”) which modify chromatin structure and regulate DNA-dependent processes. The recognition of H3K9me2/ 3 PTMs is commonly mediated by the Heterochromatin Protein 1 (HP1) family of chromodomain (CD) containing proteins which are present in a variety of eukaryotic organisms including filamentous fungi (Kumar and Kono, 2020). HP1 proteins have a conserved architecture comprised of an N-terminal chromodomain (CD) and a C-terminal chromo-shadow domain (CSD) separated by a flexible hinge region. Recognition of H3K9me2/3 is achieved by the CD. Structural analyses have demonstrated that CDs fold in to a threestranded β-sheet with an adjacent helix and that methyl-lysine coordination is achieved by a “cage” formed by the sidechains of three conserved aromatic residues (Jacobs and Khorasanizadeh, 2002; Nielsen et al., 2002). The CSD mediates homodimerization and provides a docking surface for the interaction of additional proteins with PxVxL and related motifs (Brasher et al., 2000; Cowieson et al., 2000; Huang et al., 2006). Cryo EM analysis has revealed that HP1 dimers are capable of bridging between adjacent nucleosomes (Machida et al., 2018) and further HP1 self-interactions are proposed to facilitate the formation of oligomeric structures which compact nucleosome arrays and provide a platform for Histone H3K9 KMTase complexes S. pombe Clr4 and N. crassa DIM-5 H3K9 KMTases function in the context of Cul4 ubiquitin ligase complexes, which are called CLRC (Clr4 methyltransferase complex) and DCDC (DIM-5-7-9 CUL-4 DDB1), respectively (Hong et al., 2005; Horn et al., 2005; Jia et al., 2005; Li et al., 2005; Lewis et al., 2010a; Lewis et al., 2010b). Mutations in the genes encoding CLRC/DCDC subunits result in the loss of H3K9methylation in both organisms. This indicates that the integrity of these complexes is required for the catalytic activity of the KMTase and recent work has shown that CLRC ubiquitylates histone H3 on lysine 14 (H3K14ub), promoting H3K9me by Clr4 (Oya et al.). This H3K14ub-H3K9me “crosstalk” mechanism has not yet been investigated in Z. tritici but homologs of CLRC/DCDC subunits are present (Figure 1). Furthermore, Suv39H1 is associated with CUL4 in human cells (Yang et al., 2015) and Frontiers in Genetics 04 frontiersin.org Fraser and Whitehall 10.3389/fgene.2022.1058741 Cbx2 has two sequences in its N-terminal region that conform to a proposed CSD binding motif Φx(V/P)x(L/M/V) (Huang et al., 2006). It will therefore be important to determine whether or not Cbx2 functions in a HDAC complex with the HP1 homolog Cbx1. the recruitment of additional chromatin regulatory complexes (Canzio et al., 2011; Kumar and Kono, 2020). Many species have multiple HP1 paralogs (e.g. humans and mice have three, Drosophila has five and S. pombe has two) but filamentous fungi often have a single isoform. The sole HP1 ortholog in Z. tritici, Cbx1, binds to both H3K9me2 and me3 in vitro and is enriched at H3K9me-marked loci in vivo (Fraser et al., 2022). Transcriptomic analysis indicates that Kmt1 and Cbx1 regulate the expression of highly similar sets of protein encoding genes. Surprisingly, loss of cbx1 does not result in a global increase in transcripts derived from TEs and unlike Δkmt1 mutants, Δcbx1 strains are not sensitive to abiotic stresses and exhibit only a moderate reduction in virulence in wheat infection assays (Fraser et al., 2022) (Table 1). The different phenotypes associated with loss of the HP1 (Cbx1) and the H3K9 methyltransferase (Kmt1) are suggestive of additional H3K9me readers in Z. tritici and indeed a second CD domain protein (Cbx2) that binds to H3K9me has recently been identified (Fraser et al., 2022). In contrast to the conserved HP1 protein, Cbx1, homologs of Cbx2 are restricted to species in just a few fungal families (mainly the Mycosphaerellaceae and Teratosphaeriaceae). Cbx2 is unusual in that it contains two CDs located in the C-terminal region both of which have the conserved “aromatic cage” residues (Fraser et al., 2022). The remaining region lacks any other obvious protein domains and is predicted to be largely unstructured. Mutation of cbx2 alone does not result in marked growth defects either in planta or in vitro. However, combining deletions in cbx1 and cbx2 mimics the sensitivity of Δkmt1 strains to abiotic stresses consistent with the downstream function of H3K9me PTMs being mediated by a combination of these CD proteins (Fraser et al., 2022). Furthermore, Cbx1 and Cbx2 play redundant roles in the silencing of some Kmt1-regulated genes. In vitro analysis of Cbx2 indicates that it shows a strong preference for trimethylated H3K9 peptides. This is potentially significant as analysis of H3K9me2 and me3 marks in S. pombe have revealed that they demarcate functionally different types of heterochromatin that are associated with different levels of transcriptional silencing (Jih et al., 2017). While the role of Cbx2 in the establishment and or maintenance of constitutive heterochromatin remains to be determined, it is interesting to note that it shares features with the N. crassa heterochromatin regulator CDP-2 (Honda et al., 2012). Both proteins have CDs located in their C-terminal regions with much of the remaining sequence predicted to be unstructured. Furthermore, alignment of Cbx2 and CDP-2 homologues has revealed a small block of homology in the N-terminal region. CDP-2 is a subunit of a HP1-containing histone deacetylase complex called HCHC which functions in parallel with a HP1DNA methyltransferase complex (HP1-DIM2) to establish and maintain heterochromatin in N. crassa. The N-terminal region of CDP-2 has a PxVxL-like motif which is required for interaction with the CSD of N. crassa HP1 (Honda et al., 2012). Interestingly, Z. tritici Frontiers in Genetics H3K9me3 and DNA cytosine methylation DNA cytosine methylation (5 mC) is an additional feature of constitutive heterochromatic loci in many organisms. The deposition of 5 mC has been well studied in N. crassa and is mediated by the DNA methyltransferase (DNMT) DIM-2 which is targeted to H3K9me3-marked genomic loci through a direct interaction with HP1 (Honda and Selker, 2008). While many Z. tritici isolates (including the commonly studied reference strain IPO323), lack functional copies of dim2 and thus 5 mC, several Iranian Z. tritici isolates have been identified which have a functional dim2 allele and high levels of 5 mC at TEs (Möller et al., 2021). Comparison of the genomic patterns of H3K9me3 and 5 mC revealed the co-localization of these modifications suggesting an interaction between Cbx1 (HP1) and Dim2 similar to that observed in N. crassa. In fungi, 5 mC is mainly associated with TEs and is involved in a genome defence mechanism called RIP (RepeatInduced Point mutation) which is underpinned by the deamination of 5 mC to thymine leading to the accumulation of C to T transitions in repeated elements (Gladyshev, 2017). Accordingly, the presence of functional Dim2 increased mutation rates associated with Z. tritici TEs (Möller et al., 2021). Mechanisms of H3K27me3 deposition and recognition in Z. tritici The deposition and recognition of H3K27me3 in metazoans is mediated by Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), respectively. PRC2 type complexes contain a H3K27 KMTase, together with other core subunits: EED, SUZ12, and RBBP4/7 (Laugesen et al., 2019). A variety of other ‘accessory’ proteins are associated with mammalian PRC2 complexes at substoichiometric levels. The EZH2 KMTase is catalytically inactive in the absence of the other PRC2 subunits and structural studies have revealed that it undergoes a conformational change in the presence of SUZ12 and EED which results in the correct positioning of the methyl donor (S-adenosyl methionine) and the ε-amino group (Jiao and Liu, 2015). PRC1 functions as a reader of H3K27me3 and is thought to bring about transcriptional silencing by directing the compaction of chromatin and the monoubiquitination of histone H2A on lysine 119 (H2AK119Ub) (Piunti and Shilatifard, 2021). Mammals have a variety of PRC1 type complexes which vary in their subunit composition with binding to H3K27me3 being mediated by one 05 frontiersin.org Fraser and Whitehall 10.3389/fgene.2022.1058741 of five chromobox (CBX) proteins which are orthologs of the Drosophila CD protein Polycomb (Pc) (Piunti and Shilatifard, 2021). PRC2 subunits are generally conserved in fungi that mediate H3K27 trimethylation including Z. tritici (Ridenour et al., 2020). An exception is the Cryptococcus neoformans PRC2 complex which apparently lacks a SUZ12 equivalent (Dumesic et al., 2015). Functional analyses of the putative Z. tritici EED (Mycgr3G109560), SUZ12 (Mycgr3G96052) and RBAP46/48 (Mycgr3G110427) subunits are currently lacking. Nonetheless, it appears that Z. tritici possesses a PRC2 complex and that the fundamentals of H3K27me3 deposition are conserved with metazoans (Figure 1). In stark contrast, PRC1 subunits have not been identified in fungi and the mechanisms by which this PTM mediates its biological downstream effects are poorly understood. Despite H3K27me3 being present in a variety of fungal species, to date only one fungal CD protein- Cryptococcus neoformans Ccc1-has been identified that is capable of specifically binding to this PTM (Dumesic et al., 2015). Ccc1 is a component of the PRC2 complex and its loss causes the redistribution of H3K27me3 to regions occupied by H3K9me indicating that Ccc1 restrains PRC2 activity to correct regions of the genome (Dumesic et al., 2015). The degree to which this is a feature of fungal PRC2 complexes remains to be determined as orthologs of Ccc1 have not been identified in other species. Analysis of a Z. tritici reference genome indicates that it encodes a relatively limited repertoire of CD proteins with the potential to recognise heterochromatic histone PTMs. While the HP1 proteins of some organisms recognise H3K27me3 (Turck et al., 2007; Yale et al., 2016), neither Cbx1 nor Cbx2 show any specificity for H3 peptide tails methylated on K27 in vitro (although an in vivo role cannot be completely discounted) (Fraser et al., 2022). A major role for any of the remaining hypothetical CD proteins (termed Cbx3-Cbx6) in the recognition of H3K27me3 is also unlikely as these proteins lack critical conserved caging aromatic amino acids and/or are encoded by genes on the accessory chromosomes. Some also exhibit similarity to retroviral CD-containing integrases suggesting they are remnants of retrotransposable elements (Fraser et al., 2022). Recent studies have provided exciting insights into mechanisms of H3K27me3 recognition and silencing independent of PRC1. Using a forward genetic screen in N. crassa, Wiles and colleagues identified EPR-1, a protein with BAH (bromo-adjacent homology) and PHD (plant homeodomain) motifs that is required for transcriptional repression at H3K27me3 marked genes. EPR-1 associates with H3K27me3 marked loci in a manner which is dependent upon the BAH domain (Wiles et al., 2020). Furthermore, the BAH domain of the Fusarium graminearum EPR-1 orthlog, BP1 binds to methylated H3K27 peptides and loss of BP1 de-represses the expression of K27me3-regulated SM genes (Tang et al., 2021). EPR-1 homologs are present in a variety of eukaryotic lineages (Wiles et al., 2020) and indeed BAH-PHD domain proteins have also been linked to PRC2-mediated silencing in plants, which like fungi, lack PRC1 components (Li Frontiers in Genetics et al., 2018; Qian et al., 2018; Yang et al., 2018). The BAH domains of human BAHD1 and BAHCC1 have also been shown to function as H3K27me3 readers and BAHD1 is required for optimal silencing of H3K27me3 marked genes (Zhao et al., 2016; Fan et al., 2020; Fan et al., 2021; Xu et al., 2021). Thus BAH mediated recognition of H3K27me3 is conserved across fungi, plants and animals suggesting that BAH represents a more ancient class of Polycomb silencing (Wiles et al., 2020). Characterisation of a putative Z. tritici EPR-1 homolog (Mycgr3G109489) has not yet been reported but it will be important to determine its role in the stability of accessory chromosomes and the control of effector gene expression. Discussion While it has been established that heterochromatic PTMs have an important influence on the genome evolution, effector gene expression and virulence of Z. tritici (Möller et al., 2019; Soyer et al., 2019; Meile et al., 2020; Habig et al., 2021), there are a number of questions surrounding their deposition, maintenance and function that remain to be resolved. Firstly, how are Kmt1 and Kmt6 KMTases targeted to specific regions of the genome? Little is known about the recruitment of Ezh2/ Kmt6 type enzymes in fungi and even in relatively well-studied mammalian systems, understanding of PRC2 recruitment is incomplete (Guo et al., 2021). In S. pombe, genomic targeting of Clr4 (H3K9me KMTase) is linked to the RNAi machinery (Martienssen and Moazed, 2015) but no functional link between RNAi and heterochromatin has been established in Z. tritici. Secondly, what are the mechanisms by which constitutive heterochromatin domains are spread and maintained through successive cell divisions in filamentous fungi such as Z. tritici? Unlike their counterparts in metazoans and fission yeast, the Suvar39/Clr4 KMTases of filamentous fungi do not have a recognisable CD in the N-terminal region. This is significant because for human Suv39H1 and S. pombe Clr4 the interaction of the CD with H3K9me2/3 is required for the spreading and maintenance of heterochromatic domains (Zhang et al., 2008; Muller et al., 2016). Thirdly, do all of the phenotypes associated with deletion of kmt1 and kmt6 result from loss of histone modifications? Mammalian Suv39H1 and Ezh2 KMTases both have nonhistone substrates (Rodriguez-Paredes and Lyko, 2019; Weirich et al., 2021) and the possibility that this is the case for Z. tritici Kmt1 and Kmt6 needs to be considered. Finally, to what extent does the remodelling of heterochromatin during infection regulate the expression of effector genes (Soyer et al., 2019; Meile et al., 2020) and what are the environmental cues and signalling pathways that control this process? Here it is worth noting that the activity of eukaryotic TEs is often upregulated in response to environmental stresses (Pappalardo et al., 2021; Ito, 2022) and the colonization of host tissue is a stress-inducing process (Sanchez-Vallet et al., 2018). Indeed, 06 frontiersin.org Fraser and Whitehall 10.3389/fgene.2022.1058741 Acknowledgments the upregulation of a large number of Z. tritici TE families coincides with the development of symptoms on wheat leaves (Fouche et al., 2020). Therefore, stress-responsive TEs could be an important facet of the controls that shape the chromatin environment of effector genes and thus the interaction of Z. tritici with its host. We thank Zoë Gardiner for comments on the manuscript. 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. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. 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. Funding CJF was supported by a PhD studentship from the Newcastle Liverpool Durham BBSRC Doctoral Training Partnership (BB/ M011186/1). References Fan, H., Lu, J., Guo, Y., Li, D., Zhang, Z. M., Tsai, Y. H., et al. (2020). 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Associations of Dietary Patterns With Brain Morphology in Children: Results From a Prospective Population-Based Study
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Associations of dietary patterns with brain morphology in children: Results from a prospective population-based study Yuchan Mou  Erasmus MC Elisabet Blok  Erasmus MC Monica Barroso  Erasmus MC Pauline W. Jansen  Erasmus MC Tonya White  Erasmus MC Trudy Voortman  (  Research Article Posted Date: June 7th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1622356/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 European Journal of Epidemiology on May 8th, 2023. See the published version at https://doi.org/10.1007/s10654-023-01012-5. Page 1/36 Abstract Dietary patterns in childhood have been associated with child neurodevelopment and cognitive performance, while the underlying neurobiological pathway is unclear. We aimed to examine associations of dietary patterns in infancy and mid-childhood with pre-adolescent brain morphology. We included 1888 and 2326 children with dietary data at age one or eight years, respectively, and structural neuroimaging at age 10 years in the Generation R Study. Measures of brain morphology were obtained using magnetic resonance imaging. Dietary intake was assessed using food-frequency questionnaires, from which we calculated diet quality scores based on dietary guidelines and from which derived dietary patterns using principal component analyses. Associations were examined using regression models adjusted for potential confounding and controlled for multiple testing. At age one year, children with higher adherence to dietary patterns labeled as ‘Snack, processed foods and sugar’ had smaller cerebral white matter volume (B = -4.30, 95%CI -6.92, -1.68). At age eight years, higher adherence to diet patterns labeled as ‘Whole grains, soft fats and dairy’ was associated with a larger total brain (B = 8.93, 95%CI 4.54, 13.32), and larger cerebral gray matter volumes (B = 5.17, 95%CI 2.86, 7.48). Children with higher diet quality and better adherence to a ‘Whole grains, soft fats and dairy’ dietary pattern at age eight years showed greater brain gyrification and larger surface area, clustered primarily in the dorsolateral prefrontal cortex. In conclusion, dietary patterns in early- and mid-childhood are associated with differences in brain morphology. These findings may explain part of the previously reported relation between dietary patterns and neurodevelopment in children. Introduction Diet is an important modifiable factor that modulates brain development by supplying energy and nutrients. In particular, differences in dietary intake in childhood may be crucial for optimal brain development, as the structural and functional complexity of the brain develops rapidly throughout childhood and adolescence [1]. Previous studies have revealed associations between specific nutrients and neurodevelopment in children [2–4], however, these studies do not account for the process by which individual nutrients interact with each other in a synergistic and complex manner in the human body. Therefore, investigating overall diet by looking at dietary patterns can complement the understanding of the association of nutrition with brain development. Overall dietary patterns, which are often quantified by predefined indices (e.g., diet quality score) or empirically derived from dietary data, are closely related to eating behaviors in the real world setting by considering how foods and nutrients are combined [5]. Examples that are often studied are: prudent dietary patterns, often featured as high whole grains, fruits and vegetables intake or western-like dietary patterns, characterized by high intakes of refined grains, saturated fat, and sugar. Previous studies have shown that dietary patterns are established in early childhood and remain relatively stable into adulthood, with less healthy patterns showing the strongest stability [6–8]. With ongoing global nutrition transitioning towards unhealthy diets [9], more than half of children have poor-quality diets in low-, middle-, and high-income countries [10–13]. Page 2/36 Page 2/36 Page 2/36 Dietary patterns in childhood are likely to have long-term effects on health, including brain development. A growing body of research has identified temporal associations between dietary patterns and children’s cognitive development. For instance, a better diet quality [14] or a prudent dietary pattern at different ages through childhood is linked to higher IQ scores of children and adolescents [15], even when accounting for socioeconomic status. In contrast, children and adolescents with higher adherence to a western-like dietary pattern showed diminished cognitive functioning and lower IQ scores [16, 17]. Neuroimaging studies have identified positive links between IQ and alterations in both global and regional brain volumetric measures in children measured using structural magnetic resonance imaging (MRI) [18, 19], although the influence of nutrition on the neurobiological underpinnings of cognitive performance has been less well studied. Neurodevelopmental disorders are also found to be associated with undesirable dietary patterns. Introduction Recent studies have shown that children with autistic traits and attention deficit hyperactivity disorder symptoms had a lower diet quality [20, 21]. Likewise, these neurodevelopmental disorders have been associated with brain morphology [22–24]. Taken together, those associations suggest a potential neurobiological pathway underlying the associations of dietary patterns with neurodevelopmental outcomes. Dietary effects on neurodevelopment may be global or specific to certain brain regions. The hippocampus and amygdala may be particularly sensitive to the effects of specific dietary patterns, such as high-fat diet diets. The hippocampus is involved in integrating multimodal information and is critical for memory, learning, and is also involved in appetitive, digestive and learned eating behaviors [25]. The amygdala has been implicated in feeding, the reward systems associated with eating, and modulating food consumption [26]. Animal studies have found that a western diet has adverse effects on hippocampal dependent memory function and neurogenesis. Additionally, western diets also induce inflammation in the hippocampus [27, 28], and enhancing amygdala-dependent, cue-based memory in juvenile mice [29]. While these animal studies provide important information related to differences in dietary patterns to brain development, it is unclear whether different dietary patterns are associated with the volumetric differences of hippocampus and amygdala in children. Only one recent study in children investigating the effect of a western diet on hippocampal and amygdala volume did not observe an association. However, they found that increased fat consumption was related to a smaller hippocampal volume [30]. This study has a small sample size and the western diet was calculated by combining percentage of daily calories from fat and sugar, which may limit the generalizability of the findings. Within this context, we aimed to examine the prospective association of dietary patterns in infancy and mid-childhood with brain morphology assessed at the age of 10 years in a large population-based cohort. Based on evidence from animal studies, we examined the relationship between dietary patterns and two regional brain volumes (hippocampus and amygdala). Due to the paucity of information on the association of overall diet with brain morphology in the literature, we used an exploratory approach that involves global, subcortical volumetric measures (total brain, cerebral white matter, cerebral gray matter, hippocampus and amygdala) and surface-based brain measures (gyrification index, surface area and cortical thickness). Study design and population This study was embedded in The Generation R Study, an ongoing population-based prospective cohort from early fetal life onward in Rotterdam, the Netherlands. The detailed information on the general design of the Generation R Study has been described in detail elsewhere [31]. Of 9749 eligible pregnant women with a delivery date between April 2002 to January 2006, 7893 mother-child dyads consented for the postnatal follow-up. The study was approved by the Medical Ethics Committee of Erasmus Medical Center, Rotterdam. Written informed consent was obtained from all participating children and their parents. Dietary information was obtained using validated food frequency questionnaires (FFQs), which were completed, generally by the mother, for 3629 children at one year-of-age (response rate: 71.3%) [32] and for 4733 children at eight years-of-age (response rate: 61.8%) [12]. Structural T1-weighted images were obtained when children were 10 years [33]. We excluded children who did not visit the neuroimaging research center, or did not provide consent for the MRI scan. For those with valid dietary data at age one year, images were available for 2303 children, and for those with dietary data at eight years of age, images were available for 2813 children. Further, children were excluded if images could not be reconstructed, had poor quality, if major incidental findings were found, or if the gyrification index could not be calculated (Fig.1). The final study population comprised 1888 children for analysis of diet at age one year and 2326 children for analysis of diet at age eight years. Introduction We used two approaches to define dietary patterns: a priori diet quality score based on national dietary guidelines, and a posteriori dietary patterns derived as principal components in the Page 3/36 Page 3/36 Page 3/36 population. For global and subcortical volumetric measures, we hypothesized that higher adherence to a western-like dietary pattern and lower diet quality score would be associated with smaller total brain volume at 10 years of age, and would be associated with smaller hippocampal and amygdala volume. We expected that positive associations would be found for a prudent dietary pattern or a better diet quality. For surface-based measures, we did not have prior hypotheses. The findings of this study will facilitate future hypotheses on overall diet and brain development, as this has yet to be studied in children. Dietary patterns Both a priori and a posteriori methods were used to evaluate dietary patterns. A priori Diet-Quality Scores (DQS) at ages one and eight years were quantified on the basis of Dutch dietary guidelines [12, 32]. Ten food groups were considered at each age, with intake cut-offs based on age-specific dietary recommendations. Specifically, DQS at one year-of-age (DQS-1y) includes scores for vegetables (≥100 g/d); fruit (≥150 g/d); bread and cereals (≥70 g/d); rice, pasta, potatoes, and legumes (≥70 g/d); dairy (≥350 g/d); meat, poultry, eggs, and meat substitutes (≥35 g/d); fish (≥15 g/d); oils and fats (≥25 g/d); candy and snacks (≤20 g/d); and sugar-sweetened beverages (≤100 g/d) [32]. DQS for children at eight years-of-age (DQS-8y) includes fruit (≥150g/d); vegetables (≥150g/d); whole grains (≥90g/d); fish (≥60g/wk); legumes (≥84g/wk); nuts (≥15g/d); dairy (≥300g/d); oils and soft or liquid margarines (≥30g/d); sugar-containing beverages (≤150g/d); and high-fat and processed meat (≤250g/wk) [12]. To determine the DQS for each participant, the ratio of reported and recommended intake for each food group was first calculated. Then a summary score was calculated by summing the individual food group scores. The overall diet quality scores range from 0 to 10, with higher scores reflecting a higher adherence to the dietary guidelines. Both DQS-1y and DQS-8y were validated for intake of macronutrients and micronutrients among children participating in the Generation R Study [12, 32]. A posteriori dietary patterns were derived using principal components analysis (PCA). Food items from the FFQ at age one year were classified into 27 food groups (Supplemental Table 2) in line with previous studies [37, 38], and food items from the FFQ at age eight years were grouped into 26 food groups (Supplemental Table 3). The varimax rotation method was used to obtain the orthogonal components with the aim of minimizing the covariance between the components. The choice of numbers of dietary patterns to extract was based on the scree plot and Kaiser criterion test. Consequently, the dietary patterns at ages one and eight years with eigenvalues of 1.3 or larger were considered as the most common dietary patterns in the study population. Factor loadings were obtained for each food group, indicating the correlation between the food group and each dietary pattern. Dietary pattern scores for each participant were calculated by summing the food group intake weighted by the corresponding factor loading. Dietary assessment Page 4/36 Dietary intake was assessed when children were approximately one year-of-age (median 12.9 months, IQR: 12.7, 13.9) and eight years of age (median 8.2 years, IQR: 8.0, 8.2) using two age-specific validated semi-quantitative FFQs. The FFQs included food items based on foods that were frequently consumed by 9-18 months-old and 2-to-12 year-old children in national food consumption surveys in the Netherlands [34, 35], resulting in 221 items for age one year and 71 items for age eight years. Each FFQ contains questions on frequency of consumption, portion size, the types of food items and corresponding food preparation methods over the past 4 weeks. The information was converted into grams of individual food items per day based on standardized portion sizes using SAS VoVris (Vovris V2.4, TNO, 1999-2006). Total Page 4/36 Page 4/36 energy and nutrient intake were calculated using reference data from the Dutch Food Composition Tables (NEVO 2001). The FFQ at age 1 year was evaluated for nutrient intakes against three 24-hour recalls in a representative sample of 32 Dutch children aged 14 months (r ranging from 0.36 to 0.74) [32]. The FFQ at age 8 years was validated for energy intake (r = 0.62) using the doubly labeled water method among 4 to 6 years Dutch children (n = 30) [36]. energy and nutrient intake were calculated using reference data from the Dutch Food Composition Tables (NEVO 2001). The FFQ at age 1 year was evaluated for nutrient intakes against three 24-hour recalls in a representative sample of 32 Dutch children aged 14 months (r ranging from 0.36 to 0.74) [32]. The FFQ at age 8 years was validated for energy intake (r = 0.62) using the doubly labeled water method among 4 to 6 years Dutch children (n = 30) [36]. Dietary patterns The scores were standardized with higher scores reflecting higher similarity to the extracted dietary patterns. Brain morphometry assessments Page 5/36 At the age of 10 years (median 9.9, IQR 9.7, 10.1), the children were invited to the MRI center. Prior to the MRI procedure, the children participated in a mock MRI session. MR images were acquired on a 3.0 Tesla GE Discovery MR750w MRI system (General Electric Healthcare, Milwaukee, WI, USA) scanner using an 8- channel head coil. The high-resolution T­1-weighted sequence were obtained using a 3D coronal inversion recovery fast spoiled gradient recalled (IR-FSPGR, BRAVO) sequence (TR = 8.77 ms, TE = 3.4 ms, TI = 600 ms, NEX = 1, flip angle = 10°, field of view = 220 × 220 mm, number of slices = 230, in-plane resolution = 1.0 mm3) [33]. At the age of 10 years (median 9.9, IQR 9.7, 10.1), the children were invited to the MRI center. Prior to the MRI procedure, the children participated in a mock MRI session. MR images were acquired on a 3.0 Tesla GE Discovery MR750w MRI system (General Electric Healthcare, Milwaukee, WI, USA) scanner using an 8- channel head coil. The high-resolution T­1-weighted sequence were obtained using a 3D coronal inversion recovery fast spoiled gradient recalled (IR-FSPGR, BRAVO) sequence (TR = 8.77 ms, TE = 3.4 ms, TI = 600 ms, NEX = 1, flip angle = 10°, field of view = 220 × 220 mm, number of slices = 230, in-plane resolution = 1.0 mm3) [33]. At the age of 10 years (median 9.9, IQR 9.7, 10.1), the children were invited to the MRI center. Prior to the MRI procedure, the children participated in a mock MRI session. MR images were acquired on a 3.0 Tesla GE Discovery MR750w MRI system (General Electric Healthcare, Milwaukee, WI, USA) scanner using an 8- channel head coil. The high-resolution T­1-weighted sequence were obtained using a 3D coronal inversion recovery fast spoiled gradient recalled (IR-FSPGR, BRAVO) sequence (TR = 8.77 ms, TE = 3.4 ms, TI = 600 ms, NEX = 1, flip angle = 10°, field of view = 220 × 220 mm, number of slices = 230, in-plane resolution = 1.0 mm3) [33]. Volumetric segmentation and cortical reconstruction were processed using FreeSurfer version 6.0 analysis suite (https://surfer.nmr.mgh.harvard.edu/) with standard processing procedure which have been described previously [39]. Global and regional volumes, including total brain, cerebral white and gray matter, hippocampus and amygdala volumes were measured. For surface-based analyses, cortical thickness, surface area and gyrification were quantified. Brain morphometry assessments The quality of cortical reconstructions was visually inspected, and images were removed in case of insufficient quality [39]. Statistical analysis Characteristics of the study population were described as mean (SD) for continuous variables with normal distribution, median (IQR) for continuous variables with a skewed distribution, or percentages for categorical variables. We examined individual associations of a priori and a posteriori dietary patterns with brain morphology using multiple linear regression models considering analysis of global, subcortical brain morphometry and surface-based morphometry. For analysis of global and subcortical brain morphometry, the outcomes of interest were total brain, cerebral white matter, cerebral gray matter, hippocampal and amygdala volumes. Next, the associations of a priori and significant a posteriori dietary patterns with surface-based morphometries, including cortical thickness, surface area and gyrification, were examined. We used two models to examine the associations. Model 1 included child sex and age at neuroimaging assessment. Model 2 was further adjusted for maternal education, household income, child ethnic background, child energy intake, child BMI at the age of 10 years, maternal diet quality, smoking, alcohol use, folic acid use, maternal psychopathology symptoms during pregnancy. For analyses including the hippocampus and amygdala, intracranial volumes were additionally included in model 2 to test whether associations are independent of a global effect. To reduce potential bias due to missing values on covariates, we applied multiple imputations by generating five independent datasets with 50 iterations using the MICE package in R. The results of pooled analyses are presented. The analyses of surface-based morphometry were performed using QDECR package [47] in R version 3.6.3 and all other statistical analyses were carried out using R version 4.0.3 (R Foundation for Statistical Computing, Vienna, Austria). Two-sided α < 0.05 was considered statistical significance. In the analyses of global and regional brain morphometry, correction for multiple testing was performed using the Benjamini-Hochberg approach [48] for 9 dietary patterns (4 for age one year; 5 for age eight years) per 5 primary outcomes (45 tests in total) with a false discovery rate (FDR) of 0.05. In analyses of surface-based morphometry, the Gaussian Monte Carlo Simulations with a cluster- wise correction were used to correct for multiple testing. The cluster-forming threshold was set to p = 0.001, which corresponds to a false-positive rate of 0.05 [49]. Bonferroni correction were further applied for each brain hemisphere (p < 0.025 cluster-wise). We performed additional analyses to test the robustness of the results. First, information on maternal and child characteristics between respondents and non-respondents with FFQ and neuroimaging data was compared. Covariates Several child and maternal characteristics were considered as potential confounders based on prior literature [12, 40-43]. Child sex and date of birth were derived from medical records filled in by obstetricians and community midwives. Self-reported questionnaires during pregnancy were used to collect information on maternal education and household income, maternal psychopathology symptoms and lifestyle-related factors during pregnancy, including smoking status, alcohol use, and folic acid supplement use. Maternal highest education level was dichotomized into low (ranging from no education up to lower vocational training) and high (higher vocational training/university) educational level. Household income was categorized into < 1200€, 1200 - 2200€ and > 2200€ per month. Smoking during pregnancy was categorized into never smoking, smoking until pregnancy was known, and continued smoking. Alcohol use during pregnancy was categorized into never drinking, drinking until pregnancy was known and continued drinking. Folic acid use was categorized into no use during embryogenesis, started the first 10 week and started periconceptional. Maternal psychopathology symptoms were assessed at the third trimester by the Brief Symptom Inventory [44], from which the global severity index was calculated. Maternal dietary intake during pregnancy was measured by food frequency questionnaires, from which diet quality scores were calculated based on dietary guidelines [45]. Child ethnic background was determined based on the country of birth of the parents, and grouped into Dutch, non-Dutch Western, and non-Dutch non-Western, in line with previous work in the same cohort [46]. Child energy intake was estimated from food frequency questionnaires at ages one and eight years old. Height and weight were measured by trained staff at the research center when children were 10 years old. Child’s sex- and age- specific BMI (kg/m2) standard deviation (SD) scores were calculated using Dutch reference growth curves. Intracranial volumes were extracted from FreeSurfer metrics. Page 6/36 Statistical analysis Second, we repeated multiple regression analyses restricted to children with a Dutch ethnic background, as the FFQs were developed and validated for Dutch children. Population characteristics Population characteristics Page 7/36 Page 7/36 The characteristics of study participants is reported in Table 1. The majority of mothers was highly educated, had a high household income, and had a healthy lifestyle during pregnancy. The majority of children had a Dutch ethnic background. The information of missing values of covariates is listed in Supplemental Table 1. Dietary patterns The characteristics of diet quality scores and dietary patterns among children at one and eight years-of- age are shown in Table 2 and Table 3, respectively. The mean DQS-1y was 4.3 (±1.4) (Table 1). With PCA, three dietary patterns were identified, as shown in Table 2, which were named after the three food groups with the highest factor loadings: (1) ‘Vegetables, potatoes and grains’ dietary pattern; (2) ‘Snacks, processed foods and sugar’ dietary pattern; (3) ‘Butter and margarines, whole grains and dairy’ dietary pattern. Together, these three dietary patterns explained 28.1% of the variation in food intake of one-year-old children. The mean DQS-8y was 4.5 (±1.2) (Table 1). Four dietary patterns were identified by PCA, as shown in Table 3, which were named as: (1) ‘Snacks, potatoes and processed foods’ dietary pattern; (2) ‘Fish, vegetables and fruit’ dietary pattern; (3) ‘Whole grains, soft fats and dairy’ dietary pattern; (4) ‘Meat replacement, legumes and nuts’ dietary pattern. In total, the four dietary patterns explained 28.8% of the variation in food intake of eight-year-old children. Correlations between a priori and a posteriori dietary patterns were calculated by the Pearson correlation coefficient and reported in Supplemental Table 4. DQS-1y is highly correlated with the ‘Vegetables, potatoes and grains’ dietary pattern at the same age (r = 0.69). DQS-8y is moderately associated with the ‘Whole grains, soft fats and dairy’ dietary pattern at age eight years (r = 0.56). Associations of a priori and a posteriori dietary patterns with global and subcortical brain morphometry Table 4 reports the associations of dietary patterns with global brain volumes. A higher adherence to the ‘Snacks, processed foods and sugar’ dietary pattern at the age of one year was negatively associated with total brain (-7.14 cm3/SD; 95% CI: -12.79, -1.49) and cerebral white matter volumes (-4.30 cm3/SD; 95% CI: -6.92, -1.68) at age 10 years, while a higher adherence to the ‘Butter and margarines, cereals and dairy’ dietary pattern at age one year was positively associated with cerebral gray matter volume at age 10 years (2.57 cm3/SD; 95% CI: 0.20, 4.94). Only the association of cerebral white matter volume remained significant after correction for multiple testing. Page 8/36 A higher adherence to the ‘Snacks, potatoes and processed foods’ dietary pattern at the age of 8 years was negatively associated with total brain (-7.63 cm3/SD; 95% CI: -13.19, -2.06) and cerebral gray matter volumes (-4.07 cm3/SD; 95% CI: -7.00, -1.14) at age 10. Conversely, a higher adherence to the ‘Whole grains, soft fats and dairy’ dietary pattern was positively associated with total brain (8.93 cm3/SD; 95% CI: 4.54, 13.32), cerebral white matter (2.95 cm3/SD; 95% CI: 0.91, 4.99) and cerebral gray matter volumes (5.17 cm3/SD; 95% CI: 2.86, 7.48). A higher DQS-8y was positively associated with cerebral gray matter volume (2.30 cm3/SD; 95% CI: 0.15, 4.45). The association of the ‘Whole grains, soft fats and dairy’ dietary pattern with total brain and cerebral gray matter volume remained present after multiple testing correction. No associations were observed between all dietary patterns at age one or eight years and hippocampal or amygdala volume at age 10 years (Table 5). In order to examine if associations observed for cerebral white and cerebral gray matter volumes were independent of a global effect, we additionally adjusted for intracranial volume in model 2. Only the association of the ‘Snacks, processed foods and sugar’ dietary pattern at one year with cerebral white matter volume remained significant (-1.44 cm3/SD; 95% CI: -2.84, -0.04). Associations of a priori and a posteriori dietary patterns with surface- based brain morphometry The cortical regions that were associate with DQS-8y and a ‘Whole grains, soft fats and dairy’ dietary pattern at eight years of age were illustrated in Fig.2 and Fig.3. Regions in model 2 were considered statistically significant. Higher DQS-8y was positively associated with gyrification in regions of the frontal, parietal and temporal lobes, including the rostral middle frontal, superior frontal, caudal middle frontal, precentral, postcentral, supramarginal, superior temporal, and transverse temporal gyrus in the right hemisphere, and caudal middle frontal, rostral middle frontal, parsopercularis, superior parietal, supramarginal, post central, paracentral, precuneus and posterior cingulate gyrus in the left hemisphere (Fig.2A). A higher adherence to the ‘Whole grains, soft fats and dairy’ dietary pattern at age eight years was positively associated with gyrification in small regions of fusiform, parahippocampal, inferior temporal, rostral middle frontal, superior frontal gyrus in the right hemisphere (Fig.2B). The positive association of higher DQS-8y with surface area was found in small clusters in rostral middle frontal and superior frontal regions in the left hemisphere (Fig.3A). Likewise, positive associations of a higher adherence to the ‘Whole grains, soft fats and dairy’ dietary pattern at age eight years with surface area was observed in small clusters in prefrontal and occipital lobe, including superior frontal, superior parietal and lateral occipital regions in the right hemisphere, and superior temporal, middle temporal and superior frontal regions in the left hemisphere (Fig.3B). No associations were found between cortical thickness with DQS-8y or the ‘Whole grains, soft fats and dairy’ dietary pattern at eight years. In addition, we did not find significant associations of diet quality at the age of one year with either of the three surface-based cortical measures. Specific information on the associated brain regions and their p values are reported in the Supplemental Table 5. Additional analyses Page 9/36 The comparison of characteristics between respondents and non-respondents to FFQ and neuroimaging showed that respondents had a higher socioeconomic status, a higher percentage of folic acid usage before or during pregnancy, a better diet quality during pregnancy, and a higher percentage of children with a Dutch ethnic background (Supplemental Table 6). In general, the analyses including only children with a Dutch ethnic background showed stronger effect estimates and found more statistically The comparison of characteristics between respondents and non-respondents to FFQ and neuroimaging showed that respondents had a higher socioeconomic status, a higher percentage of folic acid usage before or during pregnancy, a better diet quality during pregnancy, and a higher percentage of children with a Dutch ethnic background (Supplemental Table 6). In general, the analyses including only children with a Dutch ethnic background showed stronger effect estimates and found more statistically significant associations, compared to the whole study population. Specifically, a higher adherence to the ‘Snacks, potatoes and processed foods’ at eight years was associated with larger total brain and cerebral gray matter volume; a higher adherence to the ‘Whole grains, soft fats and dairy’ at eight years was associated with a larger total brain, cerebral white matter and cerebral gray matter volumes. While the effect size of association between the ‘Snacks, processed foods and sugar’ dietary pattern at age one year and cerebral white matter in the additional analysis was comparable to the main analysis, it was no longer significant (Supplemental Table 7 and 8). Discussion This large population-based prospective cohort study explored associations of different dietary patterns in infancy and in mid-childhood with global, regional brain volumes and surface-based brain morphometry when children were 10 years of age. Diet quality at one and eight years-of-age as assessed by adherence to dietary guidelines was low to moderate. Global brain volumes at age 10 were negatively associated with dietary patterns characterized by high intake of snack and processed foods at age one and age eight years. Interestingly, global brain volumes were positively associated with a dietary pattern characterized by whole grains, soft fats and dairy at age eight years. No volumetric differences were found in the hippocampus or amygdala for dietary patterns at one and eight years-of-age. Higher DQS-8y or higher adherence to the aforementioned pattern with high intake in whole grains, soft fats and dairy at age eight years was associated with a greater gyrification and larger surface area in widespread areas of brain, with significant areas primarily clustered in association cortices, notably in the prefrontal cortex. In the following discussion, we compared the dietary patterns high in snacks and processed foods intake with western-like dietary patterns, while the dietary patterns high in whole grains intake were compared with prudent dietary patterns due to their high factor loadings for those food groups. Overall, our findings suggest that having a prudent dietary pattern in school age, specifically one rich in whole grains, soft fat and dairy, is linked to larger global brain volumes, whereas consuming a western-like dietary pattern in infancy and school age is associated with lower global brain volume. Our study provides novel information that extend earlier studies examining the association of human overall diet with brain health in childhood. Page 10/36 Our findings of the association between brain morphology and children’s dietary patterns in early- and mid-childhood, especially those related to a prudent dietary pattern and a western-like dietary pattern, complement previous studies showing that overall dietary patterns are associated with school attainment [50, 51] and cognitive performance [15–17, 52, 53] in children. Considering that total brain volume [54], cerebral gray matter volume [55] and white matter microstructure [56] have been associated with cognitive ability of children, our results suggest that neuroanatomical correlates may underlie the Page 10/36 association between dietary patterns and cognitive development in children. Discussion Furthermore, our results indicated that a prudent or better diet quality was associated with brain regions functionally implicated in appetite regulation. Among those regions, most clustered in the dorsolateral prefrontal cortex (DLPFC). The prefrontal cortex is a complex region, but is notably involved in dopamine-mediated executive function, regulation of reward, and the inhibition of impulsive behaviors. Specifically, the DLPFC is associated with satiety, food craving, and executive functioning [57, 58]. A randomized controlled trail (RCT) in healthy young men with normal BMI found increased neuronal activity in right DLPFC reduced overall caloric intake and diminished self-reported appetite scores [59], which may suggest DLPFC involvement in food intake-related control mechanisms. However, it is unclear to what extent the functional connectivity translates into differences in brain morphology, although theories of gyrification support a relationship between folding patterns of the brain and increased connectivity [60]. Caution is needed regarding the translation of functional connectivity studies with the observed association between dietary patterns and brain morphology. Among all significant associations, a prudent dietary pattern at age eight years was positively associated and a western-like dietary pattern at age one year was negatively associated with global brain volumes at age 10 years after multiple testing correction. The association of ‘Snacks, processed foods and sugar’ dietary pattern at one year with cerebral white matter volumes persisted after controlling for intracranial volume, suggesting that cerebral white matter development may be specifically susceptible to a western- like diet specifically in infancy. A possible mechanistic interpretation is that overall diet influences brain development if adherence to a dietary pattern with a higher risk of nutrient inadequacy occurs during a period of high need, considering the rapid rate of brain development within early life [2, 61]. The majority of white matter myelination occurs during the first 2 years of life [62]. Meanwhile, research has shown that children with a western-like dietary pattern were exposed to excess fat and sugar intake or inadequate nutrient intake, such as omega-3 polyunsaturated fatty acids [63], which serves as an important nutrient for nerve cell myelination and has shown beneficial effect on cognitive impairment [64, 65]. Contrary to the animal studies, our findings did not support our hypothesis that overall diet is associated with hippocampal and amygdala volumes. We are aware of only one study investigating the effects of a western diet on hippocampal and amygdala volume in five- to nine-year-old-children. Discussion Stadterman et al [30] reported a link between percentage of daily calories from fat, but not western diet, with an isolated decreased left hippocampal volume, but no relationship was found with the right hippocampal or amygdala volume. Their study calculated western diet as a summed percentage of daily calories from fat and sugar and the sample size was small (n = 21), however, it represents a promising step towards understanding the impact of western-like diet on hippocampus and amygdala development in children. We build upon their study investigating the association between a western-like diet and the left hippocampus but found no association. Our large sample size and the population-based sample, coupled with our approach to derive western-like dietary patterns, likely better resembles actual eating habits, although we did not observe evidence for any effects. Future research with a focused hypothesis on the hippocampus and the amygdala is needed to ascertain their association with western-like diet in children. Page 11/36 Page 11/36 Moreover, considering the evidence from population-based studies in adults, which suggest that the left hippocampus may be more vulnerable to western-like diets [66, 67], further investigation into individual effects on hippocampus in each hemisphere is warranted. Such studies preferably follow children into adolescence and adulthood, so that potential long-term effects can be observed. Although the etiology of the relationships between dietary patterns and neurodevelopment remains unclear, there have been potential mechanisms proposed. First, the effect of dietary patterns on brain morphology may be mediated by epigenetic mechanisms. Diet, as an epigenetic regulator, affects multiple genes expression at levels of transcription, translation and post-translational modification. Subsequently, the variation in gene expression regulated by nutrition influences several neurobiological processes, including neurogenesis, synaptic plasticity and neuronal connectivity [68, 69], which may lead to rearrangement of brain structure. Second, differences in dietary patterns can induce differences in metabolic changes underlying brain morphology. For example, brain-derived neurotrophic factor (BDNF) is related to the morphological variation of the hippocampus and prefrontal cortex [70], and can be influenced by diet. High-fat diets have been found to increase oxidative stress and further interfere with the level of BDNF [71]. Regarding healthy dietary patterns, a randomized control trial study in adults found a higher plasma BDNF levels in an experimental group with a Mediterranean diet intervention compared to a control group [72]. Furthermore, decreased BDNF has been linked to cognitive decline [71]. Discussion Evidence from those RCTs is intriguing and denotes a potential causal link between nutrition and brain development. However, some RCT studies that provide supplements of a single nutrient or food in certain groups of children failed to detect changes in cognitive performance [78, 79]. It is noteworthy that none of RCTs considers overall diet in their study design, which may partly explain the inconsistency in findings. This could be explained by an adapted hypothetical scenario in which the effects of nutrient deficiency and poor-quality diet may show an interacting effect on children’s cognitive development [2], thus reducing the effect of nutrient supplementation. Moreover, it is largely unknown whether the effect of nutrition on brain development could be detected by objective anatomical measurement, like brain volumetric alteration. We filled in the gap of lack of evidence in a healthy pediatric population with a long follow-up period. The strengths of our study are the population-based prospective design, availability of multiple covariates, and large-scale neuroimaging in children. In addition, we defined dietary patterns using a priori diet quality score and a posteriori dietary patterns based on food groups, which captured unrelated eating patterns in the population. Therefore, the results of our study provide a public health message on the relationships of dietary patterns and brain development in children. The main limitation is the design of the study. Although longitudinal, the design is in essence cross- sectional, as we lack repeated measures of diet and brain imaging. Thus, our results cannot infer causality between dietary patterns and brain morphometry. Future studies with repeated measurements of dietary patterns and brain morphology, preferably those with an intervention component, are necessary to better understand the direction of the association. Another limitation involves assumptions associated with calculating the a priori and a posteriori dietary patterns analysis, which can influence the interpretation and limit the comparability of the study with others. Such assumptions include how food items were clustered into food groups, the number of principal components to be retained. Additionally, dietary patterns are likely to be a part of lifestyle behaviors, and thus there is the potential for residual confounding. However, after adjusting for BMI at the age of 10 years in the model, dietary patterns were still significantly associated with brain morphology. Several other limitations should also be acknowledged. First, the use of FFQs to quantify dietary intake is subject to measurement error [80]. Discussion While these studies were performed in adults, there may be expectations of even greater differences in children, considering the high energy consumption associated with neurodevelopment. Further, the evidence showing dietary patterns were associated with cognitive performance in children, suggests shared brain metabolic pathways that underlie brain morphological changes in children and adults. Last, diet could affect the gut microbiota which in return alter the host’s physiological responses and associated structural adaption. Short-chain fatty acids (SCFAs) are the most studied metabolites produced by microbes. Foods high in dietary fiber, such as fruits, vegetables and whole grains, increase the levels of SCFAs through gut microbial fermentation [73]. Results from animal studies indicated that SCFAs might influence gut-brain communication and brain function directly or indirectly through neurochemical pathways [74]. By reporting associations of dietary patterns in early and mid-childhood with global and regional brain volumes, our findings underline the need for nutrition research related to child development, notably in middle childhood. Nutrition has been hypothesized as an aspect of the experience-dependent environment that can influence neurodevelopment [75]. However, most studies on nutrition and brain development have focused on early life nutrition, while few studies have examined children in the ‘forgotten years.’ In fact, dietary patterns change drastically from milk-based during infancy to omnivore patterns in childhood. Previous work from our group showed that diet quality at one year-of-age changes considerably by eight years-of-age [12], indicating that relative stability of dietary patterns may occur after the age of one year. Another longitudinal study including five European countries suggests that dietary patterns are established between one and two years and remain stable to eight years of age [8]. In addition, brain metabolism associated with neurodevelopment is high, with a considerable need of energy. This changes at around the age of four to five years, coincident with the slower rate of growth Page 12/36 Page 12/36 during that age. Overall, changes in dietary patterns across childhood, highlight the importance of investigating the effect of diet not only in infancy, but also in mid-childhood. Most dietary interventions for brain development to date have targeted micronutrient supplementation, showing positive effects of nutritional supplementation in certain cognitive domains in nutrient-deficient children [76]. Some interventions which used food supplementation (e.g., fortified foods) in early childhood [2] or in children with atypical neurodevelopment [77] have shown short-term effects on cognitive and motor development. Discussion However, the FFQs used in our study were validated against three 24h recalls in Dutch children at age one year and against the doubly labelled water method in Dutch children at age eight years, and had sufficient capacity of ranking participants with regard to energy intake. Moreover, we adjusted our models for energy intake to mitigate the effect of measurement error in the FFQs that individuals tend to misreport their intake of food and beverages in the same direction. Fourth, although we adjusted for several Page 13/36 Page 13/36 covariates including socioeconomic status, maternal psychopathological symptoms, lifestyle and child BMI, we cannot rule out genetic and unmeasured environmental confounding because of the observational nature of the study. Lastly, the non-response analyses suggested a possible selection bias, which may limit the generalizability of the study. covariates including socioeconomic status, maternal psychopathological symptoms, lifestyle and child BMI, we cannot rule out genetic and unmeasured environmental confounding because of the observational nature of the study. Lastly, the non-response analyses suggested a possible selection bias, which may limit the generalizability of the study. Acknowledgements The Generation R Study is conducted by Erasmus Medical Center in close collaboration with the School of Law and the Faculty of Social Sciences at the Erasmus University, Rotterdam; the Municipal Health Service, Rotterdam area; and the Stichting Trombosedienst & Artsenlaboratorium Rijnmond (Star-MDC), Rotterdam. We gratefully acknowledge the contribution of participating mothers, general practitioners, hospitals, midwives, and pharmacies in Rotterdam, the Netherlands. Conclusion We found a relationship between early-life and mid-childhood dietary pattern and later brain morphological using a prospective population-based study of child development, which suggests that healthy diet may benefit neurodevelopment, which has an important public health message. The findings of our study suggest that a prudent dietary pattern in mid-childhood is positively associated with global and widespread differences in pre-adolescent brain morphometry. Adherence to a western-like dietary pattern in infancy and school age is differentially associated with cerebral white matter, cerebral gray matter and total brain volume, suggesting that the timing of neurodevelopmental processes may play an important role in understanding the effect of overall diet. With the observed temporal associations, our findings highlight the potential of overall dietary patterns to be associated with brain morphology in children, which may explain part of the previously demonstrated relation between dietary patterns and neurodevelopment and cognitive performance in children. However, further research with repeated measurements of dietary patterns and brain morphology is needed to better understand the direction of the association. Author contributions Yuchan Mou, Tonya White and Trudy Voortman designed research; Pauline W. Jansen, Tonya White, Trudy Voortman provided essential materials (databases); Yuchan Mou performed statistical analysis; Yuchan Mou wrote the paper; Elisabet Blok, Monica Barroso, Pauline W. Jansen, Trudy Voortman and Tonya White provided consultation regarding the analyses and interpretation of data; Yuchan Mou and Trudy Voortman had primary responsibility for final content. All authors read and approved the final manuscript. The authors have no competing interests to declare that are relevant to the content of this article. The authors have no competing interests to declare that are relevant to the content of this article. Funding The general design of the Generation R Study is made possible by finical support from the Erasmus MC, University Medical Center, Rotterdam, Erasmus University Rotterdam, the Netherlands Organization for Health Research and Development (ZonMw), Netherlands Organization for Scientific Research (NWO), Ministry of Health, Welfare and Sport and Ministry of Youth and Families. Yuchan Mou is supported by China Scholarship Council (CSC) PhD Fellowship for her PhD study in Erasmus Medical Center, Rotterdam, the Netherlands. The scholarship file number is 201806240125, CSC URL: http://www.csc.edu.cn/. Pauline Jansen is supported by a grant from ZonMw (Mental Health Care Pauline Jansen is supported by a grant from ZonMw (Mental Health Care Page 14/36 Page 14/36 Page 14/36 Research Program - Fellowship 636320005). The neuroimaging data collection and image processing is supported by ZonMw TOP Grant 91211021 to Tonya White. Supercomputing resources were supported by the NWO Physical Sciences Division (Exacte Wetenschappen) and SURFsara (Cartesius compute cluster, www.surfsara.nl). The funders had no role in the study design, data collection, management, analysis and interpretation of data, and preparation or writing the manuscript. Ethics approval The study was approved by the Medical Ethics Committee of Erasmus Medical Center, Rotterdam. Consent to participate Written informed consent was obtained from all participating children and their parents. Written informed consent was obtained from all participating children and their parents. References 1. Casey BJ, Getz S, Galvan A. 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Table 2 Characteristics of the a priori- and a posteriori-derived dietary patterns, identified among children at age one year (n = 3629) 1, 2 Tables Table 1 Characteristics of study population Page 21/36 Study sample for dietary patterns at one year (N = 1888) Study sample for dietary patterns at eight years (N = 2326) Maternal characteristics at enrollment       Educational level (Low), N (%) 548 (29.0%) 758 (32.6%)   Household income per month, N (%)         < 1200 € 187 (9.9%) 221 (9.5%)     1200 – 2200 € 372 (19.7%) 512 (22.0%)     > 2200 € 1329 (70.4%) 1593 (68.5%)   Alcohol use during pregnancy, N (%)         Never 640 (33.9%) 821 (35.3%)     Until pregnancy was known 274 (14.5%) 342 (14.7%)     Continued 974 (51.6%) 1163 (50.0%)   Smoking during pregnancy, N (%)         Never 1484 (78.6%) 1840 (79.1%)     Until pregnancy was known 187 (9.9%) 212 (9.1%)     Continued 215 (11.4%) 274 (11.8%)   Folic acid use, N (%)         No 255 (13.5%) 391 (16.8%)     Started the first 10 weeks 598 (31.7%) 730 (31.4%)     Started periconceptional 1035 (54.8%) 1205 (51.8%)   Psychopathological symptoms, median (IQR) 0.1 (0.1, 0.3) 0.1 (0.1, 0.3)   Diet quality scores during pregnancy (SD) 7.9 (1.5) 7.8 (1.5)       Child characteristics       Age at the neuroimaging assessment, median (IQR), years 9.9 (9.7, 10.0) 9.9 (9.7, 10.2) Dutch 1346 (71.3%) 1568 (67.4%)     Non-Dutch Western 151 (8.0%) 223 (9.5%)     Non-Dutch non-Western 393 (20.8%) 535 (23.0%)   Diet quality scores, mean (SD) 4.3 (1.4) 4.5 (1.2) Values are mean (SD) for continuous variables with a normal distribution, medians (IQR) for continuous variables with a skewed distribution, or valid numbers N (%) for categorical variables. Missing data of covariates (Supplemental Table 1) were imputed with multiple imputation (M = 5 imputations). Values are mean (SD) for continuous variables with a normal distribution, medians (IQR) for continuous variables with a skewed distribution, or valid numbers N (%) for categorical variables. Missing data of covariates (Supplemental Table 1) were imputed with multiple imputation (M = 5 imputations). Tables Table 2 Characteristics of the a priori- and a posteriori-derived dietary patterns, identified among children at age one year (n = 3629) 1, 2 Table 2 Characteristics of the a priori- and a posteriori-derived dietary patterns, identified among children at age one year (n = 3629) 1, 2 Page 23/36 A posteriori-derived dietary patterns A posteriori-derived dietary patterns A posteriori-derived dietary patterns Food groups 3 Mean intake (g/d) DQS-1y Vegetables, potatoes and grains Snacks, processed foods and sugar Butter and margarines, whole grains and dairy Vegetable 56 + 0.79 0.13 0.04 Potatoes 35 + 0.69 0.10 0.07 Pasta, rice and other grains 27 + 0.59 0.16 0.01 Vegetable oils 1 + 0.53 0.35 0.03 Composite meals 22 Not included 0.45 0.00 0.04 Meat 10 + 0.41 -0.04 0.26 Fish and fish products 9 + 0.40 0.12 0.07 Children's meals 70 Not included -0.38 -0.23 -0.03 Legumes 5 + 0.37 0.21 -0.05 Fruit 152 + 0.27 -0.25 0.20 Savory snacks 3 - 0.11 0.61 0.08 Refined cereals 17 Not included -0.01 0.58 0.14 Soups and bouillons 17 Not included 0.07 0.52 -0.20 Sugar and confectionery 29 - 0.01 0.50 0.43 Eggs 2 + 0.20 0.44 -0.01 Sauces and condiments 2 Not included 0.28 0.41 0.15 Other fats 2 + 0.24 0.36 0.17 High-fat dairy products 52 + 0.05 0.25 0.09 Non-sugar- containing beverages 69 Not included 0.16 0.22 -0.07 Nuts and seeds 1 Not 0.07 0.18 0.17 Soy products 5 Not included 0.02 0.06 0.01 Butters and margarines 8 + 0.08 0.06 0.60 Whole grains 56 + 0.16 -0.41 0.53 Formula and breastfeeding 416 + 0.03 0.02 -0.53 Meat products 14 + 0.27 0.09 0.52 Low-fat dairy products 166 + 0.05 0.06 0.51 Sugar- containing beverages 194 - -0.07 0.23 0.50             Eigenvalues     4.0 1.9 1.6 Variance explained, % 4     10.9 9.1 8.1 1 Factor loadings with and absolute value ≥ 0.20 are shown in bold. 1 Factor loadings with and absolute value ≥ 0.20 are shown in bold. 2 Total sample with FFQ data at one year. Total sample with FFQ data at one year. 3 Included food items in the food groups are presented in Supplemental Table 2. Total explained variance by all three a postereri derived patterns is 28.1%. 4 Total explained variance by all three a postereri derived patterns is 28.1%. Tables Table 3 Characteristics of the a priori- and a posteriori-derived dietary patterns, identified among children at age eight years (n = 4733) 1,2 Page 25/36 Page 25/36 Page 25/36 A posteriori-derived dietary patterns A posteriori-derived dietary patterns A posteriori-derived dietary patterns Food groups 3 Mean intake (g/d) DQS-8y Snacks, potatoes and processed food Fish, vegetables and fruit Whole grains, soft fats and dairy Meat replacement, legumes and nuts Snacks and fast food 21 Not included 0.61 -0.07 -0.14 -0.02 Potatoes 30 Not included 0.60 0.08 0.14 0.11 Sauces 5 Not included 0.53 -0.06 -0.04 -0.04 Composite and ready-to- eat meals 66 Not included 0.48 0.14 0.20 0.15 Refined cereals and grains 47 Not included 0.45 0.14 -0.39 0.01 Processed meat 42 - 0.45 0.12 0.25 -0.43 Sugar and confectionery products 216 - 0.43 -0.12 0.03 -0.04 Sugar- containing beverages 186 - 0.35 -0.03 -0.15 0.01 Low- and moderate-fat fish 5 + -0.05 0.61 -0.11 -0.04 Vegetables 92 + 0.15 0.55 0.34 0.13 Fat fish 10 + 0.08 0.50 -0.06 -0.04 Fruit 121 + -0.14 0.45 0.28 0.02 Low-sugar- containing beverages 314 Not included -0.03 0.41 0.02 0.06 Hard fats 1 Not included -0.14 0.35 -0.12 0.25 White meat, unprocessed 14 Not included 0.24 0.32 -0.16 -0.32 Eggs 10 Not included 0.21 0.23 -0.18 0.15 Whole grains 98 + -0.13 0.11 0.76 0.13 Soft fats 12 + 0.15 -0.19 0.62 -0.15 Low-fat dairy products 201 + -0.03 0.04 0.35 -0.32 Meat replacement products 4 Not included -0.05 0.03 0.19 0.59 Legumes 9 + 0.33 0.19 -0.02 0.37 Red meat, unprocessed 10 Not included 0.31 0.22 0.00 -0.37 Nuts and nut butters 6 + 0.08 0.01 0.20 0.36 High-fat dairy products 35 + 0.05 0.15 -0.07 0.32 Soy drinks 9 Not included 0.00 0.02 -0.06 0.23 Porridge 2 Not included 0.04 0.00 -0.09 0.14               Eigenvalues     2.5 1.9 1.7 1.4 Variance explained, % 4     9.2 7.0 6.9 5.8 1 Factor loadings with and absolute value ≥ 0.20 are shown in bold. 2 Total sample with FFQ data at eight years 1 Factor loadings with and absolute value ≥ 0.20 are shown in bold. 1 Factor loadings with and absolute value ≥ 0.20 are shown in bold. 2 Total sample with FFQ data at eight years. 3 Included food items in the food groups are presented in Supplemental Table 3. 4 Total explained variance by all four a postereri derived patterns is 28.8%. Tables 4 Total explained variance by all four a postereri derived patterns is 28.8%. Tables Table 4 Association of dietary patterns at ages one and eight years with global brain volumes at age 10 years 1 Table 4 Association of dietary patterns at ages one and eight years with global brain volumes at age 10 years 1 Page 27/36 Model 1     Model 2     B 95% CI p value B 95% CI p value Total brain volume (cm3)             Diet at one year             Principal components               Vegetables, potatoes and grains 0.24 -4.23, 4.71 0.9164 -2.24 -6.81, 2.33 0.3358   Snacks, processed foods and sugar -17.03 -21.85, -12.22 0.0000 -7.14 -12.79, -1.49 0.0134   Butter and margarines, whole grains and dairy 10.37 5.92, 14.81 0.0000 4.35 -0.15, 8.84 0.0580 DQS-1y 7.26 3.04, 11.47 0.0007 1.52 -2.73, 5.77 0.4836               Diet at eight years             Principal components               Snacks, potatoes and processed foods -11.62 -15.46, -7.77 0.0000 -7.63 -13.19, -2.06 0.0073   Fish, vegetables and fruit 0.20 -3.67, 4.07 0.9187 -1.67 -5.61, 2.27 0.4048   Whole grains, soft fats and dairy 18.13 14.29, 21.97 0.0000 8.93 4.54, 13.32 0.0001*   Meat replacement, legumes and nuts -3.74 -7.68, 0.21 0.0634 -3.51 -7.38, 0.37 0.0764 DQS-8y 8.75 4.97, 12.53 0.0000 3.64 -0.45, 7.74 0.0812               Cerebral white matter volume (cm3)             Diet at one year Total brain volume (cm3) Butter and margarines, whole grains and dairy 3.41 1.38, 5.44 0.001 1.22 -0.86, 3.31 0.2501 DQS-1y 3.18 1.26, 5.09 0.0012 1.10 -0.87, 3.07 0.2743               Diet at eight years             Principal components               Snacks, potatoes and processed foods -4.15 -5.9, -2.4 0.0000 -2.53 -5.11, 0.06 0.0553   Fish, vegetables and fruit 0.47 -1.28, 2.23 0.5979 -0.14 -1.97, 1.69 0.8800   Whole grains, soft fats and dairy 6.02 4.26, 7.77 0.0000 2.95 0.91, 4.99 0.0046   Meat replacement, legumes and nuts -1.85 -3.64, -0.06 0.0427 -1.83 -3.63, -0.03 0.0463 DQS-8y 3.02 1.3, 4.74 0.0006 1.32 -0.59, 3.22 0.1754               Cerebral gray matter volume (cm3)             Diet at one year             Principal components               Vegetables, potatoes and grains -0.1 -2.46, 2.26 0.9351 -1.35 -3.76, 1.06 0.2706   Snacks, processed foods and sugar -8.52 -11.06, -5.97 0.0000 -2.52 -5.50, 0.46 0.0974   Butter and margarines, whole grains and dairy 5.83 3.48, 8.17 0.0000 2.57 0.20, 4.94 0.0334 DQS-1y 3.36 1.14, 5.59 0.0031 0.27 -1.96, 2.51 0.8113               Diet at eight years             Principal components 0.58 Whole grains, soft fats and dairy 10.34 8.32, 12.36 0.0000 5.17 2.86, 7.48 0.0000*   Meat replacement, legumes and nuts -1.25 -3.33, 0.84 0.2404 -1.07 -3.11, 0.97 0.3040 DQS-8y 5.19 3.19, 7.19 0.0000 2.30 0.15, 4.45 0.0358 1 The effect estimates represent the difference in cubic centimeters for brain volumes per 1 SD higher score on the dietary pattern. Tables Table 5 Association of dietary patterns at ages one and eight years with regional brain volumes at age 10 1 Table 5 Association of dietary patterns at ages one and eight years with regional brain volumes at age 10 years 1 Page 30/36 Page 30/36 Model 1     Model 2     B 95% CI p value B 95% CI p value Hippocampal volume (mm3)             Diet at one year             Principal components               Vegetables, potatoes and grains 19.86 -11.86, 51.58 0.2196 10.27 -18.63, 39.17 0.4861   Snacks, processed foods and sugar -63.86 -98.36, -29.37 0.0003 6.82 -29.15, 42.79 0.7100   Butter and margarines, whole grains and dairy 59.37 27.77, 90.97 0.0002 21.55 -6.92, 50.02 0.1378 DQS-1y 46.69 16.78, 76.6 0.0022 9.75 -17.16, 36.66 0.4774               Diet at eight years             Principal components               Snacks, potatoes and processed foods -56.85 -85.27, -28.44 0.0001 -14.82 -51.65, 22 0.4298   Fish, vegetables and fruit 3.92 -24.58, 32.42 0.7875 -10.46 -36.29, 15.37 0.4271   Whole grains, soft fats and dairy 69.85 41.25, 98.46 0.0000 -17.52 -46.61, 11.58 0.2378   Meat replacement, legumes and nuts -2.66 -31.73, 26.41 0.8575 14.17 -11.3, 39.63 0.2755 DQS-8y 27.6 -0.36, 55.56 0.0530 -27.24 -54.48, 0.00 0.0500               Amygdala volume (mm3) Hippocampal volume (mm3) Tables Model 1 was adjusted for child sex and age when brain imaging was assessed. Model 2 was additionally adjusted for maternal education, household income, child ethnic background, child energy intake, child BMI measured at the age of 10 years, maternal diet quality during pregnancy, smoking during pregnancy, alcohol use during pregnancy, folic acid use, and maternal psychopathological symptoms during pregnancy. Statistical significance (p < 0.05) in model 2 is indicated with bold. 1 The effect estimates represent the difference in cubic centimeters for brain volumes per 1 SD higher score on the dietary pattern. Model 1 was adjusted for child sex and age when brain imaging was assessed. Model 2 was additionally adjusted for maternal education, household income, child ethnic background, child energy intake, child BMI measured at the age of 10 years, maternal diet quality during pregnancy, smoking during pregnancy, alcohol use during pregnancy, folic acid use, and maternal psychopathological symptoms during pregnancy. Statistical significance (p < 0.05) in model 2 is indicated with bold. * denotes the associations which remained statistically significant after the Benjamini Hochberg correction for multiple testing (45 tests) with a FDR ≤ 0.05. * denotes the associations which remained statistically significant after the Benjamini Hochberg correction for multiple testing (45 tests) with a FDR ≤ 0.05. Amygdala volume (mm3) Butter and margarines, whole grains and dairy 33.1 16.41, 49.78 0.0001 7.69 -7.33, 22.7 0.3154 DQS-1y 29.6 13.82, 45.38 0.0002 8.04 -6.15, 22.22 0.2666               Diet at eight years             Principal components               Snacks, potatoes and processed foods -27.37 -41.74, -13.01 0.0002 -11.21 -29.65, 7.23 0.2334   Fish, vegetables and fruit 2.96 -11.45, 17.36 0.6871 -4.93 -17.99, 8.13 0.4592   Whole grains, soft fats and dairy 43.46 29.04, 57.89 0.0000 2.11 -12.58, 16.79 0.7785   Meat replacement, legumes and nuts -9.25 -23.94, 5.44 0.2170 1.07 -11.85, 13.98 0.8712 DQS-8y 16.92 2.79, 31.04 0.0189 -8.49 -22.09, 5.11 0.2210 1 The effect estimates represent the difference in cubic centimeters for brain volumes per 1 SD higher score on the dietary pattern. Model 1 was adjusted for child sex and age when brain imaging was assessed. Model 2 was additionally adjusted for maternal education, household income, child ethnic background, child energy intake, child BMI measured at the age of 10 years, maternal diet quality during pregnancy, smoking during pregnancy, alcohol use during pregnancy, folic acid use, maternal psychopathological symptoms during pregnancy and child intracranial volume. 1 The effect estimates represent the difference in cubic centimeters for brain volumes per 1 SD higher score on the dietary pattern. Model 1 was adjusted for child sex and age when brain imaging was assessed. Model 2 was additionally adjusted for maternal education, household income, child ethnic background, child energy intake, child BMI measured at the age of 10 years, maternal diet quality during pregnancy, smoking during pregnancy, alcohol use during pregnancy, folic acid use, maternal psychopathological symptoms during pregnancy and child intracranial volume. 1 The effect estimates represent the difference in cubic centimeters for brain volumes per 1 SD higher score on the dietary pattern. Model 1 was adjusted for child sex and age when brain imaging was assessed. Model 2 was additionally adjusted for maternal education, household income, child ethnic background, child energy intake, child BMI measured at the age of 10 years, maternal diet quality during pregnancy, smoking during pregnancy, alcohol use during pregnancy, folic acid use, maternal psychopathological symptoms during pregnancy and child intracranial volume. Figures Page 32/36 Figure 1 Study population flow chart Study population flow chart Page 33/36 Figure 2 Figure 2 The association of diet quality and a ‘Whole grains, soft fats and dairy’ dietary pattern at eight years-of- age with gyrification in children Figure 2 The association of diet quality and a ‘Whole grains soft fats and dairy’ dietary pattern Figure 3 The association of diet quality and a ‘Whole grains, soft fats and dairy’ dietary pattern at eight years-of- age with surface area in children The association of diet quality and a ‘Whole grains, soft fats and dairy’ dietary pattern at eight years-of- age with surface area in children Figure 3 legends: LH, left hemisphere; RH, right hemisphere. Model 1 was adjusted for child sex and age when brain imaging was assessed. Model 2 was additionally adjusted for maternal education, household income, child ethnic background, maternal diet quality during pregnancy, smoking during pregnancy, alcohol use during pregnancy, folic acid use, maternal psychopathological symptoms, and BMI measured at the age of 10 years. Colored clusters represent regions of the brain that were positively associated with diet quality (A) or a ‘Whole grains, soft fats and dairy’ dietary pattern (B) that remained after the cluster- wise correction for multiple comparisons (p < 0.001). SupplementalMaterialsDietBrian3mayfinal.pdf Figure 2 The association of diet quality and a ‘Whole grains, soft fats and dairy’ dietary pattern at eight years-of- age with gyrification in children Page 34/36 Page 34/36 Figure 2 legends: LH, left hemisphere; RH, right hemisphere. Model 1 was adjusted for child sex and age when brain imaging was assessed. Model 2 was additionally adjusted for maternal education, household income, child ethnic background, maternal diet quality during pregnancy, smoking during pregnancy, alcohol use during pregnancy, folic acid use, maternal psychopathological symptoms, and BMI measured at the age of 10 years. Colored clusters represent regions of the brain that were positively associated with diet quality (A) or a ‘Whole grains, soft fats and dairy’ dietary pattern (B) that remained after the cluster- wise correction for multiple comparisons (p < 0.001). Page 35/36 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementalMaterialsDietBrian3mayfinal.pdf Page 36/36
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Assigning Backbone NMR Resonances for Full Length Tau Isoforms: Efficient Compromise between Manual Assignments and Reduced Dimensionality
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Introduction [17,18], demonstrating that tau plays a more direct role in neurodegeneration than originally thought. First isolated as microtubule-associated proteins [1], the tau protein family is comprised predominantly of six isoforms that are alternately spliced from a single gene [2,3]. Localized chiefly to the axons in both the central and peripheral nervous systems tau functions primarily to facilitate and stabilize microtubule poly- merization [4–6]. Aside from its primary role, tau is implicated in many different cellular processes including axon development [7], signal transduction [8] and mediation of microtubule-membrane interactions [9]. Perhaps one of the most important cellular functions of tau is to mediate signals for various kinases. There are numerous phosphorylation sites within tau that help regulate its many functions, including binding to microtubules [10–13]. There are two general domains in the primary structure of tau. The N-terminal or projection domain protrudes from the microtubule surface while the C-terminal or assembly domain, which contains either three or four semi-conserved microtubule- binding motifs, remains associated with the microtubule surface [19]. The six distinct isoforms are formed from alternate splicing of exons 2 and 3 in the N-terminal domain, and exon 10 in the microtubule-associated region (Fig. 1). In solution tau is an intrinsically disordered protein (IDP), lacking a well-defined tertiary structure. Since IDPs do not form compact well-ordered structures they cannot be typically crystal- lized in isolation. In contrast, nuclear magnetic resonance (NMR) spectroscopy has proven particularly useful for the study of IDPs in solution [20,21]. Indeed numerous NMR studies have looked at the structure of the various isoforms of tau and their cellular interactions [11,22,23]. For example, we have previously studied the properties of a microtubule-binding repeat containing fragment of tau (Tau K19) both in solution and when interacting with lipid surfaces [24]. Subsequently, a number of NMR studies of tau fragments [25,26] and more recently of full length tau isoforms [27–29] have been reported. Due to the unstructured nature of the protein NMR spectra of tau protein have sharp resonances but lack the high degree of spectral dispersion While tau plays many roles in normal cellular function it is most studied for its role in neurodegeneration. Tau is the primary component in neurofibrillary tangles (NFT), a pathological hallmark of a group of diseases called tauopathies that includes Alzheimer’s disease (AD), progressive supranuclear palsy and Pick’s disease among others [14]. Abstract Tau protein is the longest disordered protein for which nearly complete backbone NMR resonance assignments have been reported. Full-length tau protein was initially assigned using a laborious combination of bootstrapping assignments from shorter tau fragments and conventional triple resonance NMR experiments. Subsequently it was reported that assignments of comparable quality could be obtained in a fully automated fashion from data obtained using reduced dimensionality NMR (RDNMR) experiments employing a large number of indirect dimensions. Although the latter strategy offers many advantages, it presents some difficulties if manual intervention, confirmation, or correction of the assignments is desirable, as may often be the case for long disordered and degenerate polypeptide sequences. Here we demonstrate that nearly complete backbone resonance assignments for full-length tau isoforms can be obtained without resorting either to bootstrapping from smaller fragments or to very high dimensionality experiments and automation. Instead, a set of RDNMR triple resonance experiments of modest dimensionality lend themselves readily to efficient and unambiguous manual assignments. An analysis of the backbone chemical shifts obtained in this fashion indicates several regions in full length tau with a notable propensity for helical or strand-like structure that are in good agreement with previous observations. Editor: Ramo´n Campos-Olivas, Spanish National Cancer Center, Spain Editor: Ramo´n Campos-Olivas, Spanish National Cancer Center, Spain Received February 15, 2012; Accepted March 8, 2012; Published April 18, 2012 Received February 15, 2012; Accepted March 8, 2012; Published April 18, 2012 Copyright:  2012 Harbison 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 work was supported by NIH/NIA grants AG025440 and AG019391 (Dr. Eliezer) and GM066354 (NYSBC - New York Structural Biology Center,) (www. nih.gov) and NSF (National Science Foundation) agreement DMR-0454672 (NYSBC) (www.nsf.gov). 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: dae2005@med.cornell.edu PLoS ONE | www.plosone.org Assigning Backbone NMR Resonances for Full Length Tau Isoforms: Efficient Compromise between Manual Assignments and Reduced Dimensionality Nicholas W. Harbison1, Shibani Bhattacharya2, David Eliezer1* 1 Tri-Institutional Program in Chemical Biology, Department of Biochemistry, Weill Cornell Medical College, New York, New York, United States of America, 2 New York Structural Biology Center, New York, New York, United States of America Nicholas W. Harbison1, Shibani Bhattacharya2, David Eliezer1* 1 Tri-Institutional Program in Chemical Biology, Department of Biochemistry, Weill Cornell Medical College, New York, New York, United States of America, 2 New York Structural Biology Center, New York, New York, United States of America Nicholas W. Harbison1, Shibani Bhattacharya2, David Eliezer1* Citation: Harbison NW, Bhattacharya S, Eliezer D (2012) Assigning Backbone NMR Resonances for Full Length Tau Isoforms: Effic Manual Assignments and Reduced Dimensionality. PLoS ONE 7(4): e34679. doi:10.1371/journal.pone.0034679 Introduction The core of NFT is comprised solely of tau in various aggregate forms including, paired helical filaments and straight filaments [15,16]. Though the role of these filaments in the progression of the diseases remains unclear, genetic mutations in the tau gene were discovered in patients suffering from frontotemporal dementia and parkinsonism (FTDP) April 2012 | Volume 7 | Issue 4 | e34679 1 Efficient Manual NMR Assignment of Full Length Tau Figure 1. Diagram of the full-length tau protein, Tau441 showing the domain structure, alternately spliced exons (green) and the microtubule-associated repeats (R1–R4). Tau352 is the shortest full-length form of tau with no alternately spliced regions. TauK19 containing R1, R3 and R4 is shown above. doi:10.1371/journal.pone.0034679.g001 Figure 1. Diagram of the full-length tau protein, Tau441 showing the domain structure, alternately spliced exons (green) and the microtubule-associated repeats (R1–R4). Tau352 is the shortest full-length form of tau with no alternately spliced regions. TauK19 containing R1, R3 and R4 is shown above. doi:10.1371/journal.pone.0034679.g001 We employed G-Matrix Fourier Transformation NMR (GFT- NMR), one of several recently developed reduced-dimensionality approaches to acquiring NMR data that uses phase-sensitive joint sampling of the indirect dimension to produce subspectra onto which a G-matrix is applied prior to fourier transformation [33,34]. GFT has been applied to several experiments to allow for higher-dimensional data acquisition through the mathematical linear combinations of the subspectra [35–37]. We report the use of both established and new GFT acquisition sequences (unpub- lished) to obtain de novo resonance assignments for three tau constructs: tau K19, Tau352 (full length tau with all alternatively spliced regions absent), and Tau441 (the longest tau isoform), without the use of bootstrapping assignments obtained from small fragments [31] or of fully automated computational analyses [32]. The method presented here is a good compromise between these two previously demonstrated approaches. characteristic of well-ordered proteins. This causes resonance overlap which becomes even more pronounced when trying to study the full-length isoforms (Tau441). One way to overcome the resonance overlap in spectra of longer tau isoforms is to selectively label specific amino acids to obtain partial spectra that can be added together [30]. A second method involves the use of splicing information extracted from spectra collected using smaller fragments of the protein [31]. A third method is to use higher dimensionality experiments, which however can demand prohib- itive NMR data acquisition times. Introduction A solution to this problem has appeared in the past few years in the form of reduced dimensionality NMR experiments (RDNMR), which permit for the acquisition of high-dimensionality, and therefore well- dispersed, NMR data using shorter acquisition times. Most recently, very high dimensionality (6D and 7D) RDNMR data were used in combination with automated analysis to produce backbone resonance assignments for full length tau [32]. Here we report the use of lower dimensionality RDNMR experiments combined with manual analysis to accomplish the same goal. Manual analysis is difficult with very high dimensionality data, yet is often desirable for intervention, verification, and correction of assignments. Samples Recombinant proteins were expressed in Escherichia coli cells transfected with plasmids for TauK19 (gift from Drs. Peter Lansbury and Kenneth Kosik), Tau352 or Tau441 (gifts from Dr. Table 1. Acquisition Parameters of GFT-NMR Experiments. Table 1. Acquisition Parameters of GFT-NMR Experiments. Experiment name Linear combination of shifts (relative to carriers) measured in GFT TauK19{ Dimension: complex points/sw (ppm) Tau3521 Dimension: complex points/sw (ppm) Tau4411 Dimension: complex points/sw (ppm) {(4,3)D HNNCabCa V0(13Ca i/i21) 6 V1(13Ca i/i21) V0 (13Ca i/i21) 6 V1(13Cb i/i21) 15N:25/23 13C:55/80 15N:50/23 13C:64/80 15N:50/23 13C:64/80 {(4,3)D CabCa(CO)NHN V0(13Ca i21) 6 V1(13Ca i21) V0 (13Ca i21) 6 V1(13Cb i21) 15N:20/23 13C:50/80 15N:50/23 13C:64/80 15N:50/23 13C:64/80 (4,3)D HNNCaCO V0(13C9 i) 6 V1(13Ca i) V0(13C9 i21) 6 V1(13Ca i21) 15N:25/23 13C:75/65 15N:36/23 13C:75/60 Did not collect {(4,3)D HNNCOCa V0(13C9 i21) 6 V1(13Ca i21) V0 (13Ca i21) 6 V1(13C9 i21)* 15N:25/23 13C*:75/65 15N:36/23 13C:75/60 Did not collect (4,3)D HNN(Ca)NHN V0(15Ni+1) 6 V1(1Hi+1) V0(15Ni21) 6 V1(1Hi21) Did not collect 15N (t2):32/23 15N (t3):55/40 15N (t2):32/23 15N (t3):55/40 (4,3)D HNN(COCa)NHN V0(15Ni+1) 6 V1(1Hi+1) Did not collect 15N (t2):32/23 15N (t3):55/40 Did not collect {Data collected at 600 MHz. 1Data collected at 900 MHz. {Experimental details published in reference [35]. *For this experiment V(Ca) was detected in quadrature. doi:10.1371/journal.pone.0034679.t001 April 2012 | Volume 7 | Issue 4 | e34679 2 Efficient Manual NMR Assignment of Full Length Tau Figure 2. 13C/1H strip plots from 3D (left) and (4,3)D (right; additive experiment) HNCACB experiments, at 15N chemical shifts of 125.69 ppm and 125.67 ppm, respectively, for Tau K19 K257T. Shown are the spin systems for residues Gln307 and Glu338 (corresponding to tau352 residues 218 and 249) which can be better and more accurately resolved and identified using the GFT linear combinations. Positive peaks are shown in black and negative peaks are in red. doi:10.1371/journal.pone.0034679.g002 Efficient Manual NMR Assignment of Full Length Tau Figure 2. 13C/1H strip plots from 3D (left) and (4,3)D (right; additive experiment) HNCACB experiments, at 15N chemical shifts of 125.69 ppm and 125.67 ppm, respectively, for Tau K19 K257T. Shown are the spin systems for residues Gln307 and Glu338 (corresponding to tau352 residues 218 and 249) which can be better and more accurately resolved and identified using the GFT linear combinations. Positive peaks are shown in black and negative peaks are in red. doi:10.1371/journal.pone.0034679.g002 Guy Lippens), all under the control of a T7 promoter. Samples The FTDP- linked point mutation K257T was introduced into the TauK19 isoform using a stratagene mutagenesis kit. The proteins were purified as follows: cells were lysed by sonication in a solution containing 3 M Urea, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 10 mM Tris at pH 8.0 followed by ultracentrifugation at 40,000 rpm in a Beckman ultracentrifuge using a Ti 50.2 rotor. The supernatant was further purified using a streptomycin sulfate precipitation before being dialyzed against 25 mM Tris, 20 mM NaCl, 1 mM EDTA and 1 mM DTT and applied to a cation- exchange column and eluted by a NaCl gradient. Fractions containing tau were pooled, dialyzed against 5% acetic acid and purified on either a C18 (TauK19) or C4 (Tau352, Tau441) reverse-phase HPLC column using an acetonitrile gradient with 1% trifluoroacetic acid. The purified protein was lyophilized and stored at 220uC. Purity was verified by SDS-PAGE. 1% trifluoroacetic acid. The purified protein was lyophilized and stored at 220uC. Purity was verified by SDS-PAGE. NMR samples for TauK19 were prepared by resuspending lyophylized protein in PBS buffer (100 mM NaCl and 10 mM Na2HPO4 with 10% D20 at pH 7.4) and samples for larger isoforms in Tris buffer (REF) (25 mM Tris, 50 mM NaCl, 2.5 mM EDTA, 5 mM DTT, 5% D20 at pH 6.8). NMR samples for TauK19 were prepared by resuspending lyophylized protein in PBS buffer (100 mM NaCl and 10 mM Na2HPO4 with 10% D20 at pH 7.4) and samples for larger isoforms in Tris buffer (REF) (25 mM Tris, 50 mM NaCl, 2.5 mM EDTA, 5 mM DTT, 5% D20 at pH 6.8). PLoS ONE | www.plosone.org Data Collection All spectra were collected on Bruker AVANCE 600 and 900 MHz spectrometers equipped with z-axis gradient TCI CryoProbes (New York Structural Biology Center). For backbone resonance assignments of each protein a series of (4,3) GFT experiments were acquired in conjunction with standard suite of triple resonance experiments [38]. Details of the experiments are PLoS ONE | www.plosone.org April 2012 | Volume 7 | Issue 4 | e34679 3 Efficient Manual NMR Assignment of Full Length Tau Figure 3. Correlation plot of the Ca chemical shift values from the (4,3)D HNCACB experiment versus those from the standard 3D HNCACB for tauK19 K257T. The lease squares fit has a slope of 1.001 and an R2 value of 0.999. doi:10.1371/journal.pone.0034679.g003 Figure 3. Correlation plot of the Ca chemical shift values from the (4,3)D HNCACB experiment versus those from the standard 3D HNCACB for tauK19 K257T. The lease squares fit has a slope of 1.001 and an R2 value of 0.999. doi:10.1371/journal.pone.0034679.g003 values with only minor deviations seen in the aspartic acid region (Fig. 3). provided in Table 1. All of the experiments were collected with 1024 points, a spectral width of 10 ppm and an offset of 4.7 ppm in the 1H dimension. ( g ) Having confirmed that the K19 data from the GFT based experiments proved reliable, we collected an expanded set of GFT experiments at 900 MHz for the shortest and longest physiological tau isoforms, Tau352 and Tau441. Full assignments for Tau352 were initially made by ‘‘walking’’ through the (4,3)D HNNCabCa spectra (Fig. 4). Due to the high level of sequence redundancy coupled to the random coil structure in tau, just utilizing this standard practice did not provide an unambiguous set of assignments. We then refined the assignments further by using a linear combinations of amide nitrogen and proton shifts obtained from the intra- and inter-residue connectivities in the (4,3)D HNN(Ca)NHN and (4,3)D HNN(COCa)NHN experiments respec- tively (Table 1). The ability to correlate the 15N chemical shift of the ith residue with those of the i21 and i+1 residues in the HNCANNH type experiments [39] provides a crucial link in the assignment strategy of unfolded and/or large proteins [40]. After splitting the GFT data two distinct resonances, one for i21 and one for i+1, are present for each ith residue in the two subspectra. Data Collection Once the i+1 peak has been identified from the (4,3)D HNN(COCa)NHN experiment the 15N chemical shift for residue i21 in the f3 dimension (in ppm) is extracted simply by averaging the corresponding i21 15N shifts from the sub-spectra. For example, in Figure 5 the position in the f3 dimension of the peaks corresponding to the i21 residue (His94) are at 121.71 ppm and 116.34 ppm for the additive (panel D) and subtractive (panel E) combinations respectively. Averaging these values yields a 15N chemical shift of 119.03 which corresponds exactly to that of His94 (119.06 PPM) in the (4,3)D HNNCabCa experiment (panels A–C). The same holds true for i+1 peak with 15N shift of 123.56 ppm calculated from GFT data matching the Cab correlations at 123.59 ppm in the nitrogen dimension of the Data Analysis Data was processed with NMRPipe and analyzed using NMRViewJ. To prepare the secondary chemical shift plots, Ca chemical shift values were extracted from both the additive and subtractive (4,3)D HNNCabCa experiments. These values were then averaged to give the correct chemical shift for each residue. The expected random coil shift for each residue was then subtracted from the experimental chemical shift to obtain the secondary shift. Results In order to test the efficacy of applying GFT to the tau system, (4,3)D HNNCabCa and (4,3)D CabCa(CO)NHN spectra were acquired, at 600 MHz, for tau K19 with the K257T mutation enabling assignments for 98% of 1HN, 15N and Cab atoms for this construct. One of the major advantages in using the GFT-based experiments is that many peaks that were difficult to resolve in the standard 3D HNCACB are now better differentiated (Fig. 2) allowing for an increased level of both precision and confidence in the assignments [33]. This results from the fact that in general a linear combination of the chemical shifts of several nuclei is less likely to be degenerate between residues as illustrated in Figure 2. In non-GFT multidimensional experiments typically the resolution of overlapped peaks in the indirect dimensions is limited by the digital resolution, which becomes a significant issue for data acquired at higher fields. The overall agreement between the 3D and (4,3)D data is shown by a linear fit on a correlation plot of the chemical shifts obtained from the GFT versus standard HNCACB experiments. The plot shows good agreement between the GFT PLoS O April 2012 | Volume 7 | Issue 4 | e34679 PLoS ONE | www.plosone.org 4 Efficient Manual NMR Assignment of Full Length Tau Figure 4. 13C/1H strip plots showing inter-residue connectivities for residues Val128, Ser129, and Lys130 f additive (A) and subtractive (B) data for Tau352 at, at 15N chemical shifts of (1) 122.31 ppm (2) 120.14 pp Assignments were made by walking through the data from i to i21 spin systems. Positive peaks are shown in black and Dashed lines show peak connectivities. doi:10.1371/journal.pone.0034679.g004 Figure 4. 13C/1H strip plots showing inter-residue connectivities for residues Val128, Ser129, and Lys130 from the (4,3)D HNCACB additive (A) and subtractive (B) data for Tau352 at, at 15N chemical shifts of (1) 122.31 ppm (2) 120.14 ppm and (3) 124.10 ppm. Assignments were made by walking through the data from i to i21 spin systems. Positive peaks are shown in black and negative peaks are in red. Dashed lines show peak connectivities. doi:10.1371/journal.pone.0034679.g004 (4,3)D HNNCabCa experiment. By applying the N15 chemical shifts to build and confirm overlapping fragments comprised of i21, i, and i+1 residues we were successful in eliminating much of the uncertainty in assignments that arose, primarily, from sequence overlap and allowing for 97% completion of backbone assignments of Tau352 (Fig. PLoS ONE | www.plosone.org Results 6). same assignment strategy as with Tau352, using data from (4,3)D HNNCabCa and (4,3)D HNN(Ca)NHN experiments to determine the spin system connectivities, we were able to obtain 85% complete assignment of the backbone resonances 1HN, 15N and Ca for each residue (not counting the leading proline in pro-pro couplets, which can be assigned manually using other experiments, as noted in [32]), though that figure increases to 92% when including partial assignments of 1HN and either 15N or Ca chemical shifts (Fig. 7). Some partial assignments result from sequence repetition (i.e Ala89-Ala91) though most are caused by With confidence in this assignment strategy we moved on to making assignments for Tau441. The presence of all three alternately spliced exons increases the spectral overlap consider- ably though the quality of the data remains high. Following the PLoS ONE | www.plosone.org April 2012 | Volume 7 | Issue 4 | e34679 5 Efficient Manual NMR Assignment of Full Length Tau Figure 5. 13C/1H strip plots at 15N chemical shifts of 119.06 ppm (A), 116.36 ppm (B), and 123.59 ppm (C) extracted from the (4,3)D HNCACB experiment for Tau441 (only the additive strips are shown) corresponding to residues His94, Thr95, and Glu96. Dashed lines indicate the inter-residue connectivities. Assignments were verified using the additive (D) and subtractive (E) linear combinations of the (4,3)D HNNCANNH data as shown by 15N/1H strip plots below at the 15N chemical shift of Thr95 at 116.58. doi:10.1371/journal.pone.0034679.g005 Figure 5. 13C/1H strip plots at 15N chemical shifts of 119.06 ppm (A), 116.36 ppm (B), and 123.59 ppm (C) extracted from the (4,3)D HNCACB experiment for Tau441 (only the additive strips are shown) corresponding to residues His94, Thr95, and Glu96. Dashed lines indicate the inter-residue connectivities. Assignments were verified using the additive (D) and subtractive (E) linear combinations of the (4,3)D HNNCANNH data as shown by 15N/1H strip plots below at the 15N chemical shift of Thr95 at 116.58. doi:10.1371/journal.pone.0034679.g005 spectral overlap in one of the spectra, especially when dealing with charged residues (Asp, Glu, Lys). extended elements. Figure 8B shows the secondary shifts derived from our current GFT data for tau K19, plotted together with secondary shifts from previously published studies of the K19 construct [24]. PLoS ONE | www.plosone.org Results The two sets of secondary shifts are in good agreement, and both show the originally noted three regions of positive secondary shifts indicating three regions of helical propensity, confirming the accuracy of the GFT-derived shifts. Figure 8A shows the secondary shifts for Tau352. As we can see, in addition to the regions of helical propensity previously noted in the microtubule-binding repeats of K19, several other regions of tau Though tau is unstructured in solution, residual secondary structural propensities can be determined by plotting the secondary chemical shifts or the difference between the experi- mental chemical shift and the expected random coil value. In particular, Ca secondary chemical shifts are very sensitive to the w and y dihedral angles and are good reporters of the protein secondary structure [41,42]. Positive shifts are indicative of a- helical propensity while negative shifts represent b-strand or PLoS ONE | www.plosone.org April 2012 | Volume 7 | Issue 4 | e34679 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org 6 Efficient Manual NMR Assignment of Full Length Tau Figure 6. 15N/1H HSQC spectrum for free state tau352 with peak assignments given by residue numbers for the longest tau isoforms (tau441). The highly overlapped region (box) is enlarged. doi:10.1371/journal.pone.0034679.g006 Figure 6. 15N/1H HSQC spectrum for free state tau352 with peak assignments given by residue numbers for the longest tau isoforms (tau441). The highly overlapped region (box) is enlarged. doi:10.1371/journal.pone.0034679.g006 including Alzheimer’s disease and Frontemporal Dementia. Despite being a relatively large protein, containing 441 residues in its longest form, tau contains no well-structured domains, and the entire polypeptide is highly dynamic and disordered in solution. Tau has been the subject of NMR studies for several years, mostly applied to smaller fragments of the protein, but only recently has technology advanced sufficiently to allow the study of full-length tau constructs in more detail. Previous studies of the full-length protein reported partial backbone NMR resonance assignments of full-length constructs of tau obtained using selective labeling of specific amino acids [30] or full assignments obtained by bootstrapping from or splicing assignments of smaller constructs [31]. More recently, an automated assignment method using very high dimensionality (6D and 7D) experiments acquired using RDNMR methods such as automatic projection reconstruc- tion spectroscopy (APSY) was reported allowing nearly complete backbone assignments to be obtained that compared well with the previously reported results [32]. Results 352 exhibit significant groupings of positive secondary shifts, indicating a preference for helical formations. In addition, as might be expected, there are some regions of negative shifts around residues 151–158, 170–189, and 214–230 which are all found in the proline-rich region of the protein, as well as at residues 306–311, which correspond, as previously noted, to the VQIVYK PHF6 motif originally identified by the Mandelkows [43]. Figure 7. Primary sequence of Tau441 showing residues with complete Ca, HN, N assignments in blue and residues with HN and Ca or HN and N assignments in red. Residues in green can be logically assigned based on sequence overlap while the prolines highlighted in yellow cannot be assigned using these experiments. doi:10.1371/journal.pone.0034679.g007 Discussion NMR has long been a valuable tool in the study of protein structure, but is particularly valuable in the study of non-native protein states and especially of intrinsically disordered proteins – i.e. those that do not form well-defined native structure when isolated in solution. A well-known representative of this class of proteins is the microtubule-associated protein tau, which is of particular interest for its role in several neurodegenerative diseases, Figure 7. Primary sequence of Tau441 showing residues with complete Ca, HN, N assignments in blue and residues with HN and Ca or HN and N assignments in red. Residues in green can be logically assigned based on sequence overlap while the prolines highlighted in yellow cannot be assigned using these experiments. doi:10.1371/journal.pone.0034679.g007 April 2012 | Volume 7 | Issue 4 | e34679 PLoS ONE | www.plosone.org 7 7 Efficient Manual NMR Assignment of Full Length Tau Here we report the use of RDNMR (specifically of G-matrix comparable (93% assignment reported) to the Figure 8. CSI plots for Tau352 (A) full sequence and (B) microtubule binding region (bars) overlaid with data for Ta Positive deviations are indicative of a-helical conformations while negative deviations correspond to b-sheet structure. doi:10.1371/journal.pone.0034679.g008 Efficient Manual NMR Assignment o Figure 8. CSI plots for Tau352 (A) full sequence and (B) microtubule binding region (bars) overlaid with data for TauK19 (red line). Positive deviations are indicative of a-helical conformations while negative deviations correspond to b-sheet structure. doi:10.1371/journal.pone.0034679.g008 Figure 8. CSI plots for Tau352 (A) full sequence and (B) microtubule binding region (bars) overlaid with data for TauK19 (red line). Positive deviations are indicative of a-helical conformations while negative deviations correspond to b-sheet structure. doi:10.1371/journal.pone.0034679.g008 Here we report the use of RDNMR (specifically of G-matrix fourier transform techniques) with a relatively modest dimension- ality (4D) to obtain de novo backbone assignments for both the shortest and longest physiological tau isoforms: Tau352 and Tau441. Using two experiments acquired over only a few days we are able to make reliable protein assignments for 97% of Tau352 and 92% of Tau441 in a matter of weeks. The results are comparable (93% assignment reported) to the computer-aided APSY method, and this approach is considerably more efficient than the originally reported fragment bootstrapping method, which took several months to years to obtain and required many experiments on many constructs. PLoS ONE | www.plosone.org Efficient Manual NMR Assignment of Full Length Tau lower S/N of larger proteins and spectral artifacts, the general reliability of automatically generated assignments based on higher dimension data has not been established with absolute certainty, although excellent results obtained for the case of tau [32]. b-sheet structure is strongest at the previously identified PHF6 motif, as well as in the polyproline region, between the microtubule binding repeats and the helix preferring C-terminal region, and at the very N-terminus of the molecule, again consistent with observations made using the bootstrapped assignments. g [ ] Comparison of the GFT-NMR generated chemical shifts and secondary shifts with data previously published from standard experiments (Fig. 8B) shows an excellent level of agreement confirming the accuracy of shifts obtained using the GFT method. Indeed, GFT methods may be expected to provide more accurate shifts since the linear combinations increase the dispersion of the data and thereby decrease overlap. Our Ca secondary shifts calculated using the GFT-based assignments suggest that several regions of tau beyond the formerly identified microtubule-binding repeat regions exhibit a preference for helical structure. These include the C-terminal ,20 residues of the protein, which was also observed to populate helical conformations in a study of a 187- residue C-terminal fragment of the protein [25], short stretches around residues 200 and 240, and about 70 residues at the N- terminal end of the molecule starting at residue ,20. Helicity at position 240 has been observed in studies of a fragment containing this region (unpublished data) and is stabilized by CDK2/CycA3 phosphorylation [44]. Helicity in the N-terminal region is evident in the analysis of the bootstrapped assignments of the full length protein [29]. Interestingly, the I1 and I2 alternatively spliced inserts interrupt the relatively contiguous helical propensity observed for tau352, suggesting that splicing can influence the structural properties of tau. Evidence in our data for extended or Obtaining residue-specific NMR backbone resonance assign- ments is a crucial step in the detailed structural characterization of disordered proteins. The chemical shifts obtained from assign- ments of full-length tau isoforms report directly on the secondary structure propensities of the protein as described above and in previous reports [29]. Furthermore, the availability of such assignments has resulted in important new information regarding the interaction of the protein with microtubules [45], the effects of phosphorylation [44,46], and key features in tau aggregation [47]. References 16. 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Drewes G, Ebneth A, Preuss U, Mandelkow EM, Mandelkow E (1997) MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption. Cell 89: 297–308. 29. Mukrasch MD, Bibow S, Korukottu J, Jeganathan S, Biernat J, et al. (2009) Structural polymorphism of 441-residue tau at single residue resolution. PLoS Biol 7: e34. doi:10.1371/journal.pbio.1000034. 14. Garcia ML, Cleveland DW (2001) Going new places using an old MAP: tau, microtubules and human neurodegenerative disease. Curr Opin Cell Biol 13: 41–48. 30. Efficient Manual NMR Assignment of Full Length Tau Our results demonstrate a rapid and accessible method, avoiding either laborious studies of multiple sub-fragments or highly automated methods employing less accessible very high dimen- sionality data, easily executed on any modern spectrometer and requiring minimal computing power, for obtaining such assign- ments for large disordered proteins. Author Contributions Conceived and designed the experiments: NH SB DE. Performed the experiments: NH SB. Analyzed the data: NH. Wrote the paper: NH SB DE. Conceived and designed the experiments: NH SB DE. Performed the experiments: NH SB. Analyzed the data: NH. Wrote the paper: NH SB DE. Discussion Our approach facilitates manual intervention where necessary to check connectivities, resolve ambiguities, and confirm and correct assignments. Owing to April 2012 | Volume 7 | Issue 4 | e34679 PLoS ONE | www.plosone.org April 2012 | Volume 7 | Issue 4 | e34679 8 Efficient Manual NMR Assignment of Full Length Tau 31. Lippens G, Sillen A, Smet C, Wieruszeski J-M, Leroy A, et al. (2006) Studying the natively unfolded neuronal Tau protein by solution NMR spectroscopy. Protein Pept Lett 13: 235–246. 40. Freuh D, Sun Z, Vosburg D, Walsh C, Hoch J, et al. 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Kim S, Szyperski T (2003) GFT NMR, a new approach to rapidly obtain precise high-dimensional NMR spectral information. J Am Chem Soc 125: 1385–1393. doi:10.1021/ja028197d. 34. Szyperski T, Atreya HS (2006) Principles and applications of GFT projection NMR spectroscopy. Magn Reson Chem 44 Spec No: S51–60. doi:10.1002/ mrc.1817. 44. Sibille N, Huvent I, Fauquant C, Verdegem D, Amniai L, et al. (2011) Structural characterization by nuclear magnetic resonance of the impact of phosphoryla- tion in the proline-rich region of the disordered Tau protein. Proteins- doi:10.1002/prot.23210. 35. Atreya HS, Szyperski T (2004) G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment. Proc Natl Acad Sci USA 101: 9642–9647. doi:10.1073/pnas.0403529101. 45. Fauquant C, Redeker V, Landrieu I, Wieruszeski J-M, Verdegem D, et al. (2011) Systematic identification of tubulin-interacting fragments of the microtubule-associated protein Tau leads to a highly efficient promoter of microtubule assembly. J Biol Chem 286: 33358–33368. doi:10.1074/ jbc.M111.223545. 36. Xia Y, Zhu G, Veeraraghavan S, Gao X (2004) (3,2)D GFT-NMR experiments for fast data collection from proteins. J Biomol NMR 29: 467–476. doi:10.1023/ B:JNMR.0000034352.75619.3f. J 37. Atreya HS, Eletsky A, Szyperski T (2005) Resonance assignment of proteins with high shift degeneracy based on 5D spectral information encoded in G2FT NMR experiments. J Am Chem Soc 127: 4554–4555. doi:10.1021/ja042562e. j 46. References Smet C, Leroy A, Sillen A, Wieruszeski J-M, Landrieu I, et al. (2004) Accepting its random coil nature allows a partial NMR assignment of the neuronal Tau protein. Chembiochem 5: 1639–1646. doi:10.1002/cbic.200400145. 15. Wischik CM, Novak M, Edwards PC, Klug A, Tichelaar W, et al. (1988) Structural characterization of the core of the paired helical filament of Alzheimer disease. Proc Natl Acad Sci USA 85: 4884–4888. PLoS ONE | www.plosone.org April 2012 | Volume 7 | Issue 4 | e34679 9 Efficient Manual NMR Assignment of Full Length Tau Efficient Manual NMR Assignment of Full Length Tau Bibow S, Ozenne V, Biernat J, Blackledge M, Mandelkow E, et al. (2011) Structural impact of proline-directed pseudophosphorylation at AT8, AT100, and PHF1 epitopes on 441-residue tau. J Am Chem Soc 133: 15842–15845. doi:10.1021/ja205836j. 38. Cavanagh J (2007) Protein NMR spectroscopy. Academic Pr. 39. Weisemann R, Ru¨terjans H, Bermel W (1993) 3D triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins. J Biomol NMR 3: 113–120. 47. Bibow S, Mukrasch MD, Chinnathambi S, Biernat J, Griesinger C, et al. (2011) The dynamic structure of filamentous tau. Angew Chem Int Ed Engl 50: 11520–11524. doi:10.1002/anie.201105493. 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Feature Interaction in Smart Environments
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Feature Interaction in Smart Environments Luis Emanuel Neves Jesus Federal University of Bahia Institute of Mathematics: Universidade Federal da Bahia Instituto de Matematica Daniela Barreiro Claro (  dclaro@ufba.br ) Universidade Federal da Bahia Instituto de Matematica https://orcid.org/0000-0001-8586-1042 Tatiane Nogueira Rios Federal University of Bahia Institute of Mathematics: Universidade Federal da Bahia Instituto de Matematica Research Article Keywords: Feature Interaction, Internet of Things, Automate Detection Posted Date: April 13th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-398452/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Jesus et al. RESEARCH Feature Interaction in smart environments Luis E.N. Jesus1 , Daniela B. Claro1* and Tatiane N. Rios2 * Correspondence: dclaro@ufba.br FORMAS Research Group, Computer Science Department, Federal University of Bahia, Salvador-BA, Brazil Full list of author information is available at the end of the article 1 Abstract The Internet of Things (IoT) connects many devices daily together in the same environment. Each device may follow the set of rules from a static environment. A static environment is usually controlled by an expert who knows all the necessary rules to provide this environment. The violation of one rule can cause a feature interaction. A feature interaction occurs when two or more devices generate instability in an environment. In a dynamic environment like IoT, devices’ inclusion, and exclusion make it impossible for an expert to maintain all these rules up-to-date. It is necessary to provide an automatic solution to avoid violating these rules and maintain the environment’s good performance. Thus, this work introduces a new approach to detect a feature interaction in dynamic environments automatically. Almost all previous work provide static rules defined by an expert in a controlled environment to detect an interaction. However, this is not possible in dynamic environments because of the number of device interactions and the number of device connections in/out, which grow exponentially in IoT environments. We started with a lightweight systematic review to better position our research, and then we identified one gap to provide our solution. Thus, our method learns to detect the interactions based on data analysis and then automatically predict the device detections in IoT environments. Datasets were manually annotated. Experiments were performed, and results provide evidence that automatic detection of a set of device interactions is possible in similar or either in complementary domains. Keywords: Feature Interaction; Internet of Things; Automate Detection Introduction With the growth of the Internet, more devices are plugged and published over the world [25]. Internet infrastructure optimizes some routines and enables users to achieve some requirements outlining a new technological reality. Some envisioning systems are now available through the Internet due to the composition of devices, such as remote lock operation, home appliances operation via a network, and remote control of smart environments. This new reality empowers the connection among devices transparently, providing a new system of devices called the Internet of Things (IoT). Due to the diversity of smart devices, IoT defines a set of physical objects embedded with sensors and actuators, connected by wireless networks [7]. These devices are plugged into independent domain systems, such as industry, transport, health, and smart environments. Such devices’ exponential growth is estimated to reach over 75 billion smart devices in 2025 [1]. The evolution of the Internet has contributed to an increase in complex information systems. Such complexity is related to the communication and interaction between numerous components that occur statically and dynamically, requiring an Jesus et al. Page 2 of 23 effort to provide solutions with total interoperability [32]. However, the disordered growth of IoT allows the combination of devices that can generate unforeseen interactions or some side effects classified as: desirable and undesirable. These effects are called feature interaction [56]. Desirable features interactions occur when an effect modifies a behavior that improves some functionality [57]. For example, transaction management and database locking system cooperate to ensure data atomicity, consistency, isolation, and durability. Thus, it can be classified as a desirable feature interaction [20]. Undesirable feature interaction occurs when the effect provides an unstable behavior or an inconsistent data in a system [40]. Kolberg et al [27] treat undesirable feature interaction when a resource, when interacting with other resources, generates unexpected behavior in the environment. For example, a residence made up of security and entertainment services that share the DVD player within the security service. The process of recording a camera in the DVD player starts due to security rules. However, the entertainment service is triggered to record a TV program simultaneously, disabling the security setting. Such interaction causes an effect of assumption violation, since the activation of the entertainment service disables the security services, violating some rules, thus causing an undesirable feature interaction [56]. Features interactions have been a challenge for a long time [9]. Finding solutions that identify and solve such interactions is crucial to providing stable and intelligent systems capable of matching user behavior. Such solutions are subject to a critical step, which is the management of feature interactions[10]. A feature interaction occurs when the resource behavior is influenced by the presence of another resource (or a set of other resources) [6]. It is possible to identify a feature interaction when rules and interactions analyze sole resource behaviour. However, such a case can degrade due to resource failure or intervention from other resources. To avoid such scenarios, a FI can occur where there are at least two resources. Batory et al [8] discuss an intelligent building scenario with fire and flood controls with fire sensors on every ceiling and water sensors on every floor. In a given situation, when a sensor detects a fire, sprinklers are activated. On the other hand, observing the water on the floor, the flood sensor is activated, and sprinklers are deactivated. Such interaction between fire and flood controls generates, in this scenario, a conflict of interest that causes an untreated feature interaction [56]. This problem can let the building on fire and, as a consequence, physical and material damage. Several studies analyze feature interaction problems, but no one has done an exhaustive literature review focusing on IoT systems. However, our study is performed with a constraint: time[55]. Other systematic mapping studies have been conducted at the same time constraint. They adjectivally denominate such kind of systematic review as rapid systematic studies [54]. In this work, we follow the definition provided by Turner et al [55], designating our approach as a Lightweight Systematic Mapping Study (LSMS). Our LSMS provides a study on feature interactions within IoT to position our research solution in the state of the art. Seven research questions regarding feature interactions in IoT were defined. Our period to retrieve articles was between 2003 and 2019. This period was based on the systematic mapping conducted by Soares et al [53], carried on the software product Jesus et al. Page 3 of 23 line. As a result, 20 studies analyze feature interactions, and all of them were classified according to detection, resolution, or general analysis. Our findings identified some gaps on feature interaction in smart environments. Such gaps motivated us to focus on the automatic detection of feature interaction within a smart environment. Our approach concerns a machine learning model to predict a feature interaction in a given environment automatically. Datasets from smart environments were manually annotated to provide a classification of feature interaction. We validate our model against a set of manual rules designed by the experts. New desirable and undesirable interactions were found by our model. Such findings were validated against our definition of feature interaction, obtaining satisfactory and valid results. This paper is organized as follows. Section 2 presents the formal concepts of feature interactions in IoT. Section 3 describes our LSMS. Section 4 discusses some related work. Section 5 identifies some research gaps. Section 6 introduces our feature interaction detection model. Section 7 describes our experiments’ setup. Section 8 presents our findings. Section 9 discusses some of our results. Finally, section 10 concludes and show some envisioning work. Formal definitions The term feature interaction emerged in the 1980s to describe everyday situations in telecommunications[8]. Since then, this term has been spread to several areas, such as software engineering, smart grid, automotive, IoT, and smart environments [49]. The literature presents many definitions of feature interaction. Such definitions address aspects that vary from resource behavior from the user’s point of view. Some definitions are described as follows: • A feature interaction occurs when we integrate two or more resources to produce a new product, but together they do not work as intended [24]; • A feature interaction is the way a resource or a set of resources modifies or influences another resource in its behavior in the system [30]; • A feature interaction is a situation in which two or more characteristics exhibit unexpected behavior that does not occur when the characteristics are used in isolation [5]; • A feature interaction occurs when a combination of specific characteristics has an unexpected influence on performance [52]; and • A feature interaction is the combination of two or more services that perform correctly and individually to obtain unexpected results when combined [31]. As far as there is still no consensus regarding the definition of a feature interaction, we redefine it from the point of view of smart environments. Feature Interaction for smart environments Considering the previous definitions of feature interactions [22] and the characteristics of IoT, the main factor for FI in IoT is the resource [11, 51]. We redefine the resource considering the context of IoT as follows: Definition 1 (Resource (R)) is a composition of devices (D) with expected behaviors (Ce) that individually meet the needs of the system: Jesus et al. Page 4 of 23 D, Ce |= R (1) A feature interaction occurs when two or more resources R1 and R2 interact considering that R1 and R2 satisfies each individual behavior in isolation and R1 and R2 do not satisfy the expected behavior when combined, that is: (D1 , Ce1 |= R1 ) ∧ (D2 , Ce2 |= R2 ) 6→ D1 ∧ D2 , Ce1 ∧ Ce2 , R1 ∧ R2 (2) Consider the smart home scenario, with the following features: fire control (R1) and window control feature (R2). The resource R1 is composed of two devices (smoke sensor (D1) and window (D2)), for an expected behavior (fire (Ce1)). The expected result of Ce1 is the opening of D2 in case of smoke detection by D1). The resource R2 is composed of devices (D2 and light sensor (D3)) and two expected behaviors (airy environment (Ce2) and night security (Ce3)). The expected result of Ce2 is opening of D2 in case of detection of luminosity D3. The expected result of Ce3 is the closing of D2 in case of lack of light by D3. The resources R1 and R2 alone have the expected behaviors achieved. In the event of interaction between the R1 and R2 resources, an undesirable feature interaction of the type Multiple Action Interaction is generated, compromising at least one of the resources and the D2 is in an undefined state. Figure 1 exemplifies the occurrence of an undesirable feature interaction in the smart home scenario. The fire and window control features work well in isolation. However, in the interaction between resources, a feature interaction can be generated. Based on this circumstance, if a fire occurs at night, the fire control triggers the window to open. However, because it is in the night, the window control triggers the window’s closing to maintain nighttime safety behavior. This interaction ends up interfering with the functioning of the window and, consequently, with the smart home’s security. Definition 2 (Feature Interaction in Smart environments) A feature interaction can occur in a smart environment when two or more resources (Ri ) interact, causing unexpected behavior; that is, a resource affects the behavior of the other resources and, consequently, the behavior of the environment. Type of FI Regarding the type of interaction, a feature interaction can be classified into four types [27]. In this work, only the Multiple Action Interaction (MAI) was carried out. • Multiple Action Interaction (MAI): occurs when two resources try to control the same device. Depending on the rules from the environment, this interaction can compromise at least one resource or device, which can remain in an indefinite state; Jesus et al. Page 5 of 23 Figure 1 Undesirable Feature Interaction. Considering a dynamic environment with thousands of resources (Ri ) and devices (Dj ) moving automatically in and out, the detection of a feature interaction may be hard to be followed by an expert. In this context, an investigation in the literature was carried out to evaluate methods and techniques adopted to detect a feature interaction. Our Lightweight Systematic Mapping This systematic mapping study aimed to provide an overview of the detection of FI area and to discover the gaps, authors, research groups, and its trends [44, 45]. Some systematic reviews had imposed restrictions. Turner et al [55] proposed a set of procedures to develop and document the conduction of systematic literature reviews emphasizing stakeholders with time constraints. Such particularity imposes a restriction to conduct the whole SMS process. Such SMS has achieved positive results, and it is called the Lightweight Systematic Mapping Study. Other approaches adopted similar restrictions but with different names. Rapid systematic reviews[54] aim to offer simplified alternatives to the traditional systematic review process. Some of the peculiarities in the rapid review process are focused on reaching the set of research questions. In contrast to the classic approach that focuses on more rigid steps, these approaches have provided a systematization to carry out an overview if a specific domain. Thus, in this work, we adopted the Lightweight Systematic Mapping Study (LSMS). Methodology Our LSMS is divided into 3 phases: planning, conduction and documentation as depicted in Figure 2. Jesus et al. Page 6 of 23 The planning phase includes the preliminary scope of the literature and aims to identify and refine the objectives of the study. A protocol is developed, and so the criteria for selecting articles. The protocol serves as the basis for the execution of the conduction and documentation phases. The protocol design was built and validated into two-fold: a) with researchers from the IoT domain and b) a manual execution of the protocol to observe which papers were retrieved. Figure 2 Our phases of LSMS (adapted from [44]). The conduction phase deals with the execution of the mapping, and it is responsible for searching and selecting papers based on the research questions defined in the protocol. For this execution, two strategies for searching articles were defined: automatic search and manual search. Additional filtering was employed according to the inclusion and exclusion criteria [44]. The documentation phase defines a classification and information extraction scheme to answer the planning phase’s research questions. Results from the detailed analysis of each primary study are presented on this lightweight systematic mapping. Our LSMS results were divided into quantitative and qualitative answers. Due to space reduction, our quantitative results were placed into Appendix . Research Questions The methodology followed to construct the research question was based on [47], adapting the PIOC method: P Population: IoT; I. Intervention: Detection of feature interaction; O Result: Methods and techniques for detecting features interaction; C Application: Smart environments. Jesus et al. Page 7 of 23 This LSMS aims to identify feature interaction approaches in IoT, defined by the combination of the characteristics in PIOC. Consequently, seven secondary questions were identified, grouped into two-folds: three questions with quantitative aspects and four questions with qualitative aspects. The quantitative aspects focus on providing some statistical knowledge of FI on IoT. On the other hand, qualitative aspects aims to analyze the detection methods already proposed in the literature, how they were evaluated, and the domains in which FI in IoT was employed. RQ1. Quantitative Questions RQ1a. Which countries are researching feature interaction in IoT? Conduct a survey of the countries working with feature interaction in IoT; RQ1b. How many articles have been published in conferences and journals? Collect the mediums which are getting more publicity on FI in IoT; RQ1c. How many universities and/or research groups are working on FI in IoT? Find out which universities are researching FI in IoT. RQ2. Qualitative Questions RQ2a. What feature interaction solutions have been proposed on IoT environments? Investigate the solutions that involve the detection and management of feature interaction in IoT to bring together research communities and industries focused on such solutions; RQ2b. What are the methods employed to detect feature interaction? Categorize the methods for the detection of FI in IoT, addressing the levels of syntactic interoperability, semantic or pragmatic [50], being formalized through rules, access policies, formal notation, among others; RQ2c. What are the domains that apply FI in IoT? Examine which domains are usually provided to detect FI in IoT. This question was subdivided into three extraction possibilities: (i ) the domain, (ii ) the detection of specific or generic feature interaction, that is if the solution can be applied in other domains, and (iii ) in which domains the solutions need to consider specific contexts; RQ2d. What are the evaluation methods of feature interaction in IoT? In this question, the objective is to investigate applied empirical research (case study, controlled experiment, and formal validation). Allowing to carry out the evaluation and validate the area’s maturity in detecting feature interaction in IoT. Research Strategy A three-stage strategy to select the papers: automatic search, manual search, and full paper (Figure 4). The first stage automatic search was against a set of search engines presented in Table 1 [53]. All studies published from 2003 to 2019, including conferences, newspapers, and workshops in the field of feature interaction in the IoT were retrieved. The search string is composed of keywords according to the Kitchenham and Charters [26] method: (i ) Feature Interaction and (ii ) Internet Of Things. These words served as a basis to build the search string, adding synonyms and alternative words, applying the operators AND and OR to perform the joining of the terms, according to Table 2. Jesus et al. Page 8 of 23 Table 1 Digital search engines. ACM Digital Library IEEE Xplore Scopus Engineering village Science direct Springer http://dl.acm.org/ http://ieeexplore.ieee.org/ http://www.scopus.com/ http://engineeringvillage.com/ http://www.sciencedirect.com/ http://springer.com/ Table 2 Search string. (“feature interaction” OR “feature-interaction”) AND (“Smart” OR “Smart City” OR “Smart House” OR “Sensor” OR “Actuator” OR “Smart Home” OR “Intelligent Industry” OR “Intelligent Home” OR “Internet of Things” OR “IoT” OR “FoT” OR “IIoT” OR “FoG” OR “Sensors” OR “Actuators”) A set of 661 publications was gathered after applying the search string (Table 1). Figure 3 presents the distribution of the articles per vehicles. Most articles were from Springer, followed by Science Direct with 56.6% and 29.5%, respectively. Figure 3 Percentage of publications by search engines. All 661 publications were submitted to the inclusion and exclusion criteria, as described in Figure 4. . • Inclusion criteria – Written in English (F6); – Publications with search strings in the title, abstract or keywords (F4); – Publications at conferences or in newspapers (F3). • Exclusion criteria – Gray literature (such as tutorials and manuals) (F2); – Short articles (less than six pages) (F5); – Studies that do not have feature interaction on the IoT (F7); – Duplicate studies (F1). The analysis of the first filter was related to duplicate articles (F1). Due to the indexing process used by search engines, the F1 filter removed 52 articles. The second (F2) and the third filter (F3) removed 266 articles, 262 of which were removed from F2 and 4 from F3, composed of gray literature and articles published in workshops. Then, F4 filter was applied which corresponds to reading the title, abstract Jesus et al. Page 9 of 23 Figure 4 Filters from LSMS. and keywords that do not have feature interactions in IoT, resulting in an exclusion of 304 publications. The F5 filter, concerning short publications contributes to the removal of 6 publications. The last filter (F6) was related to publications that were not written in the English language, where there were no removals. At the end of the first stage (automatic search), 95.1% of the publications were discarded, resulting in 33 articles for the second stage (manual search). The second stage (manual search) was applied to obtain a sample of the references from the set of filtered articles. This stage was divided into (i ) manual search in conferences and (ii ) snowballing. The conferences were chosen by IoT domain. More 3 new publications were added to our approach. The snowballing [58], a manual search on the references of relevant studies, was conducted based on 36 pre-filtered publications (33 from the automatic search and 3 from the manual search). This process resulted in the selection of 2 more publications to be added. Thus, the manual search and automatic search summarized 38 articles for the third stage (full read). Jesus et al. Page 10 of 23 The third stage (full read) was tackled by the F7 filter. Publications were full reading, removing manuscripts that: (i ) are not about feature interaction in IoT; (ii ) do not have any discussion on feature interaction; or (iii ) do not provide an assessment of the approach. At the end of the full reading, 20 publications were selected, and made available at https://www.bibsonomy.org/user/fi-smart-envirn. Our findings As we described previously, our LSMS results were divided into two-fold: quantitative and qualitative results [16]. Due to space purposes, all Quantitative results were placed into Appendix . Qualitative results The qualitative assessment stage focuses on the interpretation of the study, with an emphasis on subjectivity [14]. In addition to allowing the understanding and context that influence the results from each article, it provides a reflection based on questions regarding the solutions, methods, domains and evaluation of each article. Feature interaction solutions. Different solutions were developed to deal with feature interactions in different environments on IoT. In this study, five categories were identified: • framework is a reusable mini-architecture that applies to the development of solutions in a specific and limited domain [48]; • method means the definition of systematized procedures for the description and explanation of phenomena [14]; • algorithm a sequence of computational steps that transforms inputs into outputs [12]; • architecture is a process that defines a conceptual framework of the elements of a process [41]; and • model defines the flow of activities, actions, artifacts and organization of activities to be carried out [48]. Among these categories, framework and method stood out, with 70% of the solutions (Figure 5). In a framework approach, Maternaghan and Turner [35] provided a mini-architecture by a set of rules based on access policies to the set of resources in a smart home. Pedersen et al [43] describes the method through model checking, applied in a home automation environment to finding feature interactions. Methods for detecting feature interactions. Our LSMS retrieved three main groups of methods to detect a FI in IoT: • algorithms present a sequence of computational steps that transforms input(s) into output(s) [12]; • formal notation is a set of rigorous engineering practices, along with welldefined rules that are based on mathematical theory [19]; and • rules are rules that require something to be done within the established conditions [21]. Figure 6 shows the distribution of each group. The algorithm [37] details the actions to perform a task. Jesus et al. Page 11 of 23 Figure 5 Solutions to detect features interactions. The formal notation group corresponds to methods that adopted a formal method or a Z notation to describe their approach. The rules group is based on the conceptualization of standards defined by experts. Among the papers, those that presented a variation in the application of the rules were either grouped, as they follow the same concept. IoT Domain. With the development of intelligent components and devices, several domains are perceived from the papers: • automotive is a feature pack that provides advanced functionality for a vehicle [23]; • home automation consists of managing a set of resources for a home [43]; • sensors are devices that detect signals from physical phenomena (such as thermal, electrical or magnetic radiation) and convert them into digital values [33]; • embedded systems consist of any system that has a built-in microprocessor, with the exception of equipment easily identified as computers. This definition of embedded systems includes smart objects [29]; • smart building is composed of equipment to automate constructions, with the objective of facilitating the real-time monitoring and control of each function [42]; • smart grid is an enhanced electrical grid that collaborates with information technologies for efficient electricity distribution [15]; and • smart home is a residence equipped with smart technologies designed to provide personalized services to users [34]. Figure 7 presents the most addressed domains in comparison with the solutions highlighted in the selected works. Smart home obtained 37.5% of incidences, followed by home automation with 25%. Jesus et al. Page 12 of 23 Figure 6 Detection methods of features interactions. Among the solutions, framework and method are concentrated in the domains of home automation and smart homes. The other approaches were spread into other domains. With the diversity of solutions and domains, it is possible to provide new approaches for detecting feature interaction in specific domains that can have significant results. These solutions can work with the applicability of knowledge acquisition from data. Evaluation of feature interaction. Regarding the evaluation of feature interaction, two categories were mapped based on the 20 studies: • experiment is used to support the formulation of new theories. An experiment is characterized by the composition and execution of several activities that include changing input data, parameters, programs or even their combination [36]; and • case study is an empirical investigation and comprises a comprehensive method, with the logic of data planning, collection and analysis [59]. The predominant method was the case study with 13 publications, while the experiments had 5 appearances. Two studies did not evaluate their method regarding FI aspects. Research Opportunities With the evidence from our LSMS, the methods for detecting feature interactions were mostly focused on the applicability of rules and the absence of data analysis. Looking at the scenario of smart environments, a large number of devices are connected at the same time. According to [17], in 2025, it is projected that 41.6 billion devices will be connected to the internet and will generate 79.4 zetabytes. This considerably increases the complexity of determining priority policies manually. Jesus et al. Page 13 of 23 Figure 7 Amount of solution per domain. In dynamic environments such as the Web and IoT, the inclusion and exclusion of devices make it hard to maintain manual rules up-to-date, increasing the potential for feature interactions. In such environments, with the inclusion and exclusion of devices and resources dynamically, there is a need to treat feature interactions in an automated manner. One possible way to automate is to identify patterns from the IoT datasets to detect a feature interaction[46] [3] [39] [28]. We observed other transversely gaps that must have an attention from the research community. The lack of basic artifacts for replicating the experiments, such as a benchmark of feature interaction is an important envisioning work to enable the evolution of FI area. Another aspect is concerning a general solution to different domains instead of having specific approaches to solve an isolated problem. Such solutions can carry out an automate manner to detect FI due to the exponential growth of devices in IoT systems. Finally, there is no measure concerning temporal detection of a feature interaction in IoT. This is an important measure due to the mobility in IoT environments. Concerning these research opportunities, we develop a new model to detect patterns of feature interactions in IoT datasets. We faced some challenges related to Jesus et al. Page 14 of 23 the annotated smart environments data set, the definition of FI in the context of the IoT, as well as experts to annotate the dataset. Feature Interaction Detection Model Considering our previous definition of FI in IoT, we introduce our approach with the focus on data to automate the detection of patterns. This detection enables an assessment of the existence of feature interaction and its associations. We follow the knowledge discovery process from [18] and we depicted all three stages in Figure 8. Figure 8 Method for detecting feature interaction in smart environments. The first step consists of pre-processing the data. Firstly, we identified the attributes referring to devices and resources. Secondly we analysed the sampling, the balancing and data cleaning, applying techniques to incomplete and inconsistent data. Afterwards, we did a data transformation to convert and normalized some of them[18]. The second stage identifies patterns based on interpreting the predictive and descriptive model algorithms. The predictive model aims to learn from previously labeled instances by creating a model capable of predicting unknown values. The descriptive model, on the other hand, has the function of finding patterns and trends interpretable by humans to describe the data [13]. The third step aims to consolidate the knowledge discovered with the validation and verification of conflicts related to the detected features interactions, (“desirable” Jesus et al. Page 15 of 23 or “undesirable”), based on the formalization adopted by the method or with the help of the rules defined by an expert. Next sections describe our experiments to evaluate our approach. Experiment Setup Two experiments were carried out to evaluate the detection model thru a dataset. The first experiment consists of detecting features interactions, and the second of identifying new FI and validating existing ones. The experiments were carried out in a synthetic database in the context of smart environments, in an environment capable of providing temperature, safety and pleasant energy efficiency for the possible residents and with the presence of undesirable features interactions. Synthetic Dataset Our dataset simulates a smart home made up of smart devices. An algorithm was built to input events for each device with other random events to add undesirable feature interaction data sets. Based on the LSMS, all datasets were made up of binary attributes. Alemdar et al [2] explored the data to identify the pattern behavior of their residents and served as a parameter for defining the data domain of our dataset. Our dataset consists of devices arranged in a smart home: (i ) fan, (ii ) curtain, (iii ) air conditioning, (iv ) temperature sensor, (v ) window, (vi ) door, (vii ) smoke detector, (viii ) photosensor, (ix ) anemometer, and (x ) rain sensor, totaling 10 devices, with random data events generated by the algorithm. The dataset contains 11 attributes, 10 of which refer to devices and resources, and one ‘target‘ attribute, labeled by an expert. The values of the ‘target‘ attribute of a feature interaction are determined to be desirable or undesirable. The dataset consists of 12.931 observations, divided into 6.422 by events with desirable feature interactions and 5.969 with undesirable feature interactions. Seven rules were defined by the expert Ci , (i = 1...7), as follows, to meet the user’s need. • C1 - Fan: IF temperature between 15o C and 24o C THEN Turn on the Fan; • C2 - Curtain: IF Wind speed greater than 20 km/h THEN Close the curtain; • R3 - Air conditioning: IF temperature greater than or equal 24o C THEN Turn on the Air Conditioning; • C4 - Window : IF rain THEN Close the window; • C5 - Curtain: IF temperature greater than or equal to 20o C THEN Open the curtain; • C6 - Door : IF smoke THEN Open the Door; and • C7 - Window : IF smoke THEN Open the window. Machine Learning Algorithms Two experiments were carried out: identification of FI (experiment A); and detection of rules for FI (experiment B). Both experiments generated a generic model to detect feature interaction. To identify FI, algorithms for the classification task were employed [38]: (i ) KNN, (ii ) Naive Bayes, (iii ) Decision tree, (iv ) Random Forest and (v ) SVM. Such algorithms are commonly employed to identify patterns from IoT data [3, 28, 39, 46]. Jesus et al. Page 16 of 23 To detect the rules, an algorithm for the association task were employed [18]: Apriori. This algorithm was adapted to finding new interactions of characteristics and validating the rules defined by the expert. The evaluation of the A experiment occurred through the analysis of the confusion matrix, precision, recall and F-measure. On the other hand, in experiment B, the evaluation process was based on the rules generated by the algorithm and on the comparison of them with the expert’s rules. Results are presented and discussed in the following sections. Results We present our results considering each experiment performed. Firstly, we describe the Experiment A considering the classification task and then we present our second experiment B which aims to discover the rules based on an associate task. Experiment A This experiment aims to predict the class considering if it is a desirable or undesirable FI. We provide a generic model based on a 10 fold cross-validation. Results are presented in Table 3 and we can observe that the Decision Tree based algorithm gathered the best accuracy. Table 3 Accuracy, recall and F-measure of predictive methods from our dataset. Algoritmos Random Forest Decision Tree KNN SVM Naive Bayes Accuracy 0,9883 0,9899 0,9720 0,881 0,8658 Recall 0,9997 0,9952 0,9725 0,9517 0,8635 F-measure 0,9939 0,9920 0,9722 0,9188 0,8646 We provided additional experiments to guarantee no overfitted approach. We analyze the distribution of the data in each fold of the cross-validation. Our findings make evidence that no overfitting was performed due to medians close to 1 and with low dispersion. Results from the decision tree algorithms were above 98%, and a new investigation of data distribution within a cross-validation approach was employed. Figure 9 depicts the boxplot graph with the data distribution, which the average is close to 1, and there is a little dispersion, thus achieving a good distribution. Figure 9 Distribution of the 10-folds of the algorithm Random Forest e Decision Tree Jesus et al. Page 17 of 23 Experiment B Experiment B was performed by an Associate algorithm, Apriori, which is based on finding a subset of the most frequent items in a data set [18]. The main goal of such an experiment was to compare the rule set from the dataset and the rule set defined by the expert. This algorithm enables the discovery of new feature interactions that have not been previously defined by the expert. As it is a balanced dataset, built to apply the predictive model, the extraction of the rules adopted a threshold of 0.4 and 0.8 for support and confidence parameters, respectively. Nine (9) rules (Table 4) were generated taking these thresholds, of which two of these rules are the same defined by the expert (R6 and R7 ). • R6 - Door : IF smoke THEN Open the Door (associated with rule 8 in Table 4); and • R7 - Window : IF smoke THEN Open the Window (associated with rules 1 in Table 4) of the association algorithm. Table 4 Rules generated by the association algorithm with 0.4 support and 0.8 confidence. Lines 1 and 8 indicate the aforementioned rules defined by the expert (R6 and R7 ). 1 ... 8 9 Rules Smoke =>Window ... Smoke =>Door Door =>Smoke We experimented with decreasing the threshold to catch more rules, adopting support at a rate of 0.2 and confidence at a rate of 0.6. Thus, 40 rules were generated with this new threshold. From this set of 40 rules, 3 of them were the same as those defined by the expert (R3 , R6 ,R7 ) which are (25, 32, 39) in Table 5. • R3 - Air conditioning: IF temperature greater than or equal 24o C THEN turn on the Air Conditioning (associated with rule 25 in the Table 5); • R6 - Door : IF smoke THEN Open the Door (associated with rule 39 in Table 5); and • R7 - Window : IF smoke THEN Open the Window (associated with rule 32 in Table 5). Table 5 Rules generated by the association algorithm with 0.2 support and 0.6 confidence. Rule 3 indicates an undesirable feature interaction and rules 25, 32 and 39 indicate the rules defined by the expert (R3 , R6 and R7 ). 1 2 3 ... 25 ... 32 ... 39 40 Rules Rain, Smoke =>Door Door, Rain =>Smoke Rain, Smoke =>Window ... Temperature = Warm =>Air conditioning ... Smoke =>Window ... Smoke =>Door Door =>Smoke In addition, the model detects a new undesirable feature interaction at rule 3 (in green) in Table 5, involving rain and smoke sensors. The interaction between these sensors generates a feature interaction of the type Multiple Action Interaction, since the smoke detection service triggers the window opening and the rain detection service triggers the window closure. Jesus et al. Page 18 of 23 Discussion The association and decision tree algorithms validated the rules defined by an expert. In addition to providing the detection of feature interaction in a smart environment, this approach envisions the possibility to create a generic model of feature interaction detection for different domains. Such models provided interoperability in smart environments based on data through machine learning algorithms, without relying on architecture or a set of rules defined by an expert to obtain a stable environment. Results were based on a synthetic dataset that may have generated an undesirable bias. It is important to note that the origin of this dataset is real. The problem is that the generation of real datasets must cause undesirable feature interaction, so it is necessary to set up a real environment with inclusion and exclusion of devices dynamically and then evaluate more complex rules. The dataset analysis indicated that the occurrence of feature interaction is low, in which the detection of feature interaction can be addressed in data anomaly. Another relevant aspect is related to dataset agglutination, that is, the union of different domain datasets from smart environments. The challenge stands out in combining the attributes of different domains by building a model capable of detecting feature interaction without reapplying the learning process and adjustments in the model. Conclusion We provide a lightweight systematic mapping with an overview of the feature interaction approaches in the IoT domain, delimiting the construction of a feature interaction detection model from data. The detection model used classification and association algorithms to detect feature interaction in IoT environments through data analysis. The model can help domain experts adjust the rules, mainly detecting undesirable feature interaction. The analysis for the detection of feature interaction was performed manually. In a real environment, this analysis would be carried out by an expert, a complex task when dealing with millions of thousands of devices working together. As future work, we intend to improve our approach to consider rules with a significant probability and provide a benchmark dataset to guide other works on this challenging task. Regarding the aspects inherent to the detection of feature interaction in smart environments, the present work demonstrated that it is possible, with the acquisition of knowledge from a set of data, to detect feature interaction without the presence of an expert. In addition, other transversal contributions can be listed: (i ) a detection model of feature interaction based on the data, (ii ) a data set annotated with feature interaction undesirable, and (iii ) a rule detection model with the purpose of assisting domain experts to improve the addressed environment. Appendix A Quantitative results Authors in [4] define that the data in a quantitative search must be expressed with numerical measures focusing on the relationship between variables. Based on these Jesus et al. Page 19 of 23 variables, issues related to countries, conferences, journals, universities, and research groups investigating feature interactions in the IoT were analyzed. Regarding the countries that are developing research with features interactions in IoT, 60.65% of the publications are from Asia. Each country is counted from the researcher’s publication. These results condition Japan and China (with 18 and 10 publications, respectively) as references in this research area (Figure 10). Figure 10 Number of countries with research in the area of feature interaction. Regarding the year of publication, Figure 11 shows the distribution of articles over the years. Studies were published in journals, magazines, and conferences from 2003 to 2019. In 2008 there was a higher incidence of publications, followed by the years 2007, 2011, 2015, and 2017 with two publications per year; that is, the research area is evolving. However, this evolution is timid since only 1 or 2 articles were published annually among those selected. Considering the paper venues, we observed that 65% of the publications occur in journals (Table 6). Table 6 Journals of selected articles Feature Interactions in Software and Communication Systems IX Electronic Communications of the EASST 2008 Computer Networks 2004 IEICE TRANSACTIONS on Information and Systems IET communications arXiv IEEE Transactions on Emerging Topics in Computing IEEE Communications Magazine Pervasive and Mobile Computing Computer Networks Science of Computer Programming Transactions on Emerging Telecommunications Technologies Jesus et al. Page 20 of 23 Figure 11 Number of publications per year. Chongquing University University of Stirling 8 8 Kobe University 7 NTT Network Innovation Lab. University of Waterloo 5 5 Aalborg University 4 University of Disburg-Essen Osaka Institute of tecnology 3 3 Osaka University 3 University of Calgary 2 0 2 4 6 8 Figure 12 Universities and Group researching feature interaction. In publications by universities or research groups, quantitative aspects were explored to obtain an overview of where the techniques and solutions are being developed and evolved. The evaluation process to define the institutions was based on the number of researchers linked to each institution. Figure 12 stands out: Chongquing University and University of Stirling. Jesus et al. Page 21 of 23 In the quantitative aspects, an annual variation of publications, particularly in journals, the concentration on the Asian continent, and either their universities and laboratories consolidating the evolution of the feature interaction research area in the context of IoT. Acknowledgements Not applicable Funding Not applicable List of Abbreviations IoT - Internet of Things FI - Feature Interaction LSMS - Lightweight Systematic Mapping Study MAI - Multiple Action Interaction Availability of data and materials The datasets generated and analysed during the current study are available in the FORMAS Github repository, https://docs.google.com/spreadsheets/d/1XC0k4oUniha63_TvrKxehhsMqdFkfe_s/edit#gid=1596292566 Ethics approval and consent to participate Not applicable Competing interests The authors declare that they have no competing interests. Consent for publication All authors consent for publication. Authors’ contributions LENJ analyzed and interpreted data regarding the feature interaction. DBC and LENJ discussed the formal definitions and the LSMS. TNR and LENJ performed the evaluation from machine learning algorithms. DBC and LENJ was a major contributor in writing the manuscript. All authors read and approved the final manuscript. 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Turner M, Kaur R, Brereton P (2008) A lightweight systematic literature review of studies about the use of pair programming to teach introductory programming. In: PPIG, p 21 56. Weiss M, Esfandiari B, Luo Y (2007) Towards a classification of web service feature interactions. Computer networks 51(2):359–381 57. Wilson M, Magill EH, Kolberg M (2005) An online approach for the service interaction problem in home automation. In: 2th IEEE Consumer Communications and Networking Conference (CCNC), IEEE, Las Vegas, NV, USA 58. Wohlin C (2014) Guidelines for snowballing in systematic literature studies and a replication in software engineering. In: 18th International Conference on Evaluation and Assessment in Software Engineering (EASE), p 38 59. Yin RK (2015) Estudo de Caso-: Planejamento e métodos. Bookman editora Figures Figure 1 Undesirable Feature Interaction. Figure 2 Our phases of LSMS (adapted from [44]). Figure 3 Percentage of publications by search engines. 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Deep Neural Networks and Transfer Learning on a Multivariate Physiological Signal Dataset
Bioengineering
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  Citation: Bizzego, A.; Gabrieli, G.; Esposito, G. Deep Neural Networks and Transfer Learning on a Multivariate Physiological Signal Dataset. Bioengineering 2021, 8, 35. https://doi.org/10.3390/ bioengineering8030035 Keywords: multivariate data; physiological signals; signal processing; artificial intelligence; deep neural networks; transfer learning Academic Editor: Antonio Lanata Andrea Bizzego 1 , Giulio Gabrieli 2 and Gianluca Esposito 1,2,3,* Andrea Bizzego 1 1 Department of Psychology and Cognitive Science, University of Trento, 38068 Rovereto (Trento), Italy; andrea.bizzego@unitn.it 1 Department of Psychology and Cognitive Science, University of Trento, 38068 Rovereto (Trento), Italy; andrea.bizzego@unitn.it g 2 Psychology Program, School of Social Sciences, Nanyang Technological University, Singapore 639798, Singapore; GIULIO001@e.ntu.edu.sg 2 Psychology Program, School of Social Sciences, Nanyang Technological University, Singapore 639798, Singapore; GIULIO001@e.ntu.edu.sg 3 2 Psychology Program, School of Social Sciences, Nanyang Technological University, Singapore 639798, Singapore; GIULIO001@e.ntu.edu.sg 2 Psychology Program, School of Social Sciences, Nanyang Technological University, Singapore 639798, Singapore; GIULIO001@e.ntu.edu.sg 3 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 639798, Singapore * Correspondence: gianluca.esposito@ntu.edu.sg Psychology Program, School of Social Sciences, Nanyang Technological University, Singapore 639798, Singapore; GIULIO001@e.ntu.edu.sg 3 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 639798, Singapore * Correspondence: gianluca.esposito@ntu.edu.sg Singapore 639798, Singapore; GIULIO001@e.ntu.edu.sg 3 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 639798, Singapore * Correspondence: gianluca.esposito@ntu.edu.sg 3 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 639798, Singapore * Correspondence: gianluca.esposito@ntu.edu.sg * Correspondence: gianluca.esposito@ntu.edu.sg Abstract: While Deep Neural Networks (DNNs) and Transfer Learning (TL) have greatly contributed to several medical and clinical disciplines, the application to multivariate physiological datasets is still limited. Current examples mainly focus on one physiological signal and can only utilise applications that are customised for that specific measure, thus it limits the possibility of transferring the trained DNN to other domains. In this study, we composed a dataset (n = 813) of six different types of physiological signals (Electrocardiogram, Electrodermal activity, Electromyogram, Pho- toplethysmogram, Respiration and Acceleration). Signals were collected from 232 subjects using four different acquisition devices. We used a DNN to classify the type of physiological signal and to demonstrate how the TL approach allows the exploitation of the efficiency of DNNs in other domains. After the DNN was trained to optimally classify the type of signal, the features that were automatically extracted by the DNN were used to classify the type of device used for the acquisition using a Support Vector Machine. The dataset, the code and the trained parameters of the DNN are made publicly available to encourage the adoption of DNN and TL in applications with multivariate physiological signals. Citation: Bizzego, A.; Gabrieli, G.; Esposito, G. Deep Neural Networks and Transfer Learning on a Multivariate Physiological Signal Dataset. Bioengineering 2021, 8, 35. https://doi.org/10.3390/ bioengineering8030035 bioengineering bioengineering Article Deep Neural Networks and Transfer Learning on a Multivariate Physiological Signal Dataset Andrea Bizzego 1 , Giulio Gabrieli 2 and Gianluca Esposito 1,2,3,* 1. Introduction DNNs are a family of Machine Learning methods that rely on the use of modular architectures, based on multiple nonlinear processing units (layers), to extract high-level patterns from data. Thanks to the hierarchical structure of the layers, DNN progressively obtains high-level features from low-level representations [13], thus trans- forming input data into a multi-dimensional representation useful to solve the classification task [14]. However, optimally training DNN typically requires a much larger amount of data than statistical Machine Learning models: the Transfer Learning (TL) approach over- comes this issue partially. The key idea of TL is leveraging on DNN which were already trained on different domains or datasets, and adapting them to the new task, by partially re-trained using data from the new task [15]. Thanks to the availability of large-scale data and pre-trained architectures [16,17], the TL approach boosted the adoption of DNN in several disciplines and out-performed previous state-of-art Machine Learning models and, in some cases, human expertise [18]. DNNs based on Convolutional Neural Networks (CNN) are currently the state-of- the-art models in several image classification applications that adopt an “end-to-end” paradigm: i.e., images are directly processed by the CNN without prior processing (e.g., feature extraction). The adoption of DNN and CNN in applications based on medical data (bio images and physiological signals) is rapidly growing, with a wide range of applications [19–23]. pp DNN and CNN have also been applied to physiological signals. For instance, a DNN, was successfully employed to design a biometric identification signal based on Electrocar- diogram (ECG) [24], or to identifyventricular and supraventricular ectopic beats [25]. In a work by Xu et al. [26], a DNN was effectively employed to classify the type of heartbeat patterns (e.g., normal beat, arrhythmia) from raw ECG. Similar procedures were employed on other physiological signals: Yu and Sun [27] used a DNN to classify emotions from pha- sic and tonic components of the Electrodermal Activity (EDA), while Mukhopadhyay and Samui [14] employed DNN to classify limbic movements from Electro-myogram (EMG) sig- nals. A recent review by Rim et al. [28], surveyed the application of DNN on four different Physiological signals (EMG, ECG, Electro-oculogram, and Electro-encephalogram) in both single- and multivariate datasets. The datasets were drawn from both public and private repositories and were identified based on the methodology employed to analyze the data. 1. Introduction Received: 3 February 2021 Accepted: 3 March 2021 Published: 6 March 2021 In medicine and other medical sciences, physiological recordings are widely employed to monitor and assess the health status of patients [1,2]. Since physiological signals are directly controlled by the Autonomic Nervous System, they are also less influenced by social inferences and explicit behavioral responses [3]. The analysis of multivariate physiological signals allows the investigation of psycho-physiological regulatory mechanisms of the individual from different perspectives and enables researchers to study how humans react to external stimuli and adapt to environmental changes [2,4,5]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. p g However, employing multivariate physiological signals in the research process comes at a cost: first, physiological measurements are highly sensitive to the presence of noise and artifacts; second: each type of physiological signal requires specialized hardware and tools for data collection and processing. For these reasons, the typical approach to multivariate signals analysis involves some steps of visual inspection to assess the quality of the recordings (e.g., to discriminate between usable and unusable samples), and select the most appropriate tools and procedures for analysis [6]. In an attempt to reduce the subjectivity of these steps, Artificial Intelligence (AI) methods have been recently introduced. AI was employed to automatize the phases of preprocessing and analysis of recorded signals, therefore increasing the reproducibility and reducing the likelihood of human errors. In Gabrieli et al. [6], different Machine Learning models proved to be Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/bioengineering Bioengineering 2021, 8, 35. https://doi.org/10.3390/bioengineering8030035 Bioengineering 2021, 8, 35 2 of 12 able to efficiently discriminate between usable and unusable samples in multiple infants’ fixation time studies, while numerous studies employed AI models on preprocessed physiological signals, for instance, to identify ventricular hypertrophy [7], arrhythmia [8], muscle fatigue [9], and stress [10]. able to efficiently discriminate between usable and unusable samples in multiple infants’ fixation time studies, while numerous studies employed AI models on preprocessed physiological signals, for instance, to identify ventricular hypertrophy [7], arrhythmia [8], muscle fatigue [9], and stress [10]. One of the most promising AI methods is the use of Deep Neural Networks (DNN) [11,12]. 1. Introduction Inclusion criteria for being reviewed included the employment of DNN for clinical applica- tions and emotion recognition. The use of DNN on physiological signals can effectively and efficiently support clinicians in their work, for example by identifying heart diseases [29,30] or brain disease [31–33]. However, the majority of DNN employed in physiological signals analysis target only a specific type of physiological signal. As a consequence, they are not appropriate for analyzing multivariate datasets, which are rapidly becoming the standard in medicine and neuroscience [34], and are application-specific, meaning trained models cannot be easily used for TL [35]. 2.1. Peripheral Physiological Signals 2.1. Peripheral Physiological Signals In this study, we consider six peripheral physiological signals that are commonly used in multivariate experimental settings. In this study, we consider six peripheral physiological signals that are commonly used in multivariate experimental settings. The Electrocardiogram (ECG) and Photo-plethysmogram (PPG) are both recorded to measure the cardiac activity. ECG employs surface electrodes to detect the electrical potential of cardiac cells, PPG is based on optical sensors, and it measures blood volume changes in the microvascular bed of tissue [36]. Both signals can be used to investigate the Heart Rate Variability: how the heart rate changes in response to physiological needs and external stimuli, under the control of the Autonomic Nervous System. EDA is the measurement of changes in the electrical properties of the skin that occur in response to sweat secretion. Since the sweat glands are under the control of the sympathetic nervous system, EDA is used to investigate emotional arousal [37,38]. EMG is the study of the muscles’ electrical signals, which are generated by the contraction and relaxation of muscles. Analysis of the EMG is used for both clinical and research purposes since it can be used in biomechanics, motor, and neuromuscular physiology, and emotion recognition [39–41]. Information about individuals’ respiration patterns can provide researchers with details about the status of both the central and peripheral nervous system [42]. A respiratory signal (RESP) can be recorded using an elastic band positioned around an individual’s chest to measure the relative volumetric expansion. Finally, we consider the acceleration signals (ACC) used to detect body movements which are used in clinical settings, not only to study conditions associated with impaired movement but also stereotypical motor movements in children with autism spectrum disorders [43–45]. 1.1. Aim of This Study The aim of this study is to obtain a DNN to classify multivariate data and extract physiological patterns. From the clinical perspective, we aim at setting the base for the development of computer-assisted diagnostics based on physiological signals, to keep pace with other clinical disciplines based on imaging. This study provides three main practical contributions to facilitate the adoption of DNNs for multivariate physiological signals: (i) We apply a DNN to classify six types of physiological signals: Electrocardiogram, Electrodermal activity, Electromyogram, Pho- toplethysmogram, Respiration, and Acceleration. Signals were composed into a dataset Bioengineering 2021, 8, 35 3 of 12 3 of 12 of 813 physiological signals collected from 232 subjects using four different acquisition devices. (ii) We test the efficacy of the TL approach. Based on high-level features extracted from the trained DNN, we applied a standard Machine Learning method to classify the type of device. Although standard TL approaches based on re-training of weights might be more effective, our example demonstrates that the DNN extracts signal patterns that can be ported to other domains. (iii) We make available the training dataset as well as the DNN architecture with the pre-trained weights, with the aim of encouraging and facilitating the adoption of DNN with multivariate physiological signal datasets. 2.2. Multivariate Signals’ Datasets Physiological signals used in this study were obtained from four publicly available datasets of multivariate signals: the Database for Emotion Analysis using Physiological signals (DEAP, reference [46], available at http://www.eecs.qmul.ac.uk/mmv/datasets/ deap/, accessed on 5 March 2021), the Wearable and Clinical Signals dataset (WCS, ref- erence [47], available at https://doi.org/10.21979/N9/42BBFA, accessed on 5 March 2021), a dataset used to investigate Synchrony In Dyads (SID, reference [48], available at https://doi.org/10.21979/N9/O9ADTR, accessed on 5 March 2021), and a dataset used to investigate the Perception of Implicit Aesthetic Pleasure (PIAP, reference [49], available at https://doi.org/10.21979/N9/YCDXNE, accessed on 5 march 2021). p g The DEAP dataset was created to investigate the association between physiological response and emotional stimuli and provide data for Machine Learning applications aimed at emotion recognition. From the list of peripheral signals included in the DEAP, we selected the EDA and the PPG. All signals were collected with the same sampling rate of 512 Hz using the Biosemi ActiveTwo acquisition unit. The DEAP dataset provides signals of 32 subjects. j The WCS dataset was created to compare signals collected with clinical-grade and wearable devices and to test and validate algorithms to assess the quality of the data. Specifically, in this study, we consider five different types of signals, collected with 2 different devices: (i) the FlexComp acquisition unit, operating at a sampling rate of 2048 Hz, used to collect four physiological signals: ECG, EDA, PPG, and RESP; and (ii) the Empatica Bioengineering 2021, 8, 35 4 of 12 E4, a wrist-band used to collect PPG (64 Hz), EDA (4 Hz), and ACC (32 Hz). The WCS dataset provides signals of 18 subjects in two experimental conditions: at rest (n = 18) and while performing an in-place walking (n = 18). E4, a wrist-band used to collect PPG (64 Hz), EDA (4 Hz), and ACC (32 Hz). The WCS dataset provides signals of 18 subjects in two experimental conditions: at rest (n = 18) and while performing an in-place walking (n = 18). The SID dataset was used to investigate the physiological synchrony between members of dyads with different levels of relationship. We considered three types of physiological signals (ECG, EDA, EMG) provided by the SID dataset, all collected with the FlexComp acquisition unit, operating at a sampling rate of 2048 Hz. The SID dataset provides signals of 124 subjects (62 dyads). 2.2. Multivariate Signals’ Datasets The PIAP dataset was used to investigate the perceived aesthetics of web-pages and images and it contains several physiological signals of young adults (N = 59, 33 females, Mean age = 21.52 years) while they were asked to assess the aesthetic appeal of websites and of emotional pictures. Specifically, for this dataset, we consider three types of physio- logical signals: ECG, EDA, EMG (corrugator supercilii), recorded with a sampling rate of 1000 Hz using an open-source biosignal acquisition platform [50]. In this study, a subset of 22 participants has been selected. p p Overall (Table 1), for this study, we composed a dataset with 6 types of signals from 4 acquisition devices and 232 subjects, totaling 813 samples of physiological signals. The dataset was divided into two partitions, preserving the percentage of samples for each class. The first partition (n = 609, 75% of the dataset) was used for training the AI models, the second partition (n = 204, remaining 25% of the dataset) was uniquely used for testing. Using separate partitions for training and testing AI models is a common practice to ensure unbiased evaluations of the predictive performances. Table 1. Number of samples per type of signal and source dataset, with sampling frequency (in Hz) and devices. Dataset ECG EDA EMG PPG RESP ACC N. of Samples Device DEAP - 32 (512 Hz) - 32 (512 Hz) - - 64 Biosemi WCS 36 (2048 Hz) 36 (2048 Hz) - 36 (2048 Hz) 36 (2048 Hz) - 144 Flexcomp - 36 (4 Hz) - 36 (64 Hz) - 36 (32 Hz) 108 E4 SID 128 (2048 Hz) 128 (2048 Hz) 128 (2048 Hz) - - - 384 Flexcomp PIAP 44 (1000 Hz) 26 (1000 Hz) 43 (1000 Hz) - - - 113 Bitalino Total 208 258 171 104 36 36 813 Number of samples per type of signal and source dataset, with sampling frequency (in Hz) and devices. Table 1. Number of samples per type of signal and source dataset, with sampling frequency (i mples per type of signal and source dataset, with sampling frequency (in Hz) and devices. 2.4. Deep Learning Architecture 2.4. Deep Learning Architecture 2.4. Deep Learning Architecture The DNN architecture used in this study has three sequential components (Figure 1): (i) a convolutional branch; (ii) a Long Short-Term Memory (LSTM) module; a Fully Connected Head (FCH). Figure 1. Diagram representing the architecture of the Deep Neural Network (DNN) used in this study. Left: the complete network with the three components (the Convolutional Branch, the Long Short-Term Memory module, and the Fully Connected Head) and parameters used for each layer. The input signal is processed by the DNN to output the probability of belonging to each of the six signal types considered in the study. Right: structure of a general Convolutional Block with Nin input channels Nout output channels. Figure 1. Diagram representing the architecture of the Deep Neural Network (DNN) used in this study. Left: the complete network with the three components (the Convolutional Branch, the Long Short-Term Memory module, and the Fully Connected Head) and parameters used for each layer. The input signal is processed by the DNN to output the probability of belonging to each of the six signal types considered in the study. Right: structure of a general Convolutional Block with Nin input channels Nout output channels. The convolutional branch is composed of 4 convolutional blocks, each composed of a convolutional layer (with kernel size set to 3), a normalization layer (using batch normal- ization [52]), a Rectified Linear Unit [53], and a pooling layer based on maximum (with kernel size set to 2). The convolutional layer of each block expands the number of channels: the first expands from 1 to 32 channels, the second to fourth blocks duplicate the number of channels up to 256 channels for the fourth block. The structure of the convolutional branch is directly inspired by Convolutional Neural Networks that are largely used on images and videos. The main difference is that, in our study, we use a one-dimensional, layer—which is more appropriate to process signals—instead of two-dimensional layers which are used for images. After the 4 convolutional blocks, an additional pooling layer is used to compute the average of the convoluted (multichannel) signal at 10-time points, thus obtaining a temporal sequence of 10 elements, each with 256 values (one for each channel). p q The LSTM module [54,55] is a recursive layer that is used in Neural Networks to leverage the specific properties of sequential data. 2.3. Pre-Processing To reduce the memory size of the dataset, the original signals were cropped into segments with a maximum length of 300 s. The selected segment corresponded to the first 300 s when the signal was shorter than 600 s; otherwise, to the portion between 300 to 600 s. This was to avoid the initial portions of the signal, where noise and artifacts are more frequent because the preparation of the subject or the experiment is still ongoing. In line with the end-to-end approach, only two pre-processing steps were performed: normalization and resampling. Both steps were required to comply with the DNN archi- tecture. The normalization was necessary to uniform the range of the input signal: all signals were standardized by subtracting the mean value and dividing by the standard deviation. The resampling was performed to overcome the heterogeneity of sample rates of the different signal acquisition devices. Signals were resampled with a resampling rate of 100 Hz, which was empirically selected as a trade-off: while higher sampling rates would allow for more informative signals, lower sampling rates would increase the speed of the training and processing. A key step to improve the performance of DNN is data augmentation, the procedure of enriching an existing dataset, by randomly modifying its datapoints. Data augmentation prevents the network from relying on instance-specific patterns, thus preventing overfitting and improving generalizability [51]. In our implementation, every time a signal is used to train the network, we randomly select the 10 s length portion of the signal that is actually used as input to the DNN. When used to test the network performance, the central 10 s length portion is selected instead. 5 of 12 Bioengineering 2021, 8, 35 2.5. Analytic Plan This study is composed of two separate Machine Learning experiments: the first focuses on training the DNN to classify the type of signals, the second uses the trained DNN as a feature extractor and uses a Support Vector Machine (SVM) to classify the type of device used to collect the signals. Both experiments are based on the same dataset and use the train partition to train the model, and the test partition to assess the performance of the trained model. In the first experiment, the signals in the train partition are used to optimize the parameters of the DNN. The DNN adopts an end-to-end pipeline: the signals are directly processed by the sequence of the three components to output the probability that the signal belongs to each type of signal. The type with higher probability is considered the result of the classification. The training was iterated for 400 epochs; in each epoch, all the signals in the training dataset are used, randomly divided into batches of 32 signals. The DNN parameters are optimized to minimize the Cross-Entropy Loss [56] between the true and predicted types of signal. For the optimization of the DNN parameters, we used the Adam optimizer [57], with an initial learning rate of 0.001 which was divided by 10 every 50 epochs. The performance of the DNN was evaluated using the multi-class Matthew Correlation Coefficient (mMCC) and the Confusion Matrix for both the train and test partitions [58]. In the second experiment, we provide a demonstrative example of how the previously trained DNN can be useful for TL approaches. TL relies on the fact that many of the features that are automatically extracted by the DNN can be useful for other applications. All signals in the dataset were then processed by the convolutional branch and LSTM module to obtain 100 features for each signal. These features, which are expected to synthesize patterns useful to classify the type of signal, are transferred to classify the type of device that was used to collect the signal. A SVM model, with linear kernel and regularization parameter C = 1, was trained on the features extracted from the signals of the train partition and evaluated on features from signals of the test partition. The performance of the SVM was evaluated using the mMCC and the Confusion Matrix for both the train and test partitions [58]. 2.5. Analytic Plan The implementation of the mMCC and SVM models and training of the SVM model were based on the scikit-learn Python package (v0.23.2, reference [59]). The implementation of the DNN and training methods, including the loss function and optimizer, were based on pytorch (v1.3.1, reference [60]). 2.6. Code and Data Availability The code, the dataset with pre-processed signals, and the parameters of the trained DNN are available at https://gitlab.com/abp-san-public/dl-signal-classification (accessed on 5 march 2021). 2.4. Deep Learning Architecture In the implementation used in this study, the LSTM module has an output size set to 100 and a single layer. The LSTM processes the output of the convolutional layer one element at a time: each time, the output of the LSTM is updated considering the new element in the sequence and the output obtained from the previous element. The output of the LSTM module is a vector with 100 elements, which is the result of the recursion on the last element of the sequence. Bioengineering 2021, 8, 35 6 of 12 The final component of the DNN is the FCH, which linearly combines the 100 elements from the LSTM module to output a vector of 7 elements, on which a Softmax is finally applied to compute the probability of belonging to any of the 6 types of signals. pp p p y g g y yp g The first two components (the convolutional branch and the LSTM module) are used as automated feature extractors in the TL experiment. Each signal is sequentially processed by both components, and the vector with 100 elements resulting from the LSTM module is the vector of the extracted features. 3. Results The network trained to classify the type of signal achieved a mMCC of 0.931 on the train partition and 0.938 on the test partition. The confusion matrices (Figure 2) showed a high class accuracy (>96.2%) for all types of signals, except RESP. 7 of 12 Bioengineering 2021, 8, 35 Figure 2. Confusion Matrices that show the result of the classification of the type of signal, on the train (left) and test (right) partitions. On the diagonal (green), the numbers of correctly classified samples for each type of signal. Out of the diagonal, in red, the numbers of mis-classified samples. Figure 2. Confusion Matrices that show the result of the classification of the type of signal, on the train (left) and test (right) partitions. On the diagonal (green), the numbers of correctly classified samples for each type of signal. Out of the diagonal, in red, the numbers of mis-classified samples. Overall, the reported performances were similar in both the train and test partitions, suggesting that the training process was able to efficiently detect and extract characterizing patterns and avoid over-fitting. However, we observed that the model was not able to correctly classify the RESP signals, which were confounded with EDA. The first two components of the trained network were used to extract the features for the classification of the type of device. To visualize the results of the feature extraction, we apply a dimensionality reduction on the extracted features, based on two-dimensional Principal Component Analysis (PCA). We observed (Figure 3) that the features generated by DNN efficiently separate the type of signals; however, we also recognized the lack of separation between RESP and EDA signals, which motivates the low-class accuracy for the RESP type. Figure 3. Representation of the input samples in the two-dimensional space defined by the Principal Component Analysis (PCA) of the extracted features. Left: colored by type of signal; Right: colored by type of device. Figure 3. Representation of the input samples in the two-dimensional space defined by the Principal Component Analysis (PCA) of the extracted features. Left: colored by type of signal; Right: colored by type of device. The SVM model, trained on the extracted features to predict the type of device, achieved a mMCC of 0.638 on the train partition and of 0.609 on the test partition; the confusion matrices reported similar performances in both partitions (Figure 4). Figure 4. 4. Discussion In this study, we demonstrated the use of DNN in the classification of multivariate physiological data. In particular, a key aspect of our study is that it focuses on six different types of physiological signals, instead of a limited number of physiological signals like similar studies have done [61–63]. We successfully employed DNN to classify the type of physiological signal, with a very good overall performance, demonstrated by the high mMCC on both the train and test partitions. Only one type of signal, the respiratory signal (RESP), was incorrectly classified, possibly due to the low number of samples available in the dataset. Future studies should focus on having a richer and more balanced dataset. Notably, the Convolutional branch and the LSTM components automatically learned to synthesize features that encode the relevant patterns to allow for discriminating the type of signals, as shown by the results of the PCA analysis. y Moreover, we provided a proof of concept to show how multivariate analysis can benefit from the TL approach, enabled by the use of DNN. The trained DNN was used to obtain generalized features of the signals: although created to encode signal patterns relevant to discriminate the type of signals, the extracted features were also useful to classify the type of device. Specifically, we adopted one of the simplest TL approaches: the extracted features were used to train a SVM model, achieving an overall mMCC of 0.638 on the train partition and of 0.609 on the test partition. We did not investigate how the achieved performance depends on the partitioning of signal types into the different devices in the original dataset; as well as it was out of the scope of this study to improve the performance of the TL task. We posit that, by adopting more specialized TL approaches, for instance, re-training only the FCH component, will allow better performance. The ability to classify the type of device itself may not be of immediate use. Instead, our TL implementation aimed at providing an example of how the approach facilitates the adoption of DNN in different and transversal applications based on multivariate data. Drawbacks of using TL should be considered as well, especially when applied to biological or physiological data. If features learned by DNN usually outperform hand- crafted features, they are usually harder to interpret in biological terms [64–67]. 4. Discussion When the emphasis on interpretability is a key factor, the use of TL and DNN, in general, should be paired with more standard approaches. To be efficiently transferred to other domains, DNN should extract generic patterns. This is the reason we believe that existing examples of DNN applied to physiological signals, being trained on specialized tasks, would not allow transfer learning. Our DNN, being trained on a more generic task and on a multivariate dataset, would a better candidate to be transferred to other domains. However, more studies are needed to clarify this aspect: in the literature, only a few examples made use of DNN on multivariate physiological signals, and even fewer adopt TL. Finally, the DNN can be easily improved by adding more physiological signals from other databases and by considering new types of signals, for instance, brain signals such as from Electro-encephalogram [46] and functional Near Infra-Red Spectroscopy [68,69]. 3. Results Confusion Matrices that show the result of the classification of the type of device, on the train (left) and test (right) partitions. On the diagonal (green), the numbers of correctly classified samples for each type of device. Out of the diagonal, in red, the numbers of mis-classified samples. Figure 4. Confusion Matrices that show the result of the classification of the type of device, on the train (left) and test (right) partitions. On the diagonal (green), the numbers of correctly classified samples for each type of device. Out of the diagonal, in red, the numbers of mis-classified samples. Bioengineering 2021, 8, 35 8 of 12 The device with the lowest class accuracy was the Biosemi (13.7% on train, 7.7% on test), while the most frequent classification error was attributing a signal to the FlexComp device. Again, the motivation can be found by observing the results of the PCA analysis (Figure 3): since the same device is used to collect multiple types of signals, the extracted features, which well separated the types of signals, are not as optimal to discriminate the devices. 4.2. Implications With a specific focus on multivariate physiological signals, the DNN approach adopted in this study can be easily adapted to simultaneously process multiple types of signals: convolutional branches can be used to extract a set of features from each type of signal that are then merged by the FCH [19]. The trained DNN has its immediate application with novel signal processing platforms (e.g., https://datagrok.ai, accessed on 5 March 2021), where the recognition of the type of signal is the key step to initiate or recommend the correct processing procedure. In turn, we highlight the importance of sharing the weights of trained networks and benchmark datasets; these two practices have largely contributed to the adoption of DNN in other disciplines. However, compared to other medical fields (e.g., Nuclear Medicine, Histology and Pathology, Microscopy), the use of DNN on multivariate physiological signals is still quite unexplored. This is probably due to the lack of extended datasets and paucity of foundational applications, which, as in the case of medical imaging, could then be adapted to the specific use cases and improved. By providing a benchmark dataset and a working generalized DNN, we aim at providing a significant contribution to the development and diffusion of DNN for multivariate physiological signals. The use of multivariate data in clinical applications mostly pertains to diagnostic and monitoring tasks (for instance in Intensive Care Units), where the efficacy of AI approaches can be fully exploited. It should be noted, however, that, to be reliably applied in real-time clinical applications, AI models require a delicate and demanding phase of training, where the availability of good datasets and computing resources are the key. The availability of general datasets and pre-trained models that can be adapted to more specific tasks using TL represents therefore a fundamental resource. Regarding the dataset used in this study, having different tasks and subjects from different populations is a key feature of our approach, since it allows for obtaining a more general model. In the case of clinical applications, this model can be further improved using a specialized dataset with subjects from the same population while executing the same task. 4.1. Limitations This study has some methodological issues, motivated by constraints and limitations due to the exploratory nature of the study and characteristics of the dataset. Bioengineering 2021, 8, 35 9 of 12 First, in the preprocessing steps, we resampled all signals to a sampling rate of 100 Hz. While for some signals this rate is well above the one required to obtain significant physiological information (e.g., RESP, EDA), for other signals (e.g., ECG, EMG), this likely caused information losses. However, since the purpose of the network was to classify the type of signal, not to extract clinical or physiological evidence, we deem that the potential loss of information is acceptable, considering the gain in terms of increased processing speed. However, approaches targeting diagnostic tasks should consider higher sampling rates. p g Second, we demonstrated the TL approach on the same dataset used for the main DNN, to reduce the overall complexity of the study. In reality, TL applications usually consider not only a different task or target but also different datasets. Third, compared to networks used for medical images, the neural network used in this study is not very deep: it is composed of 20 layers (including normalization and activation layers), for a total number of 274,366 parameters. As a reference, the VGG-16 network [70] contains 39 layers, for a total of 138.4 M parameters. Using a relatively low number of layers was motivated by the fact of having a low number of samples (VGG-16 was originally trained on a dataset with 1.3 M images). If required in future applications, our architecture can be properly improved, provided that a dataset with an adequate sample size is used. References 1. Bizzego, A.; Battisti, A.; Gabrieli, G.; Esposito, G.; Furlanello, C. pyphysio: A physiological signal processing library for data science approaches in physiology. SoftwareX 2019, 10, 100287. [CrossRef] 2. Wagner, J.; Kim, J.; André, E. 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Implications While we believe the dataset and the pre-trained model developed in this study represent a key contribution to the adoption of AI for clinical applications, further studies are required to provide reliable guidelines and examples about how AI and TL should be efficiently applied to achieve reliable results. 10 of 12 Bioengineering 2021, 8, 35 10 of 12 Author Contributions: Conceptualization, A.B.; methodology, A.B. and G.G.; software, A.B. and G.G.; resources, G.E.; data curation, G.G. and A.B.; writing—original draft preparation, A.B. and G.G.; writing—review and editing, A.B., G.G. and G.E.; supervision, G.E. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the 2015 NAP Start-up Grant M4081597 (GE) from Nanyang Technological University Singapore and the Ministry of Education Tier-1 Grant (GE). 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: All the data used in this study were obtained from publicly available datasets: the Database for Emotion Analysis using Physiological signals (DEAP, reference [46], available at http://www.eecs.qmul.ac.uk/mmv/datasets/deap/, accessed on 5 March 2021), the Wearable and Clinical Signals dataset (WCS, reference [47], available at https://doi.org/10.21979 /N9/42BBFA, accessed on 5 March 2021), a dataset used to investigate Synchrony In Dyads (SID, reference [48], available at https://doi.org/10.21979/N9/O9ADTR, accessed on 5 March 2021), and a dataset used to investigate the Perception of Implicit Aesthetic Pleasure (PIAP, reference [49], available at available at https://doi.org/10.21979/N9/YCDXNE, accessed on 5 March 2021). Acknowledgments: We would like to thank An An Lieu, as well as other research members of the Social and Affective Neuroscience lab (Nanyang Technological University, Singapore) as well as research members of the Affiliative Behavior and Physiology lab (University of Trento, Italy), for their assistance in this work. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References In Proceedings of the 27th European Signal Processing Conference (EUSIPCO), A Coruna, Spain, 2–6 September 2019; pp. 1–5. p p pp 11. Manzalini, A. Towards a Quantum Field Theory for Optical Artificial Intelligence. Ann. Emerg. Technol. Comput. (AETiC) Print ISSN 2019, 3, 2516-0281. [CrossRef] 12. Sánchez-Sánchez, C.; Izzo, D.; Hennes, D. Learning the optimal state-feedback using deep networks. 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Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae
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Title Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae P li k UCLA UCLA Previously Published Wo Title Genome-Wide High-Resolution Mapping of U Saccharomyces cerevisiae Permalink https://escholarship.org/uc/item/36n044f6 Journal PLOS Genetics, 9(10) ISSN 1553-7390 Authors Yin, Yi Petes, Thomas D Publication Date 2013-10-01 DOI 10.1371/journal.pgen.1003894 Peer reviewed UCLA UCLA Previously Published Wo Title Genome-Wide High-Resolution Mapping of U Saccharomyces cerevisiae Permalink https://escholarship.org/uc/item/36n044f6 Journal PLOS Genetics, 9(10) ISSN 1553-7390 Authors Yin, Yi Petes, Thomas D Publication Date 2013-10-01 DOI 10.1371/journal.pgen.1003894 Peer reviewed Abstract As in the SDSA pathway, mismatches within the heteroduplex region can be repaired to generate a conversion event. Lastly, invasion of one broken end can result in the generation of a replication structure that duplicates sequences from the other chromosome from the point of invasion to the end of the chromosome (break-induced replication, BIR; Figure 1C). Powered by the California Digital Library University of California eScholarship.org Yi Yin, Thomas D. Petes* Department of Molecular Genetics and Microbiology and University Program in Genetics and Genomics, Duke University Medical Center, Durham, North Carolina, United States of America Department of Molecular Genetics and Microbiology and University Program in Genetics and Genomics, Duke University Medical Ce States of America Abstract In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers. Citation: Yin Y, Petes TD (2013) Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae. PLoS Genet 9(10): e1003894. doi:10.1371/journal.pgen.1003894 Editor: Andre´s Aguilera, CABIMER, Universidad de Sevilla, Spain Received June 20, 2013; Accepted September 5, 2013; Published October 31, 2013 Received June 20, 2013; Accepted September 5, 2013; Published October 31, 2013 Copyright:  2013 Yin, Petes. 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: The research was supported by NIH grants GM24110 and GM52319. The funders publish, or preparation of the manuscript. Funding: The research was supported by NIH grants GM24110 and GM52319. 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: tom.petes@duke.edu * E-mail: tom.petes@duke.edu The resulting product has a region of heteroduplex DNA and mismatches within the heteroduplex can be repaired to yield a gene conversion event unassociated with a crossover (Figure 1A). Alternatively, the broken ends can both engage in pairing with the intact chromosome resulting in a double Holliday junction (Figure 1B). This structure can be resolved to yield a crossover or non-crossover. Introduction Recombination occurs in both meiotic and mitotic cells. In budding yeast, there are about 100 meiotic crossovers per cell [1]. Although mitotic recombination events in S. cerevisiae are about 105-fold less frequent than meiotic exchanges [2], homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) that occur spontaneously or that are induced by DNA damage. Yeast strains that lack HR grow more slowly than wild-type strains, and are sensitive to DNA damaging agents [3]. In HR events in diploid cells, the broken chromosome is repaired utilizing an intact sister chromatid or homolog as a template. Most organisms also have a pathway termed ‘‘non- homologous end-joining’’ (NHEJ) in which the broken ends are re- joined by a mechanism that does not require sequence homology. In diploid cells of S. cerevisiae, HR is much more important than NHEJ for repair of DNA damage [4]. We will first discuss pathways of HR, followed by a description of UV-induced DNA damage, and the recombinogenic effects of this damage. One consequence of mitotic recombination is to cause loss of heterozygosity (LOH) for markers near the initiating lesion (gene conversions) or extending distal from the initiating lesion to the end of the chromosome (crossovers and BIR events). In Figure 2, we show the repair of DSBs in diploid mitotic cells by HR involving the homolog. In Figure 2A, we show the repair of a single broken chromatid (G2 event) using the homolog as a template. The red and black colors indicate that the two homologs have single-nucleotide polymorphisms (SNPs) that allow the detection of recombination events. Figure 2A shows a crossover between chromatids 2 and 3. If chromatids 1 and 3 segregate into one daughter cell (D1), and 2 and 4 segregate into the other (D2), a reciprocal pattern of LOH would be observed. Segregation of unrecombined chromatids 1 and 4 into one cell and the DSBs can be repaired by a number of different HR pathways [5]. For all of these pathways, the broken DNA ends are processed by 59 to 39 degradation, followed by invasion of the processed chromosome end into either a sister chromatid or a homolog (Figure 1). In the synthesis-dependent strand annealing (SDSA) pathway, after strand invasion and DNA synthesis, the invading broken end is displaced and reanneals to the other broken end. October 2013 | Volume 9 | Issue 10 | e1003894 Author Summary recombined chromatids 2 and 3 into the other would not lead to LOH. These two patterns of segregation are equally frequent in yeast [6]. Our previous studies [2,7] showed that most (80%) crossovers are associated with gene conversion events (indicated by boxes in Figure 2). In Figure 2B, we show a conversion event unassociated with a crossover which produces an interstitial LOH event in one of the daughter cells. The conversion events shown in Figure 2A and 2B are termed ‘‘3:1’’ events since three of the chromatids have one type of SNP and one has the other within the boxed region. A BIR event produces a region of LOH that extends to the telomere in one but not both daughter cells (Figure 2C). Although it is clear from many previous studies that UV greatly elevates the frequency of mitotic recombination in yeast [17–23], the recombinogenic mechanism is not well understood. There are two types of models. First, it is possible that the recombinogenic lesion is generated by NER. Consistent with this model, Galli and Schiestl (1999) [20] observed that UV of G1-synchronized cells was not recombinogenic unless the cells were allowed to replicate. They concluded that the recombinogenic lesion was likely to represent an NER-associated gap that was replicated to produce the recombination-stimulating DSB. This model predicts that the gene conversion events associated with UV-treatment of G1- synchronized cells would be exclusively 3:1 conversion events (Figure 2A). In a preliminary study [7], however, we found that about half of the observed UV-induced conversions were 3:1 events and about half were 4:0 events (Figure 2D). This observation is inconsistent with the simplest form of the model proposed by Galli and Schiestl. g ( g ) The 3:1 conversion events shown in Figures 2A and 2B are expected from the repair of a single DSB generated in S or G2 of the cell cycle. In addition, since the chromosome with the DSB acts as a recipient of information derived from the intact chromosome, these conversion events have the pattern expected if the recombinogenic DSB was on the black chromosome [4]. We observed previously, however, that over half of the mitotic conversion events had a different form from that shown in Figures 2A and 2B. Author Summary In Figure 2D, we show a conversion event unassociated with a crossover in which both daughter cells have an interstitial region of LOH that is homozygous for the same SNPs; these events are called ‘‘4:0’’ conversions. We interpret 4:0 events as resulting from the repair of two broken sister chromatids in which the DSBs are located at the same positions. One simple mechanism to obtain this pattern of breakage is that the recombinogenic DSB is generated in G1, the broken chromosome is replicated, and the two resulting broken chromatids are repaired in G2 (Figure 2D). The alternative model in which the DSB is generated and repaired in G1 is ruled out because such events would not be associated with LOH for markers located distal to the conversion event [8]. If the two broken chromatids are repaired to generate conversion tracts of the same lengths, a 4:0 event is generated. If one conversion tract is longer than the other, repair of two broken sister chromatids can also generate hybrid 3:1/4:0 conversion tracts [2,7]. Our previous studies indicated that most spontaneous crossovers had conversion events consistent with a G1-initiated DSB rather than a G2-initiated DSB [9,10], and spontaneous events resembled those induced by gamma rays in G1-synchronized yeast cells [11]. An alternative model is that the unexcised dimers and other DNA lesions are the recombinogenic lesion. For example, replication forks stalled at an unexcised dimer may engage in replication re-start or be broken. Although both re-start and the repair of an S-phase DSB would be expected to involve an interaction with the sister chromatid [24], some fraction of these events could involve the homolog, resulting in LOH. Kadyk and Hartwell (1993) [21] showed that UV stimulates recombination between both sister-chromatids and homologs in NER-proficient cells. In rad1/rad1 (NER-deficient) diploids, conversions, but not crossovers, were stimulated by UV in a replication-dependent manner [21]. One complication in interpreting this result is that Rad1p is involved with multiple recombination-related reactions [25–27] in addition to its role in NER. Regardless of this ambiguity, it is likely that unexcised dimers are recombinogenic. The summary of studies performed thus far is that some fraction of UV-induced recombination events reflects lesions resulting from NER and another fraction reflects unexcised dimers. In the experiments described below, we examine mitotic crossovers and gene conversion events induced by UV in diploid cells. Introduction October 2013 | Volume 9 | Issue 10 | e1003894 1 PLOS Genetics | www.plosgenetics.org Author Summary Author Summary Nearly every living organism has to cope with DNA damage caused by ultraviolet (UV) exposure from the sun. UV causes various types of DNA damage. Defects in the repair of these DNA lesions are associated with the human disease xeroderma pigmentosum, one symptom of which is predisposition to skin cancer. The DNA damage introduced by UV stimulates recombination and, in this study, we characterize the resulting recombination events at high resolution throughout the yeast genome. At high UV doses, we show that most recombination events reflect the repair of two sister chromatids broken at the same position, indicating that UV can cause double-stranded DNA breaks. At lower doses of UV, most events involve the repair of a single broken chromatid. Our mapping of events also demonstrates that certain regions of the yeast genome are relatively resistant to UV-induced recombina- tion. Finally, we show that most UV-induced DNA lesions are repaired during the first cell cycle, and do not lead to recombination in subsequent cycles. Although most UV-induced lesions are removed quickly by this error-free process, a small fraction of the 30-nucleotide gaps are expanded by the action of Exo1p, resulting in large RPA-coated gaps [13,14]. These RPA-coated regions recruit Mec1p/Ddc2p and the 9-1-1 complex, followed by subsequent recruitment of other components of the DNA damage checkpoint [15]. In addition to checkpoints triggered by the action of Exo1p, if unrepaired lesions persist into the S-phase, single-stranded regions may also be generated during the re-start of blocked replication forks. Strong activation of Mec1p by UV is observed in S-phase cells, presumably by this mechanism [16]. recombined chromatids 2 and 3 into the other would not lead to LOH. These two patterns of segregation are equally frequent in yeast [6]. Our previous studies [2,7] showed that most (80%) crossovers are associated with gene conversion events (indicated by boxes in Figure 2). In Figure 2B, we show a conversion event unassociated with a crossover which produces an interstitial LOH event in one of the daughter cells. The conversion events shown in Figure 2A and 2B are termed ‘‘3:1’’ events since three of the chromatids have one type of SNP and one has the other within the boxed region. A BIR event produces a region of LOH that extends to the telomere in one but not both daughter cells (Figure 2C). UV-Induced Recombination in Yeast dosages of UV-C (,254 nm) are pyrimidine dimers including cyclobutane dimers (CPDs) and (6-4) photoproducts (6-4 PPs) [3]. Although CPDs can be reversed in yeast by the action of photolyase, the repair of most lesions in wild-type cells likely reflects nucleotide excision repair (NER). In NER, multiple proteins act to excise a short oligonucleotide containing the damaged bases. The resulting 30-nucleotide gap is filled in by DNA polymerase delta and/or epsilon [12], and the remaining nick is sealed by Lig1p. In yeast, as in many other organisms, UV- induced lesions are more quickly repaired in transcribed genes than in non-transcribed regions [3]. Author Summary Dissociation of the D-loop, followed by re-annealing of the two broken ends results in a region of heteroduplex (outlined in blue) with flanking markers in the parental non-crossover (NCO) configuration. B. Double-strand break repair (DSBR). Following expansion of the D-loop, pairing occurs between the displaced strand and the right end of the broken chromosome, resulting in two regions of heteroduplex. The resulting double Holliday junction can be resolved by topoisomerase-mediated dissolution or by cleavage of the Holliday junctions to yield an NCO or a crossover (CO). C. Break-induced replication (BIR). In this pathway, the right broken chromosome is lost and the left molecule invades the homologous chromosome, resulting in duplication of sequences distal to the point of invasion. doi:10.1371/journal.pgen.1003894.g001 100 UV-induced LOH events selected on chromosome V and about 400 unselected LOH events throughout the genome. We found that the unselected events were widely distributed through- out the genome with no very strong hotspots. The ribosomal RNA gene cluster, however, was significantly ‘‘cold’’ for crossovers compared to the rest of the genome. telomere of chromosome V, one homolog (shown in black in Figure 3A) has an insertion of SUP4-o, an ochre-suppressing tRNA gene. The diploid is also homozygous for the ade2-1 ochre mutation. Diploids homozygous for the ade2-1 mutation and zero, one or two copies of SUP4-o form colonies that are red, pink, and white, respectively [28]. In most of the experiments described below, G1-synchronized diploid cells were plated and immediately irradiated with UV. If the resulting DNA damage induces a crossover between the heterozygous SUP4-o gene and the centromere of chromosome V before the first cell division, a red/white sectored colony will be formed (Figure 3A). Since formation of a sectored colony requires a crossover, followed by the segregation pattern in which each daughter cell receives one recombined chromosome and one unrecombined chromosome (Figures 2A and 3A), only half of the crossovers induced in the first division following irradiation result in LOH. If the UV-induced DNA damage is not repaired in the Author Summary In G1-synchronized cells treated with high doses of UV, most of the events reflect the repair of two broken sister chromatids whereas at low doses, most events reflect repair of a single broken chromatid. We also show that UV induces crossovers more efficiently than BIR events. We mapped the distribution of about UV results in DNA lesions that are both mutagenic and recombinogenic. The primary types of lesions caused by low October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 2 UV-Induced Recombination in Yeast Figure 1. Mechanisms of homologous recombination. The two interacting DNA molecules are shown as double-stranded. The recombination event is initiated by a double-stranded DNA break (DSB) on the black molecule, and the broken ends are resected 59 to 39. In this depiction, the left processed end invades the red molecule, forming a heteroduplex. Dotted lines show DNA synthesis associated with the recombination event. A. Synthesis-dependent strand annealing (SDSA). The 39 end of the invading strand is used as a primer for DNA synthesis, extending the D-loop. Dissociation of the D-loop, followed by re-annealing of the two broken ends results in a region of heteroduplex (outlined in blue) with flanking markers in the parental non-crossover (NCO) configuration. B. Double-strand break repair (DSBR). Following expansion of the D-loop, pairing occurs between the displaced strand and the right end of the broken chromosome, resulting in two regions of heteroduplex. The resulting double Holliday junction can be resolved by topoisomerase-mediated dissolution or by cleavage of the Holliday junctions to yield an NCO or a crossover (CO). C. Break-induced replication (BIR). In this pathway, the right broken chromosome is lost and the left molecule invades the homologous chromosome, resulting in duplication of sequences distal to the point of invasion. doi:10.1371/journal.pgen.1003894.g001 UV-Induced Recombination in Yeast UV Induced Recombination in Yeas Figure 1. Mechanisms of homologous recombination. The two interacting DNA molecules are shown as double-stranded. The recombination event is initiated by a double-stranded DNA break (DSB) on the black molecule, and the broken ends are resected 59 to 39. In this depiction, the left processed end invades the red molecule, forming a heteroduplex. Dotted lines show DNA synthesis associated with the recombination event. A. Synthesis-dependent strand annealing (SDSA). The 39 end of the invading strand is used as a primer for DNA synthesis, extending the D-loop. Detection and mapping of mitotic crossovers and gene conversions This type of conversion is termed ‘‘3:1’’ since, at the site of the conversion, three of the four chromosomes of the two daughter cells (D1 and D2) have information derived from one of the homologs. If chromatids 1 and 3, and 2 and 4 co-segregate, any markers distal to the crossover will be homozygous. B. S or G2 conversion event unassociated with a crossover. If the DSB is repaired to generate a conversion unassociated with a crossover, a region of 3:1 segregation will be observed, but the markers distal to the conversion event will remain heterozygous. C. Break-induced replication. The chromosome fragment centromere-distal to the DSB is lost and the centromere-containing fragment invades and replicates the red chromosome to the telomere. D. Repair of a G1-induced DSB. A black chromosome with a G1-induced DSB is replicated to yield two sister-chromatids with breaks at the same positions. Gene conversion events are generated at the sites of repair and the repair of one of the DSBs is associated with a crossover Within the chromosomes of the two daughter cells Figure 2. Patterns of mitotic gene conversions and crossovers. Chromosomes are shown as thick lines with the two homologs in red or black. Circles indicate the centromeres and blue boxes show gene conversions. A. Conversion and crossover associated with a DSB formed in S or G2 on one of the black chromatids. Since the broken chromosome acts as the recipient in a gene conversion event [60], sequence information derived from the red chromatid is non-reciprocally transferred in conjunction with the crossover. This type of conversion is termed ‘‘3:1’’ since, at the site of the conversion, three of the four chromosomes of the two daughter cells (D1 and D2) have information derived from one of the homologs. If chromatids 1 and 3, and 2 and 4 co-segregate, any markers distal to the crossover will be homozygous. B. S or G2 conversion event unassociated with a crossover. If the DSB is repaired to generate a conversion unassociated with a crossover, a region of 3:1 segregation will be observed, but the markers distal to the conversion event will remain heterozygous. C. Break-induced replication. The chromosome fragment centromere-distal to the DSB is lost, and the centromere-containing fragment invades and replicates the red chromosome to the telomere. D. Repair of a G1-induced DSB. Detection and mapping of mitotic crossovers and gene conversions In order to determine different types of mitotic recombination and to determine whether the conversion events are of the 3:1 or 4:0 configuration, we used a method of identifying recombination events that allows the recovery of both daughter cells with the recombinant chromosomes. The system used in the present study (Figure 3) is similar to that employed previously [2,28]. Near the October 2013 | Volume 9 | Issue 10 | e1003894 October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 3 Figure 2. Patterns of mitotic gene conversions and crossovers. Chromosomes are shown as thick lines with the two homologs in red or bla Circles indicate the centromeres and blue boxes show gene conversions. A. Conversion and crossover associated with a DSB formed in S or G2 on o of the black chromatids. Since the broken chromosome acts as the recipient in a gene conversion event [60], sequence information derived from red chromatid is non-reciprocally transferred in conjunction with the crossover. This type of conversion is termed ‘‘3:1’’ since, at the site of conversion, three of the four chromosomes of the two daughter cells (D1 and D2) have information derived from one of the homologs. If chromat 1 and 3, and 2 and 4 co-segregate, any markers distal to the crossover will be homozygous. B. S or G2 conversion event unassociated wit crossover. If the DSB is repaired to generate a conversion unassociated with a crossover, a region of 3:1 segregation will be observed, but the mark distal to the conversion event will remain heterozygous. C. Break-induced replication. The chromosome fragment centromere-distal to the DSB is lo and the centromere-containing fragment invades and replicates the red chromosome to the telomere. D. Repair of a G1-induced DSB. A bl h h d d l d ld h d h b k h UV-Induced Recombination in Ye UV-Induced Recombination in Yeast Figure 2. Patterns of mitotic gene conversions and crossovers. Chromosomes are shown as thick lines with the two homologs in red or black Circles indicate the centromeres and blue boxes show gene conversions. A. Conversion and crossover associated with a DSB formed in S or G2 on one of the black chromatids. Since the broken chromosome acts as the recipient in a gene conversion event [60], sequence information derived from the red chromatid is non-reciprocally transferred in conjunction with the crossover. Detection and mapping of mitotic crossovers and gene conversions A black chromosome with a G1-induced DSB is replicated to yield two sister-chromatids with breaks at the same positions. Gene conversion events are generated at the sites of repair, and the repair of one of the DSBs is associated with a crossover. Within the chromosomes of the two daughter cells, there is a region that is derived from only the red homolog (a ‘‘4:0’’ conversion tract shown in the blue rectangle). doi:10.1371/journal.pgen.1003894.g002 G1-synchronized cells generate a red/white sectored colony rather than a tri-colored colony. Neither gene conversion events unassociated with a crossover nor BIR events on chromosome V first cell cycle but persists into subsequent cell cycles, a pink/ white/red sectored colony could be produced (Figure 3B). As described below, most of the events induced by UV treatment in G1-synchronized cells generate a red/white sectored colony rather than a tri-colored colony. Neither gene conversion events unassociated with a crossover nor BIR events on chromosome V October 2013 | Volume 9 | Issue 10 | e1003894 October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 4 UV-Induced Recombination in Yeast Figure 3. A system for detecting mitotic crossovers by a colony sectoring assay. G1-synchronized diploid cells were treated with UV a immediately plated on solid medium. The diploid is homozygous for the ade2-1 mutation, an ochre mutation that when unsuppressed results in a colony. The diploid has one copy of the ochre suppressor gene SUP4-o inserted near the telomere of chromosome IV on the black homolog. Stra with zero, one, and two copies of SUP4-o form red, pink, and white colonies, respectively. A. Crossover in G2 of the first division following irradiat A DSB in one chromatid repaired during G2 will generate a red/white sectored colony, the white sector derived from daughter cell 1 (D1) and the sector derived from daughter cell 2 (D2). B. Crossover delayed to G2 of the second division. If DNA damage induced in G1 is not repaired during first division, a pink/white/red sectored colony would be generated. The abbreviation ‘‘GD’’ indicates the granddaughter of the irradiated cell. doi:10.1371/journal.pgen.1003894.g003 Figure 3. A system for detecting mitotic crossovers by a colony sectoring assay. G1-synchronized diploid cells were treated with UV and immediately plated on solid medium. The diploid is homozygous for the ade2-1 mutation, an ochre mutation that when unsuppressed results in a red colony. Detection and mapping of mitotic crossovers and gene conversions The diploid has one copy of the ochre suppressor gene SUP4-o inserted near the telomere of chromosome IV on the black homolog. Strains with zero, one, and two copies of SUP4-o form red, pink, and white colonies, respectively. A. Crossover in G2 of the first division following irradiation. A DSB in one chromatid repaired during G2 will generate a red/white sectored colony, the white sector derived from daughter cell 1 (D1) and the red sector derived from daughter cell 2 (D2). B. Crossover delayed to G2 of the second division. If DNA damage induced in G1 is not repaired during the first division, a pink/white/red sectored colony would be generated. The abbreviation ‘‘GD’’ indicates the granddaughter of the irradiated cell. doi:10.1371/journal.pgen.1003894.g003 October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 5 UV-Induced Recombination in Yeast (1 J/m2), 1600 (5 J/m2), 5000 (10 J/m2), and 8500 (15 J/m2). The strong stimulation of mitotic crossovers by UV is consistent with previous studies [23]. (1 J/m2), 1600 (5 J/m2), 5000 (10 J/m2), and 8500 (15 J/m2). The strong stimulation of mitotic crossovers by UV is consistent with previous studies [23]. result in a red/white sectored colony. As will be shown below, such events can be detected as unselected events in cells that have a selected crossover on chromosome V. The transition between heterozygous markers and homozygous markers in the sectored colony locates the position of the crossover. To detect the position of the selected crossover on chromosome V and to detect unselected LOH events throughout the genome, we used a diploid strain (PG311) derived from mating two sequence-diverged haploid strains: W303a and YJM789 [2,7,29]. These two strains differ by about 52,000 SNPs. We detect LOH using microarrays that examine 13,000 of these SNPs [7], allowing mapping of most events to a resolution of about 1 kb. Each SNP is represented by four 25-bp probes, two with W303a sequences (Watson and Crick) and two with YJM789 sequences. At the hybridization temperature optimized for the whole probe set, W303a genomic DNA hybridizes strongly to W303a oligonucleotides with very weak cross-hybridization to the corresponding YJM789 oligonucleotides, and vice versa for YJM789 genomic sequences. Genomic DNA is isolated from each sector of red/white sectored colonies, labeled with Cy5-tagged nucleotides, and competitively hybridized to the SNP microarray with genomic DNA from the untreated strain labeled with Cy3- tagged nucleotides. Detection and mapping of mitotic crossovers and gene conversions By assaying the ratio of hybridization of the differentially-tagged samples to each oligonucleotide [7], we can readily map LOH events. The transition between heterozygous and homozygous markers should be located near the site of the recombinogenic DNA lesion. In some studies [2,4,28], the frequency of mitotic recombination events is higher in diploids that express both mating types than in diploids that express only one mating type. Consequently, we compared the frequency of red/white sectored colonies in G1- synchronized cultures of PG311 and PSL101 (the MATa/MATa progenitor of PG311). Because PSL101 cannot be synchronized in G1 using alpha pheromone, both strains were synchronized in G1 by growing the cells into stationary phase (Text S1). After treatment of the G1-synchronized cells with 15 J/m2 of UV, 0.4% (0.2–0.9%, 95% confidence limits) of the PG311 colonies formed red/white sectors compared to 0.6% (0.4–1%) of the PSL101 colonies. Although the confidence limits are wide, these results indicate that mating type heterozygosity does not have a large effect on the frequency of UV-induced mitotic crossovers in our system. In addition to red/white sectored colonies, in the irradiated samples, we also observed pink/red and pink/white/red colonies. Such colonies could represent non-reciprocal recombination events (for example, BIR events), persistence of recombinogenic DNA damage beyond the first cell cycle, or an artifact (two closely- located independent cells). To exclude sectors formed artifactually, we micromanipulated individual G1-irradiated (15 J/m2 dose) single cells to specific positions on plates with solid medium, and monitored their subsequent development to form sectored or unsectored colonies. From a total of 970 isolated irradiated single cells, we observed eleven sectored colonies of the following types: seven red/white colonies, two pink/red colonies, and two pink/ white/red colonies. From our SNP microarray analysis of the LOH patterns on chromosome V in these colonies (described in Text S1 and Figure S1), we found that all seven of the red/white colonies represented crossovers induced during the first cell cycle. The two pink/red sectored colonies reflected chromosome loss, resulting in a monosomic red sector and a pink sector. Only one of the pink/white/red colonies was a consequence of a UV-induced recombination event in the second division (Figure 3B, Figures S1 and S2). Detection and mapping of mitotic crossovers and gene conversions In summary, of the nine sectored colonies in which sectoring reflected a UV-induced crossover, eight occurred prior to the first cell division and only one occurred after the first cell division, indicating that most UV-induced DNA lesions are rapidly repaired. Figure 4 shows the analysis of one red/white sectored colony (59RW). In this figure, we show the normalized ratio of hybridization of genomic sequences to W303a- and YJM789- specific oligonucleotides on chromosome V with red lines and black lines, respectively; CEN5 is located near coordinate 152 kb. In the top part of Figure 4A, we depict the pattern of hybridization of genomic DNA isolated from the red sector. The ratio of hybridization is about 1 for all SNPs from coordinate 105 kb to the right telomere, indicating that SNPs in this region are heterozygous. In the red sector, SNPs centromere-distal to coordinate 105 kb on the left arm are homozygous for the W303a-derived SNPs whereas the genomic DNA from the white sector becomes homozygous at approximately the same position for YJM789-derived SNPs. In Figure 4B, the same recombination event is depicted at higher resolution; each square and diamond shows the level of hybridization to an individual YJM789-specific or a W303a-specific SNP, respectively. As shown in this figure, the red sector has a single transition between heterozygous and homozygous SNPs whereas the white sector has three transitions. The pattern of these transitions indicates that the crossover is associated with a 3:1/4:0 hybrid conversion tract. We used SNP microarrays to analyze 47 sectored colonies of G1-synchronized cells treated with 5, 10 or 15 J/m2 of UV (Tables S2 and S3). 80% of the colonies were from cells treated with 15 J/m2. Nine of these colonies were derived from the single- cell experiments described above. 45 of the 47 sectored colonies examined had patterns of LOH on chromosome V consistent with a reciprocal crossover on the left arm of chromosome V. In one of the two exceptional colonies, there was a loss of one copy of chromosome V. In the other colony, there were two independent conversions that resulted in LOH events that were unassociated with a crossover. These two sectored colonies were not used in our subsequent analysis of selected events on chromosome V, although data from these colonies were used to analyze unselected recombination events. Analysis of LOH events induced by UV-irradiation of cells with a dose of 15 J/m2 Most of our experiments involve UV treatment of G1- synchronized cells with 15 J/m2; the experimental parameters used for each experiment are in Table S1. PG311 is hemizygous at the MAT locus (MATa/MATa::NAT), allowing its synchronization in G1 using the alpha pheromone [11]. The synchronized cells were plated onto solid medium and immediately irradiated at doses varying between 1 and 15 J/m2. Even at the maximum dose of UV, cell viability was 70%. No sectored colonies were observed in cells that were not treated with UV. Based on our earlier study of spontaneous crossovers in the same strain [2], the rate of crossovers in untreated cells is 1.161026/division in the 120 kb interval between CEN5 and the SUP4-o marker. Relative to this rate, UV treatment stimulated sector formation by factors of 1500 In addition to the selected LOH events on chromosome V, we observed an average of eight unselected LOH events per sectored colony. As described below, our analysis of the 45 selected and 381 unselected events (300 gene conversion events unassociated with crossovers, 60 crossovers, and 21 BIR events) allowed us to determine several important features of the UV-induced recom- bination events: 1) the patterns of gene conversion in selected and October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 6 UV-Induced Recombination in Yeast Figure 4. Mapping crossovers on chromosome V by SNP arrays. Genomic DNA was isolated from the red and white sectors of a sectored colony derived from UV-treated cells. The ratio of hybridization of SNP-specific oligonucleotides (relative to the hybridization levels of DNA from a fully heterozygous strain) for each sample was measured (details in text) and is shown on the Y axis. A hybridization level of about 1 indicates that the strain was heterozygous. The X axis shows the SGD coordinates of chromosome V beginning at the left telomere. Black and red lines indicate the normalized hybridization ratio to the YJM789- and W303a-specific oligonucleotides, respectively. The crossover and conversions associated with the sectors are diagrammed with the upper and lower panels showing patterns of LOH in the red and white sectors, respectively. A. Low-resolution depiction of the LOH events in the red and white sectors. The transition is at about SGD coordinate 105 kb. B. High-resolution depiction of LOH events (same event as shown in Figure 4A). Analysis of LOH events induced by UV-irradiation of cells with a dose of 15 J/m2 Black squares show hybridization to YJM789-specific oligonucleotides and red diamonds show hybridization to W303a-specific oligonucleotides. The red sector has a single transition between heterozygous and homozygous SNPs, whereas the white sector has three transitions. The pattern of transitions is consistent with a 3:1/4:0 hybrid tract associated with a crossover. C. Summary of patterns of heterozygous and homozygous markers with the top line showing the red sector and the bottom line showing the white sector. The black, red, and green lines indicate regions homozygous for YJM789-derived SNPs, homozygous for W303a-derived SNPs, and heterozygous regions, respectively. The orange circles show the position of the centromeres. The lengths of the line segments showing the LOH region associated with the crossover and the heterozygous region centromere-proximal to the crossover are not shown to scale. doi:10.1371/journal.pgen.1003894.g004 Figure 4. Mapping crossovers on chromosome V by SNP arrays. Genomic DNA was isolated from the red and white sectors of a sectored colony derived from UV-treated cells. The ratio of hybridization of SNP-specific oligonucleotides (relative to the hybridization levels of DNA from a fully heterozygous strain) for each sample was measured (details in text) and is shown on the Y axis. A hybridization level of about 1 indicates that the strain was heterozygous. The X axis shows the SGD coordinates of chromosome V beginning at the left telomere. Black and red lines indicate the normalized hybridization ratio to the YJM789- and W303a-specific oligonucleotides, respectively. The crossover and conversions associated with the sectors are diagrammed with the upper and lower panels showing patterns of LOH in the red and white sectors, respectively. A. Low-resolution depiction of the LOH events in the red and white sectors. The transition is at about SGD coordinate 105 kb. B. High-resolution depiction of LOH events (same event as shown in Figure 4A). Black squares show hybridization to YJM789-specific oligonucleotides and red diamonds show hybridization to W303a-specific oligonucleotides. The red sector has a single transition between heterozygous and homozygous SNPs, whereas the white sector has three transitions. The pattern of transitions is consistent with a 3:1/4:0 hybrid tract associated with a crossover. C. Summary of patterns of heterozygous and homozygous markers with the top line showing the red sector and the bottom line showing the white sector. The black, red, and green lines indicate regions homozygous for YJM789-derived SNPs, homozygous for W303a-derived SNPs, and heterozygous regions, respectively. UV-Induced Recombination in Yeast In our current study, of 300 conversion events unassociated with crossovers (Classes A–G in Table S2), 167 (56%) were SCBs and 133 (44%) were DSCBs; these classifications are shown in Table S2 with the rationale for the classifications in Text S1. Of the 92 conversions associated with crossovers (selected and unselected), 35 (38%) were SCBs and 57 (62%) were DSCBs; the distribution of SCBs and DSCBs is significantly (p = 0.005) different than that observed for the conversions unassociated with crossovers. Our analysis supports the surprising conclusion that about half of the mitotic recombi- nation events stimulated by high (15 J/m2) doses of UV in G1- synchronized cells involve two sister chromatids broken at the same position. As discussed below, a different result is obtained when cells are irradiated at low (1 J/m2) UV doses. Most (41 of 45) of the selected crossovers were associated with gene conversion events. In Figure 5, we show some of the common LOH patterns observed for the selected crossovers as well as for unselected recombination events. These depictions include a crossover unassociated with gene conversion (Figure 5A), a crossover associated with a 3:1 conversion event (Figure 5B), a crossover associated with a 3:1/4:0 hybrid tract (Figure 5C), a crossover associated with a complex conversion tract (Figure 5D), a 3:1 conversion unassociated with a crossover (Figure 5E), and a BIR event (Figure 5F). It should be noted that the patterns of gene conversion in sectored colonies can be used to infer which homolog was broken to initiate the recombination event. A chromosome that receives a DSB acts as a recipient during gene conversion [4]. Thus, in the event shown in Figure 5B, in which there is a red segment opposite a green segment, we can infer that the event was initiated by breaking the YJM789-derived (black) homolog. As in our related studies [2,7,11,29], we interpret the patterns of gene conversion and crossovers shown in Table S2 in the context of known pathways of DSB repair (Figure 1). All of these pathways involve regions of heteroduplex formation and, therefore, regions of duplex DNA that may contain mismatches. In wild-type strains, mismatches are usually efficiently removed by the mismatch repair (MMR) enzymes. In general, during mitotic recombination, MMR in yeast occurs directionally, removing the mismatched bases from the strand contributed by the broken chromosome [30]. UV-Induced Recombination in Yeast unselected recombination events, 2) the lengths of gene conversion tracts associated or unassociated with crossovers, and 3) the locations of selected and unselected recombination events induced by UV. Since the frequency of selected sectored colonies in cells irradiated with 15 J/m2 was about 1%, and the selected interval on chromosome V is about 1% of the genome, we expect about one unselected crossover per irradiated cell, roughly the observed frequency (60 unselected crossovers/47 sectored colonies). repair [30], although less frequent ‘‘patchy’’ repair events have also been described [7,29]. For our study, we assume that the recombination events reflect the repair of one chromatid or two sister chromatids broken at the same position by the pathways shown in Figure 1. For the simple events, we assume non-patchy conversion-type repair and we assume that there is no branch migration of Holliday junction intermediates. Of the 300 conversion events induced by 15 J/m2 of UV, 276 were simple events, and of the 104 crossover events (most associated with conversion), 67 were simple events. The more complex events usually involve multiple transitions between heterozygous and homozygous SNPs and can often be explained as a consequence of patchy repair within a heteroduplex and/or branch migration. More detailed descriptions of complex events are given in Text S1. unselected recombination events, 2) the lengths of gene conversion tracts associated or unassociated with crossovers, and 3) the locations of selected and unselected recombination events induced by UV. Since the frequency of selected sectored colonies in cells irradiated with 15 J/m2 was about 1%, and the selected interval on chromosome V is about 1% of the genome, we expect about one unselected crossover per irradiated cell, roughly the observed frequency (60 unselected crossovers/47 sectored colonies). Patterns of gene conversion in selected and unselected recombination events. Our conclusions are based on SNP analysis of both sectors of the sectored colonies. As we have done previously [7,29], the data from each sectored colony are presented as a pair of lines. For events on chromosome V, the top line represents the pattern observed in the red sector and the bottom line represents the white sector. Within each line, the three possible outcomes for SNPs (heterozygous, homozygous for the YJM789-derived SNP, and homozygous for the W303a-derived SNP) are shown by green, black, and red segments, respectively. Within each line, transitions between different colors are designated by a small letter. UV-Induced Recombination in Yeast For example, the red sector of the colony in Figure 4C is shown as a line with two colors and one transition, whereas the white sector has three colors and three transitions. The transition labeled ‘‘c’’ is at the same position in both sectors. All recombination events, both selected and unselected, for cells irradiated with 15 J/m2 are depicted in an analogous manner in Table S2. The sectored colony in Figure 4 is 59RW and the pattern of the associated gene conversion events is given in Class I7 of Table S2. The SGD coordinates for each transition in Table S2 are given in Table S3; depictions and coordinates for one pink/white/red tri-sectored colony are shown in Tables S4 and S5. As discussed in the Introduction, the patterns of gene conversion also provide important information about the timing of the recombinogenic DNA lesions during the cell cycle. In general (exceptions discussed in Text S1), 3:1 conversion events likely reflect a single DSB producing a single broken chromatid in S or G2 of the cell cycle that was repaired in G2 (Figure 2A). Conversions with a 4:0 tract or a 4:0 segment of a more complex conversion tract likely reflect a DSB formed in G1. If such a broken chromosome is replicated to produce two broken chromatids that are then repaired in G2, a 4:0 conversion tract would be generated (Figure 2D). We interpret 3:1/4:0 hybrid tracts of the type shown in Figure 4 as reflecting a G1 DSB. Below, we will refer to the conversion events that reflect a G2 DSB as single-chromatid breaks (SCBs) and those that reflect a G1 DSB as double-sister-chromatid breaks (DSCBs). From most previous studies, we expected UV to generate SCBs. Removal of a pyrimidine dimer, followed by replication of the resulting DNA molecule before the gap was repaired would be expected to result in a G2 DSB [20]. Alternatively (or, in addition), since unrepaired pyrimidine dimers are recombinogenic [24], a DSB at the replication fork could occur; this lesion would also be expected to result in an SCB. In a preliminary study involving three UV-induced sectored colonies, however, we found about half of the events were DSCBs. Analysis of LOH events induced by UV-irradiation of cells with a dose of 15 J/m2 The orange circles show the position of the centromeres. The lengths of the line segments showing the LOH region associated with the crossover and the heterozygous region centromere-proximal to the crossover are not shown to scale. doi:10.1371/journal.pgen.1003894.g004 Figure 4. Mapping crossovers on chromosome V by SNP arrays. Genomic DNA was isolated from the red and white sectors of a sectored colony derived from UV-treated cells. The ratio of hybridization of SNP-specific oligonucleotides (relative to the hybridization levels of DNA from a fully heterozygous strain) for each sample was measured (details in text) and is shown on the Y axis. A hybridization level of about 1 indicates that the strain was heterozygous. The X axis shows the SGD coordinates of chromosome V beginning at the left telomere. Black and red lines indicate the normalized hybridization ratio to the YJM789- and W303a-specific oligonucleotides, respectively. The crossover and conversions associated with the sectors are diagrammed with the upper and lower panels showing patterns of LOH in the red and white sectors, respectively. A. Low-resolution depiction of the LOH events in the red and white sectors. The transition is at about SGD coordinate 105 kb. B. High-resolution depiction of LOH events (same event as shown in Figure 4A). Black squares show hybridization to YJM789-specific oligonucleotides and red diamonds show hybridization to W303a-specific oligonucleotides. The red sector has a single transition between heterozygous and homozygous SNPs, whereas the white sector has three transitions. The pattern of transitions is consistent with a 3:1/4:0 hybrid tract associated with a crossover. C. Summary of patterns of heterozygous and homozygous markers with the top line showing the red sector and the bottom line showing the white sector. The black, red, and green lines indicate regions homozygous for YJM789-derived SNPs, homozygous for W303a-derived SNPs, and heterozygous regions, respectively. The orange circles show the position of the centromeres. The lengths of the line segments showing the LOH region associated with the crossover and the heterozygous region centromere-proximal to the crossover are not shown to scale. doi:10.1371/journal.pgen.1003894.g004 October 2013 | Volume 9 | Issue 10 | e1003894 7 PLOS Genetics | www.plosgenetics.org PLOS Genetics | www.plosgenetics.org UV-Induced Recombination in Yeast In Figure 1A, the mismatches within the heteroduplex shown in the boxed region would usually be corrected to generate two red strands; this pattern of correction is termed ‘‘conversion-type’’ repair. Correction to generate two black strands for this heteroduplex is ‘‘restoration-type’’ repair. Also, in heteroduplexes involving multiple mismatches, all mismatches are removed from one strand with the other strand being used as a template for when cells are irradiated at low (1 J/m2) UV doses. Recombination events induced by UV in G2-enriched cells. In addition to the comprehensive analysis of UV-induced recombination events in G1-synchronized cells, we did a preliminary analysis of the recombination events stimulated by 15 J/m2 UV in cells synchronized in G2. In one experiment, we examined the colonies derived from 2093 nocodazole-treated large-budded cells that were manipulated to defined locations on solid medium before irradiation. Although pink/red, and pink/ white/red sectors were observed, we found no simple red/white sectors. The frequency of red/white sectors in G1-synchronized cells at the same radiation dose was 8/970, suggesting that the frequency of crossovers on chromosome V is at least 10-fold less in G2-irradiated cells than in G1-irradiated cells. October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 8 UV-Induced Recombination in Yeast Figure 5. Common patterns of conversions and crossovers derived from UV-treated cells. The key is described in Figure 4C. A. Simpl crossover unassociated with conversion. B. Crossover associated with a 3:1 conversion event. C. Crossover associated with a 3:1/4:0 hybrid conversio tract. D. Complex crossover/conversion event. E. 3:1 conversion unassociated with a crossover. F. BIR event. doi:10.1371/journal.pgen.1003894.g005 UV-Induced Recombination in Yeas Because of the low frequency of events on chromosome V, we e amined UV ind ced recombination in G2 cells of the strain microarrays to examine six and seven sectored colonies derived from the G2 A and G2 B e periments respecti el Altho gh e Figure 5. Common patterns of conversions and crossovers derived from UV-treated cells. The key is described in Figure 4C. A. Simple crossover unassociated with conversion. B. Crossover associated with a 3:1 conversion event. C. Crossover associated with a 3:1/4:0 hybrid conversion tract. D. Complex crossover/conversion event. E. 3:1 conversion unassociated with a crossover. F. BIR event. doi:10.1371/journal.pgen.1003894.g005 Figure 5. Common patterns of conversions and crossovers derived from UV-treated cells. The key is described in Figure 4C. A. Simple crossover unassociated with conversion. B. PLOS Genetics | www.plosgenetics.org UV-Induced Recombination in Yeast correct morphology [31]. Second, it is possible that nocodazole can induce LOH events. Third, some putative G2 cells, identified as having a doublet morphology, may not have completed the S- period. For all these reasons, we conclude that G2-enriched cells have lower levels of UV-induced recombination events than G1 cells, but we make no definitive conclusions about the nature of these events. correct morphology [31]. Second, it is possible that nocodazole can induce LOH events. Third, some putative G2 cells, identified as having a doublet morphology, may not have completed the S- period. For all these reasons, we conclude that G2-enriched cells have lower levels of UV-induced recombination events than G1 cells, but we make no definitive conclusions about the nature of these events. within the rDNA; we expected six crossovers, but observed no crossovers. By chi-square analysis, this difference is significant (p = 0.02). Further support for this conclusion will be presented below. We also analyzed whether certain chromosomal elements or sequence motifs are enriched or underrepresented within the conversion tracts of unselected recombination events. The details of this analysis are described in Text S1. When all conversion events are examined, there was a significant under-representation of long terminal repeats (LTR, p = 0.0003), and tRNA genes (p = 0.002); the p values remain significant after correction for multiple comparisons. We also examined the representation of these elements when SCB conversions and DSCB conversions were analyzed separately. In this analysis, non-coding RNAs were significantly over-represented among the DSCB class of conver- sions. The significance of these results will be examined in the Discussion. Lengths of gene conversion tracts associated or unassociated with crossovers. From the data shown in Tables S2 and S3, we derived the lengths of gene conversion tracts in the samples irradiated with 15 J/m2. We measured tract length by averaging the maximal length (the distance between heterozygous SNPs flanking the LOH region) and the minimal length (the distance between the first and last LOH SNP within the tract). For hybrid or complex tracts, regions both 4:0 and 3:1 segments were included as part of a single tracts as were short heterozygous regions within the tract. The median length of all conversion tracts (95% confidence limits in parentheses) was 5.6 kb (5.0–6.2 kb). UV-Induced Recombination in Yeast The median length of conversion tracts associated with crossovers was 7.6 kb (6.4–9.6 kb); this median length is similar to that observed previously for spontaneous crossover-associated conversions (6.1 kb; [7]). Conversion tracts unassociated with crossovers were significantly shorter than crossover-associated conversions, 4.9 kb (4.1–5.6 kb) (p,0.00001; Mann-Whitney test). As noted previously, the lengths of mitotic conversion tracts are considerably larger than the average meiotic conversion tract (2 kb; [1]). We also observed a bias in the distribution of BIR events. Of the 21 events, eight were within 50 kb of the telomeres. Based on the genome size and the number of the telomeres, the expected number of BIR events within 50 kb of the telomeres is three. The difference between the observed and expected distribution is significant (p = 0.005). The interpretation of this observation will be discussed below. Since the induction of recombination by UV is assumed to reflect the location of processed or unprocessed pyrimidine dimers and since most (68%) of the dimers formed involve TT [33], we examined the frequencies of AA/TT dinucleotides in unselected conversion events (0.218), selected conversion tracts on chromo- some V in cells irradiated with 1 J/m2 (0.217) or 15 J/m2 (0.221). These frequencies were similar to those observed for the whole genome (0.217) or the left arm of chromosome V (0.219). In contrast, the frequency of AA/TT dinucleotides in the ribosomal DNA is very significantly (p,0.0001, Fisher exact test) reduced compared to the rest of the yeast genome, 0.190 compared to 0.217. Location of LOH events induced by UV on chromosome V and throughout the genome. The pattern of UV-induced crossovers in the 119 kb interval between the SUP4-o marker and CEN5 is shown in Figure 6A. In this figure, the X-axis shows SGD coordinates between SUP4-o (33 kb) and CEN5 (152 kb). The Y- axes show the number of times individual SNPs were included within conversion tracts associated with crossovers with the blue and red lines indicating spontaneous [2] and UV-induced events, respectively. The number of times that a specific SNP will be included within a conversion tract is a function of the frequency with which recombination initiates near the SNP and the length of the conversion tract emanating from the initiating lesion [29]. To simplify the analysis of the distribution of recombination events, we determined the mid-point of each conversion tract. UV-Induced Recombination in Yeast To support the conclusion that UV treatment is less recombinogenic for the rDNA than for other genomic sequences, we analyzed UV-induced recombination in a diploid strain YYy13 with heterozygous markers flanking the rDNA cluster (HYG and URA3) and within the cluster (TRP1) (Figure 7); the TRP1 gene within the cluster is located about 230 kb from the centromere- distal end of the cluster [34]. This strain is in a different genetic background than PG311, but is a MATa deletion derivative of AMC45, a strain in which mitotic crossovers were examined previously [34]. G1-synchronized YYy13 cells were irradiated with 15 J/m2 UV and plated non-selectively on omission medium. The resulting colonies were replica-plated to medium containing hygromycin, medium lacking tryptophan, and medium lacking uracil, and the phenotypes of sectored colonies were scored. Crossovers centromere-proximal to HYG result in one HygR Trp+ Ura2 sector and one HygS Trp2 Ura+ sector, whereas crossovers between HYG and TRP1 produce one HygR Trp+ Ura2 sector and one HygR Trp2 Ura+ sector (Figures 7A and 7B). The expected sectoring pattern for a strain with a crossover in the rDNA between the TRP1 insertion and the URA3 marker is shown in Figure 7C. In addition to sectors formed by crossovers, BIR events can also produce sectored colonies (Figure S3). One distinction between crossovers and BIR events is that the Ura+ sector of crossovers is homozygous for the URA3/URA3 wild-type alleles, whereas for BIR events, the Ura+ sectors are often heterozygous ura3/URA3. These two types of Ura+ strains can be distinguished by replica- plating cells grown non-selectively to medium containing 5-fluoro- orotate (5-FOA). 5-FOA poisons cells with wild-type URA3 Analysis of UV-induced crossovers within the ribosomal DNA. To support the conclusion that UV treatment is less recombinogenic for the rDNA than for other genomic sequences, we analyzed UV-induced recombination in a diploid strain YYy13 with heterozygous markers flanking the rDNA cluster (HYG and URA3) and within the cluster (TRP1) (Figure 7); the TRP1 gene within the cluster is located about 230 kb from the centromere- distal end of the cluster [34]. This strain is in a different genetic background than PG311, but is a MATa deletion derivative of AMC45, a strain in which mitotic crossovers were examined previously [34]. G1-synchronized YYy13 cells were irradiated with 15 J/m2 UV and plated non-selectively on omission medium. UV-Induced Recombination in Yeast Crossover associated with a 3:1 conversion event. C. Crossover associated with a 3:1/4:0 hybrid conversion tract. D. Complex crossover/conversion event. E. 3:1 conversion unassociated with a crossover. F. BIR event. doi:10.1371/journal.pgen.1003894.g005 Because of the low frequency of events on chromosome V, we examined UV-induced recombination in G2 cells of the strain JSC25 in which the SUP4-o reporter gene is located near the end of chromosome IV [29]; the CEN4-SUP4-o interval is about eight- fold larger than the CEN5-SUP4-o interval. The frequency of red/ white sectored colonies in G1-synchronized JSC25 cells irradiated with 15 J/m2 was 961022 (127 sectors out of 1420 colonies). About 1% of the colonies formed red/white sectors in G2 cells synchronized with nocodazole (G2-A experiment) or in unsyn- chronized cells in which large budded cells were manipulated to defined positions before irradiation (G2-B experiment). We used microarrays to examine six and seven sectored colonies derived from the G2-A and G2-B experiments, respectively. Although we anticipated that most of the LOH events induced by G2 radiation would represent SCB conversions, among the G2-A LOH events, most (14 of 15) resembled DSCB conversions; among the G2-B LOH events, DSCB and SCB conversions were approximately equal in frequency (20 DSCB and 17 SCB). These results are difficult to interpret for a variety of reasons. First, the two protocols for obtaining G2 cells gave significantly different results; it should be pointed out that the nocodazole-induced synchronization is usually incomplete, with about 80–90% of the cells with the October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org PLOS Genetics | www.plosgenetics.org 9 UV-Induced Recombination in Yeast October 2013 | Volume 9 | Issue 10 | e1003894 UV-Induced Recombination in Yeast We then determined whether the distributions of these midpoints were significantly different in spontaneous and UV-induced events by comparing the numbers of events in four equal-sized intervals (33– 63 kb, 63–93 kb, 93–123 kb, and 123–153 kb). By Fisher exact test, the distributions of spontaneous and UV-induced events are not significantly different. In addition, the distribution of UV- induced events on chromosome V is not significantly different from a random distribution. p y g , p Analysis of UV-induced crossovers within the ribosomal DNA. To support the conclusion that UV treatment is less recombinogenic for the rDNA than for other genomic sequences, we analyzed UV-induced recombination in a diploid strain YYy13 with heterozygous markers flanking the rDNA cluster (HYG and URA3) and within the cluster (TRP1) (Figure 7); the TRP1 gene within the cluster is located about 230 kb from the centromere- distal end of the cluster [34]. This strain is in a different genetic background than PG311, but is a MATa deletion derivative of AMC45, a strain in which mitotic crossovers were examined previously [34]. G1-synchronized YYy13 cells were irradiated with 15 J/m2 UV and plated non-selectively on omission medium. The resulting colonies were replica-plated to medium containing hygromycin, medium lacking tryptophan, and medium lacking uracil, and the phenotypes of sectored colonies were scored. Crossovers centromere-proximal to HYG result in one HygR Trp+ Ura2 sector and one HygS Trp2 Ura+ sector, whereas crossovers between HYG and TRP1 produce one HygR Trp+ Ura2 sector and one HygR Trp2 Ura+ sector (Figures 7A and 7B). The expected sectoring pattern for a strain with a crossover in the rDNA between the TRP1 insertion and the URA3 marker is shown in Figure 7C. In addition to sectors formed by crossovers, BIR events can also produce sectored colonies (Figure S3). One distinction between crossovers and BIR events is that the Ura+ sector of crossovers is homozygous for the URA3/URA3 wild-type alleles, whereas for BIR events, the Ura+ sectors are often heterozygous ura3/URA3. These two types of Ura+ strains can be distinguished by replica- plating cells grown non-selectively to medium containing 5-fluoro- orotate (5-FOA). 5-FOA poisons cells with wild-type URA3 Analysis of UV-induced crossovers within the ribosomal DNA. UV-Induced Recombination in Yeast The X-axis shows the number of events that include specific SNPs with the left scale indicating the number of spontaneo events and the right scale showing the number of UV-induced events. The Y axis shows the SGD coordinates on V between the can1-100/SUP markers (near coordinate 33 kb) and CEN5 (near coordinate 152 kb). B. Location of unselected conversion and crossovers. Events on the left arm chromosome V are not shown, since these events were selected. Centromeres are shown as black circles, and SNPs are indicated as short orange ba Gene conversion tracts unassociated with crossovers are depicted by black line segments, with the length of the line approximately equivalent to t tract length. Red arrows show the positions of conversions associated with crossovers, and BIR events are indicated by green arrows. The lengths the chromosomes are shown to scale; chromosome I is about 230 kb. doi:10.1371/journal.pgen.1003894.g006 PLOS Genetics | www plosgenetics org 11 October 2013 | Volume 9 | Issue 10 | e10038 Figure 6. Patterns of selected events on chromosome V and unselected events throughout the genome. A. Selected events on the left arm of chromosome V. All events were based on red/white sectored colonies. The blue and red lines represent spontaneous [2] and UV-induced (15 J/m2), respectively. The X-axis shows the number of events that include specific SNPs with the left scale indicating the number of spontaneous events and the right scale showing the number of UV-induced events. The Y axis shows the SGD coordinates on V between the can1-100/SUP4-o markers (near coordinate 33 kb) and CEN5 (near coordinate 152 kb). B. Location of unselected conversion and crossovers. Events on the left arm of chromosome V are not shown, since these events were selected. Centromeres are shown as black circles, and SNPs are indicated as short orange bars. Gene conversion tracts unassociated with crossovers are depicted by black line segments, with the length of the line approximately equivalent to the tract length. Red arrows show the positions of conversions associated with crossovers, and BIR events are indicated by green arrows. The lengths of the chromosomes are shown to scale; chromosome I is about 230 kb. doi:10.1371/journal.pgen.1003894.g006 Figure 6. Patterns of selected events on chromosome V and unselected events throughout the genome. A. Selected events on the left arm of chromosome V. All events were based on red/white sectored colonies. UV-Induced Recombination in Yeast The resulting colonies were replica-plated to medium containing hygromycin, medium lacking tryptophan, and medium lacking uracil, and the phenotypes of sectored colonies were scored. Crossovers centromere-proximal to HYG result in one HygR Trp+ Ura2 sector and one HygS Trp2 Ura+ sector, whereas crossovers between HYG and TRP1 produce one HygR Trp+ Ura2 sector and one HygR Trp2 Ura+ sector (Figures 7A and 7B). The expected sectoring pattern for a strain with a crossover in the rDNA between the TRP1 insertion and the URA3 marker is shown in Figure 7C The map positions of unselected LOH events in cells treated with UV doses of 15 J/m2 are shown in Figure 6B. There were 60 crossovers, 21 BIR events, and 300 gene conversion events unassociated with crossovers. These events are widely distributed throughout the genome with the number of LOH events correlating strongly with the size of the chromosome (r2 = 0.90, p = 2.461028). Although none of the individual chromosomes, normalized for size, were significantly hot or cold for LOH events, the right arm of chromosome VIII had a significantly elevated frequency of crossovers as determined by chi-square analysis (p = 0.03), correcting for multiple comparisons by the method of Hochberg and Benjamini (1990) [32]. In addition, based on the total number of unselected crossovers in the dataset of Table S3 (60) and the fraction of the genome that is the rRNA gene cluster (10%), there is significant suppression of UV-induced crossovers the TRP1 insertion and the URA3 marker is shown in Figure 7C. In addition to sectors formed by crossovers, BIR events can also produce sectored colonies (Figure S3). One distinction between crossovers and BIR events is that the Ura+ sector of crossovers is homozygous for the URA3/URA3 wild-type alleles, whereas for BIR events, the Ura+ sectors are often heterozygous ura3/URA3. These two types of Ura+ strains can be distinguished by replica- plating cells grown non-selectively to medium containing 5-fluoro- orotate (5-FOA). 5-FOA poisons cells with wild-type URA3 October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 10 UV-Induced Recombination in Yeast Figure 6. Patterns of selected events on chromosome V and unselected events throughout the genome. A. Selected events on the arm of chromosome V. All events were based on red/white sectored colonies. The blue and red lines represent spontaneous [2] and UV-induc (15 J/m2), respectively. UV-Induced Recombination in Yeast The blue and red lines represent spontaneous [2] and UV-induced (15 J/m2), respectively. The X-axis shows the number of events that include specific SNPs with the left scale indicating the number of spontaneous events and the right scale showing the number of UV-induced events. The Y axis shows the SGD coordinates on V between the can1-100/SUP4-o markers (near coordinate 33 kb) and CEN5 (near coordinate 152 kb). B. Location of unselected conversion and crossovers. Events on the left arm of chromosome V are not shown, since these events were selected. Centromeres are shown as black circles, and SNPs are indicated as short orange bars. Gene conversion tracts unassociated with crossovers are depicted by black line segments, with the length of the line approximately equivalent to the tract length. Red arrows show the positions of conversions associated with crossovers, and BIR events are indicated by green arrows. The lengths of the chromosomes are shown to scale; chromosome I is about 230 kb. doi:10.1371/journal.pgen.1003894.g006 October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 11 11 UV-Induced Recombination in Yeast Figure 7. System to monitor crossovers on chromosome XII. A diploid was constructed with heterozygous markers immediately centromere- proximal to the ribosomal RNA (rRNA) gene clusters (HYG), within the rRNA gene cluster (TRP1), and immediately centromere-distal to the cluster (URA3) [34]. G1-synchronized cells were treated with UV, plated on solid medium and grown non-selectively. The resulting colonies were replica- plated to medium lacking uracil, tryptophan, or containing hygromycin as described in the text. A. Sectoring pattern expected for a crossover centromere-proximal to the HYG marker. B. Sectoring pattern expected for a crossover within the rRNA gene cluster centromere-proximal to TRP1. C. Sectoring pattern expected for a crossover within the rRNA gene cluster centromere-distal to TRP1. doi:10.1371/journal.pgen.1003894.g007 Figure 7. System to monitor crossovers on chromosome XII. A diploid was constructed with heterozygous markers immediately centromere- proximal to the ribosomal RNA (rRNA) gene clusters (HYG), within the rRNA gene cluster (TRP1), and immediately centromere-distal to the cluster (URA3) [34]. G1-synchronized cells were treated with UV, plated on solid medium and grown non-selectively. The resulting colonies were replica- plated to medium lacking uracil, tryptophan, or containing hygromycin as described in the text. A. Sectoring pattern expected for a crossover centromere-proximal to the HYG marker. B. Sectoring pattern expected for a crossover within the rRNA gene cluster centromere-proximal to TRP1. C. UV-Induced Recombination in Yeast BIR-U and BIR-H indicate that the BIR event was initiated by a DSB located on the URA3- or HYG-containing chromosome XII homolog, respectively. Since a BIR event initiated on the HYG-containing chromosome between TRP1 and URA3 does not result in sectors on any of the media, the number of events in this category (shown in parentheses) was assumed to be the same as the number on the URA3-containing chromosome. The ICR column shows the number of intrachromosomal events (intrachromatid single-strand annealing, intrachromatid ‘‘pop-outs’’ or unequal sister-chromatid exchanges) as shown in Figure S4. aTo calculate the normalized number of UV-induced events, we multiplied the number of observed events in the untreated cells by the ratio of the colonies examined for the untreated and UV-treated cells (3902/5197), and subtracted that value from the observed number of UV-treated cells in each category. doi:10.1371/journal.pgen.1003894.t001 four times less than observed for two other large chromosomal intervals (CEN12-HYG and CEN5-SUP4-o). By chi-square analysis, the rDNA is significantly colder for UV-induced crossovers than the CEN12-HYG interval (p,0.0001). sectored colony). Consequently, in the additional thirty-six sectored colonies examined, we used microarrays specific for detecting LOH on chromosome V. The depictions of the LOH events in the 1 J/m2 irradiated samples that had the same patterns as observed for the 15 J/m2 samples are shown in Table S2; the numbers of samples with specific classes of events are shown in parentheses in this table. Patterns of LOH that were unique to the 1 J/m2 samples are shown in Table S6. The coordinates for these LOH events are shown in Table S7. The distribution of the LOH events on chromosome V for the 1 J/m2 samples was not significantly different from that observed for the 15 J/m2 samples or the spontaneous events using the same ‘‘binning’’ procedure and statistical test described above. The median length of conversion events associated with crossovers on chromosome V in cells irradiated with 1 J/m2 was 4.3 kb (2.3 kb–8.2 kb; 95% confidence limits) kb. In cells irradiated with 15 J/m2, the median length of conversion tracts associated with crossovers on chromosome V was 6.7 (4.2–13 kb). The distributions of tract lengths analyzed by the Mann-Whitney test showed that these distributions were not significantly different (p = 0.12). We estimated 11 and 36 BIR events in the CEN12-HYG and rDNA, respectively (Table 1). UV-Induced Recombination in Yeast Although the frequency of BIR events per kb in the rDNA is slightly less than in the CEN12-HYG interval, the difference is not statistically significant. We found that intrachromosomal recombination events were also substantially elevated by UV, 116 events/3902 treated cells versus 6 events in 5197 cells not exposed to UV. We did not determine whether the intrachromosomal events were a consequence of unequal sister- chromatid crossovers, intrachromatid ‘‘pop-outs’’ or single-strand annealing events. A high frequency of spontaneous mitotic intrachromosomal recombination events in the rDNA was previously observed in yeast [34–37]. In summary, although UV treatment elevates the frequency of crossovers, BIR events, and intrachromosomal recombination events in the rDNA relative to that observed in unirradiated strains, the magnitude of the induction of crossovers in the rDNA is significantly less than for other regions of the genome. A striking difference was observed in the distributions of events diagnostic of SCBs and DSCBs in cells irradiated with 1 and 15 J/ m2 of UV. Of selected events on chromosome V in cells irradiated with 1 J/m2, we observed 5 crossovers unassociated with conversion, 31 SCB events, and 10 DSCB events. In contrast, in cells irradiated with 15 J/m2, most of the selected events on chromosome V were DSCB events (Figure 8). By the Fisher exact test, the difference in the numbers of SCB and DSCB events induced by the two different UV treatments is very significant (p,0.0001). The conclusion that G1-synchronized cells have different recombinogenic DNA lesions induced by different UV doses will be discussed further below. UV-Induced Recombination in Yeast Sectoring pattern expected for a crossover within the rRNA gene cluster centromere-distal to TRP1. doi:10.1371/journal.pgen.1003894.g007 In addition to determining the frequencies of UV-induced recombination events in YYy13, we also determined the frequen- cies of spontaneous events. The summary of crossovers, BIR, and intrachromosomal events is shown in Table 1. In a sample of 3902 colonies derived from G1-synchronized irradiated cells, we observed 49 crossovers in the 300 kb CEN12-HYG interval and 92 crossovers in the 1170 kb HYG-URA3 interval containing the rRNA gene cluster. After correcting for the expected number of spontaneous events and dividing the frequencies of crossovers by the size of the interval, we calculate that the frequencies of UV- induced crossovers in the CEN12-HYG and HYG-URA3 interval are 8.261025/kb and 3.861025/kb, respectively. The frequency of UV-induced crossovers in the 120 kb CEN5 to SUP4-o region is 1.561024/kb. These results demonstrate that the frequency of UV-induced crossovers within the rDNA per kb is about two to activity. Heterozygous strains form 5-FOAR papillae on medium containing 5-FOA because the wild-type allele is readily lost by mitotic recombination or mutation; in contrast, strains that are homozygous for the wild-type allele very rarely produce 5-FOAR papillae [34]. A third type of mitotic recombination that can be detected in irradiated YYy13 cells is intrachromosomal exchange. In this type of event, sectoring is observed for the TRP1 marker, but not the flanking HYG and URA3 markers (Figure S4). Loss of the TRP1 marker could reflect either single-strand annealing [4], unequal crossing-over between sister chromatids, or ‘‘pop-out’’ of TRP1 by an intrachromatid crossover flanking the insertion. In summary, by testing growth of the sectors on a variety of types of media, we can distinguish whether sectored colonies reflect crossovers, BIR events, or intrachromosomal exchanges (details in Text S1). October 2013 | Volume 9 | Issue 10 | e1003894 12 PLOS Genetics | www.plosgenetics.org UV-Induced Recombination in Yeast Table 1. Numbers of colonies with spontaneous and UV-induced crossovers between CEN12 and the ribosomal DNA and within the ribosomal DNA. UV-Induced Recombination in Yeast CEN12-HYG interval HYG-TRP1 interval TRP1-URA3 interval ICR # colonies examined RCO BIR-U BIR-H RCO BIR-U BIR-H RCO BIR-U BIR-H UV-treated cells 49 9 2 77 16 9 15 8 (8) 116 3902 Untreated cells 1 0 0 5 2 1 0 1 (1) 6 5197 Normalized UV-treateda 48 9 2 73 14 8 15 7 (7) 111 3902 In this table, we show data obtained from spontaneous and UV-induced cells derived from the strain YYy13. As shown in Figure 7 and Figure S3, from examining the segregation of markers, we can determine the location of crossover and BIR events on chromosome XII by detecting colonies that form sectors on media lacking uracil or tryptophan or media containing hygromycin. BIR-U and BIR-H indicate that the BIR event was initiated by a DSB located on the URA3- or HYG-containing chromosome XII homolog, respectively. Since a BIR event initiated on the HYG-containing chromosome between TRP1 and URA3 does not result in sectors on any of the media, the number of events in this category (shown in parentheses) was assumed to be the same as the number on the URA3-containing chromosome. The ICR column shows the number of intrachromosomal events (intrachromatid single-strand annealing, intrachromatid ‘‘pop-outs’’ or unequal sister-chromatid exchanges) as shown in Figure S4. aTo calculate the normalized number of UV-induced events, we multiplied the number of observed events in the untreated cells by the ratio of the colonies examined for the untreated and UV-treated cells (3902/5197), and subtracted that value from the observed number of UV-treated cells in each category. doi:10.1371/journal.pgen.1003894.t001 Table 1. Numbers of colonies with spontaneous and UV-induced crossovers between CEN12 and the ribosomal DNA and within the ribosomal DNA. Table 1. Numbers of colonies with spontaneous and UV-induced crossovers between CEN12 and the ribosomal DNA and within the ribosomal DNA. Table 1. Numbers of colonies with spontaneous and UV-induced crossovers between CEN12 and the ribosomal DNA and within the ribosomal DNA. In this table, we show data obtained from spontaneous and UV-induced cells derived from the strain YYy13. As shown in Figure 7 and Figure S3, from examining the segregation of markers, we can determine the location of crossover and BIR events on chromosome XII by detecting colonies that form sectors on media lacking uracil or tryptophan or media containing hygromycin. Analysis of LOH events induced by UV-irradiation of G1- synchronized cells with a dose of 1 J/m2 Analysis of LOH events induced by UV-irradiation of G1- synchronized cells with a dose of 1 J/m2 One interpretation of our observation of frequent DSCBs in G1-irradiated cells is that the repair of two very closely-spaced single-stranded DNA lesions induced by 15 J/m2 results in DSCBs in the G1-synchronized cells, whereas SCBs reflect DNA lesions on one strand. Thus, the productions of DSCBs by this mechanism would be proportional to the square of UV dosage, whereas the frequency of SCBs would be linearly proportional to the UV dosage. By this model (details to be discussed below), one might expect that a low dose of UV should have a relatively higher frequency of SCBs. Consequently, we examined the frequency and types of recombination events induced in G1-synchronized cells by 1 J/m2. As expected, the frequency of red/white sectored colonies was reduced in cells irradiated with 1 J/m2 relative to cells irradiated with 15 J/m2 (1.661023/division versus 9.461023/ division). Ten sectored colonies were examined by whole-genome microarrays. Only four unselected events were observed. This frequency (0.4 events/sectored colony) was about twenty-fold less than that observed in samples irradiated with 15 J/m2 (8 events/ UV-Induced Recombination in Yeast It can be calculated that diploid cell irradiated with 15 J/m2 have about 7500 dimers/genome [43]; if these dimers are distributed randomly, we expect about 35 closely- opposed (separated by #75 bases) dimers, enough to explain the detected DSCB events. It is likely that the number of closely- spaced dimers is greater than that determined by this calculation. Lam and Reynolds (1987) [43] found that the fraction of dimers located within 15 base pairs of each other is greater than expected from a random distribution, and this fraction is somewhat independent of UV dose. These dimers may be responsible for the DSCB events detected in strains treated with the 1 J/m2 UV dose. g ( ) [ ] A central issue is the nature of the recombinogenic DNA damage generated by UV. Based on the mechanism of NER and on the observation that unrepaired pyrimidine dimers block replication, there are two obvious potential sources of DSBs [40]. First, if a DNA molecule with an unrepaired gap resulting from NER is replicated before filling-in of the gap and ligation, the net result would be a pair of sister chromatids with a single DSB (Figure 9A). Alternatively, if a replication fork encounters an unrepaired UV-induced lesion, breakage of the fork could also result in a single broken chromatid (Figure 9B). Based on the observation that UV treatment of G1- or G2-synchronized cells was not recombinogenic unless cells were allowed to divide, Galli and Schiestl (1999) [20] suggested that cell division was required to convert DNA lesions to recombinogenic lesions, consistent with both of the possibilities described above; their assay detected only intrachromatid deletions. Kadyk and Hartwell (1993) [21] found that unrepaired UV lesions stimulate gene conversion events between homologs, but have little effect on mitotic crossovers. This conclusion may be affected by the use of the rad1 mutation to prevent dimer excision, since rad1 strains have reduced frequencies of crossovers in some assays [41]. In summary, we suggest that low doses of UV primarily result in SCBs as a consequence of replication of a chromosome with a NER-generated DNA gap in one strand, or an unrepaired dimer resulting in breakage of one arm of the replication fork. In contrast, we suggest that high doses of UV often result in DSCB events as a consequence of a G1-generated DSB, reflecting cellular enzymes acting on closely-opposed dimers. UV-Induced Recombination in Yeast As observed in our previous studies [2,7,29], the mitotic conversion tracts are long compared to those observed in meiosis, and the tracts associated with crossovers are longer than the tracts unassociated with crossovers. Most of the conversion events are explicable as a consequence of repair of one broken chromatid or two sister chromatids broken at the same position by the standard HR pathways shown in Figure 1, with only conversion-type MMR and not restoration-type MMR. About 15% of the conversion events, however, are more complex, requiring ‘‘patchy’’ repair of mismatches within a heteroduplex (mismatches corrected by both conversion-type repair and restoration-type repair within one heteroduplex), and/or branch migration of the Holliday junction. The fraction of complex conversion tracts in the current study is similar to those observed in our previous studies [7,29]. Although these events (described in detail in Text S1 and Figures S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15) are explicable by modifications of the standard models shown in Figure 1, it is possible that some of these conversion events involve a substan- tially different mechanism such as multiple template switching events during BIR. In this context, template switching during BIR has been observed in experiments in which linear DNA fragments are transformed into yeast [39]. In addition to the complex tracts, it is possible that the very long conversion tracts reflect BIR rather than mismatch repair in a heteroduplex; 16% of the conversion events unassociated with crossovers are greater than 10 kb in length, and the longest exceeds 50 kb. Finally, it should be pointed that, although single BIR events would not be expected to generate crossovers, a model for production of a crossover by a double BIR event is shown in Figure S4 of Lee et al. (2009) [2]. Based on our results and those of others, it is likely that UV produces a variety of recombinogenic lesions. In our experiments, at a low dose of UV (1 J/m2), we observed primarily SCBs, consistent with the two models shown in Figures 9A and 9B. At 15 J/m2, we observed DSCBs more frequently than SCBs. This observation supports models shown in Figure 9C and 9D that require closely-opposed lesions, and argues against the model shown in Figure 9E in which the relative fraction of DSCB and SCB events would be expected to be independent of the density of the NER tracts. UV-Induced Recombination in Yeast UV-Induced Recombination in Yeast treatment of G1-synchronized cells effectively stimulated recombi- nation between homologs. Our previous interpretation of both spontaneous and DNA damage-induced crossovers is also consistent with this conclusion [2,7,29]. We argue that most spontaneous crossovers between homologs are initiated by a DSB in G1 in one chromosome, and replication of the broken chromosome produces two sister chromatids broken at the same position. Since these lesions cannot be repaired by sister-chromatid recombination, they are repaired by recombination with the homolog. Although it is likely that DSBs formed in S or G2 are primarily repaired by sister- chromatid recombination, some DSBs generated in G2 are repaired by interaction with the homolog [11]. gene conversion events involving a single broken chromatid (SCBs). In our study, about two-thirds of the conversion events in which cells were irradiated with 15 J/m2 reflect two broken sister chromatids, but only one-quarter of the conversions reflect two broken sister chromatids in cells irradiated with 1 J/m2 (Figure 8). Thus, there is a qualitative change in the nature of the DNA lesion with increasing UV dose. In addition, since our single-cell experiments demonstrate that UV-induced lesions are recombino- genic during the first division following treatment, DSCBs cannot be explained as reflecting the segregation of a chromosome with an unrepaired G2-associated DSB from the previous division. We suggest that most DSCBs are a consequence of a DSB in G1. Although UV damage is generally regarded as an agent that produces DNA nicks rather than DSBs, a gel-based detection of the conversion of a circular chromosome to a linear chromosome indicated that a dose of 40 J/m2 produces 5 to 10 DSBs in G2- synchronized cells [42]. There are several related mechanisms by which NER could produce a DSB in G1 cells. First, the excision tracts resulting from removal of two closely-opposed dimers could result in very short (,6 bp) unstable duplex regions between the repair tracts, resulting in a DSB (Figure 9C). A second model is that, following the removal of two closely-opposed dimers by NER, one or both of the resulting short gaps is expanded by Exo1p (Figure 9D). A third related model is that the excision tract generated by NER is expanded into a large single-stranded gap that is cleaved by an endonuclease to yield the DSB (Figure 9E). Discussion Our main conclusions are: 1) UV induces high frequencies of crossovers and gene conversions in G1-synchronized yeast cells but is less recombinogenic in G2-enriched cells, 2) the LOH events induced by high doses of UV primarily involve the repair of two sister chromatids broken at the same position whereas most events induced by low doses (1 J/m2) involve repair of a single broken chromatid, 3) UV-induced LOH events are widely distributed October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 13 UV-Induced Recombination in Yeast Figure 8. Selected SCB and DSCB conversions in strains treated with 1 J/m2 and 15 J/m2. SCB and DSCB events are indicated in gray and red, respectively. doi:10.1371/journal.pgen.1003894.g008 Figure 8. Selected SCB and DSCB conversions in strains treated with 1 J/m2 and 15 J/m2. SCB and DSCB events are indicated in gray and red, respectively. doi:10.1371/journal.pgen.1003894.g008 Recombinogenic DNA lesions induced by UV As noted in previous studies, UV very effectively induces mitotic recombination in yeast [7,10,20,21,23,38]. In experiments involv- ing heteroallelic recombination in synchronized cells, UV is somewhat more recombinogenic in G1-synchronized cells than in G2-synchronized cells [18,21]; our results support these observa- tions. Kadyk and Hartwell (1992) [24] concluded that DSBs induced by X-rays in G2-synchronized cells were repaired primarily by sister-chromatid recombination, whereas X-ray throughout the genome, although some classes of repeated genes are significantly underrepresented, 4) most of the recombinogenic effects of UV in cells treated in G1-synchronized cells are manifested in the first cell cycle after irradiation, 5) in G1- synchronized cells, crossovers are induced about six-fold more frequently than BIR events, 6) about one-third of UV-induced conversion events are associated with crossovers, and 7) although UV effectively induces crossovers between homologs, chromosome rearrangements are not produced at detectable frequencies. throughout the genome, although some classes of repeated genes are significantly underrepresented, 4) most of the recombinogenic effects of UV in cells treated in G1-synchronized cells are manifested in the first cell cycle after irradiation, 5) in G1- synchronized cells, crossovers are induced about six-fold more frequently than BIR events, 6) about one-third of UV-induced conversion events are associated with crossovers, and 7) although UV effectively induces crossovers between homologs, chromosome rearrangements are not produced at detectable frequencies. October 2013 | Volume 9 | Issue 10 | e1003894 October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 14 PLOS Genetics | www.plosgenetics.org UV-Induced Recombination in Yeast Excision of a dimer results in a small gap and replication produces one broken and one unbroken sister chromatid. B. During replication of a DNA molecule with an unexcised dimer, a DSB occurs in one of the two sister chromatids. C. Excision of two closely-opposed dimers results in a short (,6 bp) unstable double-stranded region between the excision tracts. The resulting broken chromosome is replicated to form two broken sister chromatids. D. As in Figure 9C, two closely-opposed dimers are excised. One of the resulting short gaps is expanded by the 59 to 39 Exo1p nuclease (shown in green) to generate a broken chromosome. Replication of this chromosome results in two broken sister chromatids. E. The tract resulting form excision of a single dimer is expanded, leaving a large single- stranded DNA gap. An endonuclease cleaves this single-stranded region, resulting in two broken sister chromatids. doi:10.1371/journal.pgen.1003894.g009 UV-Induced Recombination in Yeast Although this expla- nation seems straightforward, we cannot exclude more complex explanations of our data. For example, it is possible that the very large number of UV-induced lesions at high doses may overwhelm the DNA repair systems, resulting in changes in the use of repair pathways. In addition, we stress that our analysis based on interhomolog recombination does not yield an estimate of the relative frequencies of UV-induced recombinogenic lesions produced in G1, S, and G2, since most recombinogenic lesions produced in S and G2 are likely repaired by sister-chromatid recombination [24], a mechanism that does not lead to LOH [7]. Both of the models discussed above predict that UV-induced DNA damage in G1-synchronized cells would produce primarily October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 15 UV-Induced Recombination in Yeast Figure 9. Mechanisms for generating UV-induced recombinogenic DSBs. At the top part of the figure, chromosomal DNA molecules are depicted as unreplicated double-stranded DNA molecules. Newly-synthesized DNA is depicted as gray dashed lines. UV-induced pyrimidine dimers are shown as triangles, and centromeres of replicated chromosomes are shown as ovals. A. Excision of a dimer results in a small gap and replication produces one broken and one unbroken sister chromatid. B. During replication of a DNA molecule with an unexcised dimer, a DSB occurs in one of the two sister chromatids. C. Excision of two closely-opposed dimers results in a short (,6 bp) unstable double-stranded region between the excision tracts. The resulting broken chromosome is replicated to form two broken sister chromatids. D. As in Figure 9C, two closely-opposed dimers are excised. One of the resulting short gaps is expanded by the 59 to 39 Exo1p nuclease (shown in green) to generate a broken chromosome. Replication of this chromosome results in two broken sister chromatids. E. The tract resulting form excision of a single dimer is expanded, leaving a large single- stranded DNA gap. An endonuclease cleaves this single-stranded region, resulting in two broken sister chromatids. doi:10.1371/journal.pgen.1003894.g009 Figure 9. Mechanisms for generating UV-induced recombinogenic DSBs. At the top part of the figure, chromosomal DNA molecules are depicted as unreplicated double-stranded DNA molecules. Newly-synthesized DNA is depicted as gray dashed lines. UV-induced pyrimidine dimers are shown as triangles, and centromeres of replicated chromosomes are shown as ovals. A. Yeast strains Most of our experiments were done with the diploid strain PG311, a hybrid that is heterozygous for about 52,000 SNPs [2]. The PG311 genotype is: MATa/MATa::NATade2-1/ade2-1 can1- 100/can1D::SUP4-o ura3-1/URA3 trp1-1/TRP1 his3-11,15/HIS3 leu2-3,112/LEU2 V9229::HYG/V9229 V261553::LEU2/V261553 GAL2/gal2 RAD5/RAD5. Additional features of this strain are described in Text S1 (Supplemental Materials and Methods). We also describe the strains JSC24,JSC25 and PSL101, all of which are isogenic to PG311 except for the specified alterations. The experiments to measure recombination within the ribosomal RNA genes were done with the diploid AMC45 that is heterozygous for markers flanking the array and within the array [34]. The diploid YYy13 is a MATa::NAT derivative of AMC45. Unlike the other strains used in our analysis, AMY45 and YYy13 are not isogenic with PG311. About 60% of the UV-induced BIR events appear to be randomly distributed, whereas the remainder have a breakpoint located within 50 kb of the telomere. We suggest that there are two types of BIR events, the ‘‘classic’’ type in which one of the chromosome fragments is lost prior to second end capture, and a second type that is initiated by degradation of one of the two homologs beginning at the telomere. In two previous studies [52,53], LOH events near the telomere were observed in strains with mutations in genes affecting telomere structure or replication (CDC13 and EST1). It was not determined in these studies whether these LOH events were crossovers or BIR events. Since the BIR events in our study were induced by UV, one interpretation of our results is that high doses of UV are associated with telomere uncapping or some other telomere defect. An alternative explanation of our observation that BIR events are enriched near the telomere is that such events are more efficiently initiated and completed than events located more internally on the chromo- some, as demonstrated by Donnianni and Symington [49]. UV-induced BIR events Most previous studies of BIR involve transforming linear fragments of DNA or using strains in which the interacting homologous sequences are flanked by non-homologous regions [4,49]. In contrast, our ability to distinguish BIR from crossovers is based on the recovery of both cells containing recombinant products. Among unselected LOH events examined in G1- synchronized cells irradiated with 15 J/m2, we observed 60 crossovers and 21 BIR events. By the microarray analysis, as described previously, we detect only half of crossovers. All BIR events, however, can be detected. We conclude, therefore, that crossovers are induced about six-fold more than BIR events. This conclusion is in agreement with previous observations of spontaneous recombination events [50], and events induced by the I-SceI endonuclease [51] performed by others. UV-Induced Recombination in Yeast UV-Induced Recombination in Yeast observed 300 conversions without an observable crossover, and 60 crossovers. Because of the pattern of chromosome segregation, we expect only half of the crossovers will lead to LOH distal to the exchange. We calculate that there are likely 240 conversions unassociated with crossovers and 120 associated with crossovers. Thus, we conclude that about one-third of the unselected conversion events are associated with a crossover, similar to our previous conclusion based on a smaller number of events [7]. over-represented in the DSCB conversion tracts (Table S9), is also higher (0.23) than the frequency for the whole genome. A more detailed discussion of the relationship between various chromo- some elements and conversion tracts (Tables S9 and S10) is given in Text S1. Although dimer formation has a simple relationship to DNA sequence, the rate of NER-mediated repair of the dimers is enhanced by transcription and reduced by chromatin silencing and other aspects of chromatin structure [45–47]. Our discussion of dimer repair will be limited to NER, since our experiments were done under conditions in which photoreactivation was prevented. In general, dimer repair is rapid in yeast with the majority of dimers being removed within two hours [48]. Our observation that UV treatment of G1-synchronized cells primarily results in recombination in the first cell cycle following radiation is consistent with efficient dimer repair. Nonetheless, Teng et al. (2011) [44] found genomic regions in which dimer repair was delayed. To test whether these long-lasting lesions could be more recombinogenic than lesions that were quickly repaired, we determined whether the chromosome regions containing the long-lasting lesions were over-represented in our unselected gene conversion tracts (details of the analysis in Text S1). There was not a significant enrichment of the regions with long-lasting lesions in our unselected gene conversion tracts. ssociation between conversion and crossovers In meiosis in S. cerevisiae, roughly half of the conversion events are associated with crossovers [1,54]. The fraction of conversions associated with crossovers varies in different studies from ,5% to 50% [4]. In the unselected events induced by 15 J/m2, we Lack of UV-induced chromosome alterations In our previous analysis of gamma ray-treated G2-synchronized diploids, we observed about two unselected chromosome aberra- tions per irradiated cell [31]. We found that most of these events reflected homologous recombination between Ty elements located at non-allelic positions. In our current study, although UV treatment induced about eight unselected LOH events per cell irradiated with 15 J/m2, we did not detect any large deletions, duplications, or translocations. The difference in these two studies is likely to reflect the total number of DSBs and other recombinogenic lesions generated by the two treatments. In the Argueso et al. study, the gamma ray dose (800 Gray) produces about 250 DSBs/cell. Based on the estimate that 40 J/m2 of UV results in 5–10 DSBs in G2-synchronized cells [42], we expect only about two DSBs/cell as the result of irradiating G1-synchronized cells with 15 J/m2. Since Ty elements, the main target for chromosome rearrangements, represents only a small fraction of the genome (2%), the likelihood of a UV-induced DSB within Ty elements is small, although DSBs located near Ty elements can also contribute to Ty-Ty exchanges [55]. Of the 360 unselected conversion and crossover events induced by UV, 14 included a Ty element; it is unclear whether these events initiated within or nearby the Ty. Although the lack of recombinogenic lesions within or near Ty elements may be sufficient to explain the dearth of chromosome rearrangements, other factors may also be important, since UV does not effectively stimulate recombination between non-allelic Ty elements [56,57]. Media and genetic methods Standard rich growth medium (YPD) and omission media were used for these experiments [58]. We also used standard conditions for tetrad analysis, transformation, and DNA isolation. Distribution of UV-induced LOH events selected (0.221) and unselected (0.218) conversion events are very close to the frequencies on the left arm of chromosome V (0.219) and the whole genome (0.217). In contrast, the frequency of these dinucleotides in the ribosomal DNA (0.19) is very significantly less (p,0.0001) than the frequency in the whole genome. Thus, the observed reduction in UV-induced recombination in the ribo- somal DNA, at least in part, may be a consequence of a reduced frequency of dimer formation. Interestingly, the two motifs that are underrepresented in the unselected conversion events (tRNA and solo LTRs in Table S9) also have frequencies of AA/TT dinucleotides that are considerably less than the genomic frequency (0.124 for tRNA genes and 0.205 for the solo LTRs). Since the tRNA genes and the solo LTRs are smaller than the average conversion tract size, however, it is unlikely that the relative lack of AA/TT dinucleotides is the only factor influencing the frequency of UV-induced conversion events that include these elements. Finally, we note that the frequency of AA/TT dinucleotides in non-coding RNA genes, which are significantly The UV-induced recombination events were broadly distribut- ed throughout the genome; no strong recombination hotspots were detected. The distribution of UV-induced genomic LOH events is expected to be a function of multiple factors such as: 1) the distribution of DNA damage, particularly the distribution of closely-opposed dimers, 2) the relative frequency of dimer repair by recombinogenic and non-recombinogenic pathways, and 3) the relative frequency of repair of recombinogenic DNA damage by sister-chromatid recombination, non-homologous end-joining, and recombination between homologs. The regions on each chromo- some that were examined for LOH events are in Table S8. It has been shown recently that the distribution of UV-induced pyrimidine dimers observed in vivo in yeast is primarily a function of the density of TT, TC, CT, and CC sequences in the genome ([44], Sheera Adar and Jason Lieb, personal communication). As previously discussed, in cells irradiated with 15 J/m2, the calculated frequencies of AA/TT dinucleotides among our October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 16 SNP microarray analysis LOH events in PG311 and related strains were detected using SNP microarrays. For each SNP, these Agilent-constructed microarrays contain four oligonucleotides, one pair that hybridizes to the YJM789-derived SNP allele and another that hybridizes to the W303a-derived SNP allele [7]. About 13,000 SNPs distributed throughout the genome were examined. A short description of the use of SNP microarrays is in the Results section and additional details are given in St. Charles et al. (2012) [7]. In brief, genomic DNA from the experimental strain was labeled with Cy5-dUTP and control DNA from the fully heterozygous strain JSC24-2 was labeled with Cy3-dUTP. The two DNA samples were then hybridized in competition to the SNP microarrays. The micro- array was examined using a GenePix scanner. By measuring the ratio of hybridization of the two differentially-labeled samples, we could determine which SNPs were heterozygous and which were homozygous. (TIF) UV-Induced Recombination in Yeast heterozygous for a gene affecting resistance to hygromycin (HYG), TRP1, and URA3. G1-synchronized cells on rich growth medium were irradiated with UV. After colonies were formed, they were replica-plated to media containing hygromycin, lacking uracil, lacking tryptophan, or containing 5-fluoro-orotate (5-FOA). On plates containing 5-FOA, strains lacking URA3 grow confluently and strains homozygous for URA3 do not grow at all. Strains that are heterozygous for the URA3 marker form 5-FOA-resistant papillae, reflecting the loss of the URA3 marker by recombination or chromosome loss in a small fraction of the cells. A. Marker segregation pattern expected as a consequence of a BIR event initiated by a DSB centromere-proximal to the HYG marker on the homolog with the HYG and TRP1 markers. B. Marker segregation pattern expected as a consequence of a BIR event initiated by a DSB centromere-proximal to the HYG marker on the homolog with the URA3 marker. C. Marker segregation pattern expected as a consequence of a BIR event initiated by a DSB between the HYG and TRP1 markers on the homolog with the HYG and TRP1 markers. D. Marker segregation pattern expected as a consequence of a BIR event initiated by a DSB between the HYG and TRP1 markers on the homolog with the URA3 marker. E. Marker segregation pattern expected as a consequence of a BIR event initiated by a DSB between the TRP1 and URA3 markers on the homolog with the HYG and TRP1 markers. F. Marker segregation pattern expected as a consequence of a BIR event initiated by a DSB between the TRP1 and URA3 markers on the homolog with the URA3 marker. (TIF) and Petes [11]. After two hours of treatment with these agents, the cells were plated on medium lacking arginine and irradiated with UV using a TL-2000 UV Translinker; doses varied between 1 and 15 J/m2. Following the UV treatment, the plates were covered with foil to prevent light-associated removal of dimers, and incubated for two days to allow the formation of sectored colonies. In some experiments, modifications of this protocol were employed as described in Supplemental Materials and Methods. Statistical analyses Figure S4 Patterns of marker segregation in strains with intrachromosomal recombination in the ribosomal DNA. The strain depicted is YYy13 and the markers are shown in the same way as in Figure S3. A. Intrachromosomal deletion. A DSB occurs near the TRP1 insertion, and the resulting processing of the broken ends results in loss of TRP1. Because the rDNA genes are repeated, the broken ends can reanneal (single-strand annealing). B. Unequal sister-chromatid recombination. A crossover between misaligned rDNA tandem arrays results in loss of the TRP1 gene from one chromatid and its duplication in another. (TIF) Most of our statistical analysis involved chi-square analysis, the Fisher exact test, or the Mann-Whitney test. These tests were done using the VassarStat Website (http://vassarstats.net/) or the functions associated with Excel. To calculate 95% confidence limits on the median, we used Table B11 of Altman (1990) [59]. Cell synchronization and UV treatment In most of our experiments, PG311 cells were synchronized in G1 using a-factor or in G2 using nocodazole as described by Lee October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org PLOS Genetics | www.plosgenetics.org 17 UV-Induced Recombination in Yeast Supporting Information Figure S1 Recombination and/or chromosome loss events leading to different types of sectored colonies. Red and white lines depict the different homologs with circles showing centro- meres and triangles showing the location of the SUP4-o insertion. The diploid is homozygous for the ade2-1 mutation and strains with zero, one, and two copies of SUP4-o produce red, pink, and white colonies respectively. A. Reciprocal crossover resulting in a red/white sectored colony. B. Chromosome loss at the first division producing a pink/red sectored colony. C. Reciprocal crossover in the first cell cycle, followed by chromosome loss in one of the daughters, producing a pink/white/red sectored colony. D. Reciprocal crossover in the second division resulting in a pink/ white/red sectored colony. Figure S5 Recombination pathway resulting in Class E1. In this figure, we show the chromosomes as double-stranded DNA molecules with terminal arrow indicating the 39 ends. Red and black lines show sequences from W303a- and YJM789-derived homologs, respectively. Dotted lines indicate DNA synthesis, and blue ovals represent centromeres. The event is initiated by a DSB in G1, resulting in two black chromatids broken at the same place. Chromatid 1 is repaired by SDSA, and Chromatid 2 by formation of a double Holliday junction (processing of the junction indicated by blue triangles). Regions of heteroduplexes (pairing of black and red strands) are shown in blue rectangles; these regions are repaired to generate either two red strands or two black strands before chromosome segregation. The net result of these events is a 3:1/4:0/3:1 conversion tract. Figure S2 LOH patterns in sectors derived from a pink/white/ red sectored colony (78PWR). We isolated purified derivatives from each sector of 78PWR and analyzed their DNA by SNP microarrays. On chromosome II, the samples derived from the white (78W) and red (78R) sectors had identical interstitial LOH events, whereas the sample derived from the pink sector (78P) had an LOH event in which one transition occurred at the same position and the second at a different position from the events in the other two sectors. This pattern is consistent with a DSCB that was repaired in the first division to generate a hybrid 4:0/3:1 tract. (TIF) UV-Induced Recombination in Yeast The UV-induced sectors were generated under experimental conditions that varied slightly (dosage, media, temperature of incubation, and plating method). The conditions under which each sectored colony was obtained are described in this table. 1G1- synchronized cells were treated with UV doses of either 5, 10 or 15 J/m2. 2Before UV treatment, synchronized cells were plated on omission medium lacking arginine with or without canavanine (CAN). 3Plates with the treated cells were incubated at either 30uC. or room temperature (R.T., approximately 23uC). 4Treated cells were either plated and allowed to form colonies without further manipulation (‘‘plating’’) or were plated on a restricted region of the plate and micromanipulated into defined positions before allowing them to form colonies (‘‘single cell’’). Other relevant details are in Text S1. (XLS) Figure S9 Generation of Class G1 by the SDSA pathway. In this figure, we show repair of a single DSB by the SDSA pathway. Mismatches formed in the heteroduplex during strand invasion are repaired. Following dissociation of the invading strand, there are two other regions of heteroduplex (boxed in blue). If the left heteroduplex is corrected to yield two red strands and the right heteroduplex is corrected to generate two black strands, a Class G1 sectored colony can be generated. Figure S10 Generation of Class G1 by branch migration of a double Holliday junction. A single DSB is repaired by forming a double Holliday junction. Following its formation, branch migration occurs (shown by blue arrow), extending the length of the right-hand heteroduplex. Patchy repair within this heterodu- plex produces the observed LOH pattern. (TIF) Table S2 Classes of UV-induced LOH events in sectored colonies resulting from treating G1-synchronized cells with a UV dose of 15 J/m2. 1Related classes of LOH events within sectored colonies were grouped. ‘‘SCB’’ indicates that the class is most readily explained as a consequence of a single-chromatid break, and ‘‘DSCB’’ indicates that the class is most readily explained as reflecting repair of two sister-chromatids broken at the same position. As described in the main text, each sectored colony is represented by a pair of lines; for selected crossovers on chromosome V, the top line shows the pattern in the red sector and the bottom line indicates the pattern in the white sector. Green, red, and black segments represent heterozygous SNPs, SNPs homozygous for the W303a-derived alleles, and SNPs homozygous for the YJM789-derived alleles, respectively. UV-Induced Recombination in Yeast Figure S7 Patterns of LOH expected as a consequence of sister chromatids with DSBs that are close together, but not at the same position. In this figure, we show conversion tracts that are propagated unidirectionally from the DSB as observed by Mitchel et al. (2010) [30]. Assuming that conversion tracts from the two DSBs are propagated independently, four patterns (Figure S7A–S7D) are expected. One- quarter of the events should result in two 3:1 conversion tracts separated by a region of heterozygous SNPs (Figure S7D). (TIF) transitions between different LOH regions were greater than 15 kb apart, we assume that two independent initiation events are involved. A. Two independent recombination events producing Class L1. In this figure, we show one DSB that is repaired, resulting in a 3:1 conversion tract associated with a crossover. The second DSB is repaired by forming a double Holliday junction and resolving the intermediate in the non-crossover mode similar to that shown in Figures 1B and S8. Restoration-type repair would result in a red patch within the black homolog and a black patch within the red homolog. B. Depiction of Class L1 showing regions of conversion in each sector. Figure S8 Recombination pathway resulting in Class F1. The event is initiated by a single DSB on Chromatid 2. Formation of a double Holliday junction results in two regions of heteroduplex. If the double Holliday junction is cleaved symmetrically to form a non-crossover, and the left heteroduplex is corrected to yield two black strands and the right heteroduplex is corrected to yield two red strands, the net result of these events will be a sectored colony with a 3:1 conversion event and a region (HOM) that is homozygous for the W303a-derived SNPs in one sector and for YJM789-derived SNPs in the other. Note that this pattern of LOH is different from that expected by a single crossover in which the LOH region extends to the end of the chromosome. (TIF) Figure S14 Generation of Class M1 by BIR. One broken black chromatid initiates a BIR event that copies a red chromatid to the end of the chromosome. (TIF) Figure S15 Generation of Class P1 by two independent repair events. One of the broken chromatids is repaired by SDSA and the second is repaired by a BIR event. (TIF) Table S1 Conditions for generating the sectors (high UV dose). (TIF) Figure S6 Recombination pathway resulting in Class E3. Symbols are identical to those used for Figure S5. Two sister chromatid breaks are repaired, one by SDSA and one by dissolution of a double Holliday junction (indicated by blue arrows). These events result in a 3:1/4:0/3:1 conversion event similar to that shown in Figure S6. The difference between the two types of conversion tracts is that homozygous regions of the 3:1 tracts are in the same sector in Class E3 and in different sectors in Class E2. (TIF) Figure S3 Patterns of marker segregation reflecting BIR events on the right arm of chromosome XII. The diploid strain YYy13 is October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 18 UV-Induced Recombination in Yeast The lengths of the line segments are not proportional to the sizes of the LOH regions. Small letters indicate transitions between heterozy- gous and homozygous regions. Classes A–G depict gene conversion tracts unassociated with crossovers. Classes H–L are crossover events. Classes M–P are BIR events. Each of the Classes L and P events contains LOH events separated by more than 15 kb. We interpret them as two independently initiated events. The two initiation regions for these two classes are indicated by black boxes or black straight lines. Class Q is a chromosome loss event. See Text S1 for a detailed description of the classes. 2The number of events belonging to each class refers to those observed in 15 J/m2-treated G1 sectors. In Classes H–L, we include the (TIF) Figure S11 Mechanism to generate Class J1. As discussed in Text S1, the homozygous portion of a 3:1 conversion tract is usually associated with the sector that is homozygous for SNPs from the same strain in the crossover LOH region. For example, in Class H3, the homozygous red portion of the 3:1 tract is in the red sector. This pattern is expected for a conversion-associated crossover (Figure S14 of [7]). In Classes J1–J4, however, the conversion event is in the ‘‘wrong’’ sector. This pattern can be explained as a consequence of repair of two DSBs as shown in this figure. In this depiction, we assume that the very small conversion tracts occur between SNPs and are, therefore, not detectable. Similar events have been observed previously (Figure S1 of [2]). (TIF) Figure S12 Mechanism to generate Class J5. In this class, a 3:1 conversion tract is separated from the crossover by a heterozygous region. This event involves the repair of two DSBs associated with the formation of two large regions of heteroduplex. Conversion- type repair of the left heteroduplex is associated with SDSA. The right heteroduplex undergoes restoration-type repair and is associated with a crossover. (XLS) Table S4 Depiction of UV-induced LOH events in 15 J/m2- treated pink/white/red sector (78PWR) from a single-cell experiment. Genomic DNA was isolated from the pink (P), white (W), and red (R) portions of a tri-colored colony, and examined by SNP microarrays. The line depictions of LOH events in each sector use the same code employed in Table S2. (XLS) Table S9 Analysis of over- or under-represented SGD-annotat- ed genome elements within unselected UV-induced conversion tracts. In this table, we summarize our analysis of associations of various SGD-annotated chromosome elements within unselected UV-induced conversion tracts. The details of this analysis are discussed in Text S1. (XLS) Table S5 SGD coordinates for transitions of UV-induced LOH events in the pink/white/red (78PWR) and pink/red (409PR) sectored colonies. The depictions of each class of event for 78PWR are shown in Table S4. All events had identical transitions in the red and pink sectors of 409PR. Transition coordinates were determined in the same way as in Table S3. (XLS) Table S10 Analysis of associations of various DNA motifs, damage-related genome elements, and transcription rates to UV- induced unselected conversion tracts. The details of this analysis and references for the various motifs are in Text S1. (XLS) Text S1 Supplemental materials and methods, and discussion. (DOC) Table S6 Classes of UV-induced selected crossovers in 1 J/m2- treated G1 sectors that were not observed in 15 J/m2-treated G1 sectors. Most of the classes of events in cells treated with 1 J/m2 were the same as for the cells treated with 15 J/m2; these classes are shown in parentheses in Table S2. Classes unique to the cells treated with 1 J/m2 are shown in this table. (XLS) Table S7 SGD coordinates for transitions in UV-induced selected crossovers in 1 J/m2-treated G1 sectors. These transitions are for events depicted in Tables S2 and S6. The table has the same structure as Tables S3 and S5. Author Contributions Conceived and designed the experiments: YY TDP. Performed the experiments: YY. Analyzed the data: YY TDP. Contributed reagents/ materials/analysis tools: YY TDP. Wrote the paper: YY TDP. (TIF) Figure S13 Model to explain Class L1 and related sectors initiated with two independent DSBs. For sectors in which October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 19 References 1. Mancera E, Bourgon R, Brozzi A, Huber W, Steinmetz LM (2008) High- resolution mapping of meiotic crossovers and non-crossovers in yeast. Nature 454: 479–485. 11. Lee PS, Petes TD (2010) From the Cover: mitotic gene conversion events induced in G1-synchronized yeast cells by gamma rays are similar to spontaneous conversion events. Proc Natl Acad Sci U S A 107: 7383–7388. p 12. Budd ME, Campbell JL (1997) The roles of the eukaryotic DNA polymerases in DNA repair synthesis. Mutat Res 384: 157–167. 2. Lee PS, Greenwell PW, Dominska M, Gawel M, Hamilton M, et al. (2009) A fine-structure map of spontaneous mitotic crossovers in the yeast Saccharomyces cerevisiae. PLoS Genet 5: e1000410. 13. Giannattasio M, Follonier C, Tourriere H, Puddu F, Lazzaro F, et al. 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Guo for comments on the manuscript. We thank all members of the Petes and Jinks-Robertson labs for discussions, and S. Jinks-Robertson, S. Kozmin, and A. Guo for comments on the manuscript. October 2013 | Volume 9 | Issue 10 | e1003894 UV-Induced Recombination in Yeast number of events belonging to each class observed in 1 J/m2- treated G1 sectors in the parentheses. number of events belonging to each class observed in 1 J/m2- treated G1 sectors in the parentheses. number of events belonging to each class observed in 1 J/m2- treated G1 sectors in the parentheses. (XLS) Table S8 SGD coordinates of the inclusive regions of each chromosome covered by the SNP microarrays and used for analyzing unselected LOH events. On the microarray, we did not include SNPs for repetitive regions. Most of these regions were in sub-telomeric locations. In this table, the first column shows the chromosome number, the second indicates the leftmost SGD coordinate represented by a SNP on the microarray, and the last column shows the rightmost SGD coordinate of the SNP on the microarray. Since selected events were located between the left telomere and centromere of chromosome V (positions 1 to 153229), this region was not used for examining unselected LOH events. Table S8 SGD coordinates of the inclusive regions of each chromosome covered by the SNP microarrays and used for analyzing unselected LOH events. On the microarray, we did not include SNPs for repetitive regions. Most of these regions were in sub-telomeric locations. In this table, the first column shows the chromosome number, the second indicates the leftmost SGD coordinate represented by a SNP on the microarray, and the last column shows the rightmost SGD coordinate of the SNP on the microarray. Since selected events were located between the left telomere and centromere of chromosome V (positions 1 to 153229), this region was not used for examining unselected LOH events. Table S3 SGD coordinates for transitions in sectored colonies induced by a UV dose of 15 J/m2 in G1-synchronized cells. For each sectored colony, we show both selected and unselected LOH events. Information about the event class, line number, and transition labels are derived from Table S2. The numbers in the leftmost two columns represent SGD coordinates for SNPs flanking each transition, beginning at the left telomere; these coordinates are based on the values used for the 2009 version. (XLS) UV-Induced Recombination in Yeast 20. Galli A, Schiestl RH (1999) Cell division transforms mutagenic lesions into deletion-recombinagenic lesions in yeast cells. Mutat Res 429: 13–26. 41. Schiestl RH, Prakash S (1988) RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination. Mol Cell Biol 8: 3619–3626. 21. Kadyk LC, Hartwell LH (1993) Replication-dependent sister chromatid recombination in rad1 mutants of Saccharomyces cerevisiae. Genetics 133: 469–487. 42. Covo S, Ma W, Westmoreland JW, Gordenin DA, Resnick MA (2012) Understanding the origins of UV-induced recombination through manipulation of sister chromatid cohesion. Cell Cycle 11: 3937–3944. 22. Lettier G, Feng Q, de Mayolo AA, Erdeniz N, Reid RJ, et al. (2006) The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae. PLoS Genet 2: e194. y 43. Lam LH, Reynolds RJ (1987) DNA sequence dependence of closely opposed cyclobutyl pyrimidine dimers induced by UV radiation. Mutat Res 178: 167– 176. 23. Nakai S, Mortimer RK (1969) Studies on the genetic mechanism of radiation- induced mitotic segregation in yeast. Mol Gen Genet 103: 329–338. 44. Teng Y, Bennett M, Evans KE, Zhuang-Jackson H, Higgs A, et al. (2011) A novel method for the genome-wide high resolution analysis of DNA damage. Nucleic Acids Res 39: e10. 24. Kadyk LC, Hartwell LH (1992) Sister chromatids are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae. Genetics 132: 387–402. 45. Hanawalt PC, Spivak G (2008) Transcription-coupled DNA repair: two decades of progress and surprises. Nat Rev Mol Cell Biol 9: 958–970. 25. Kirkpatrick DT, Petes TD (1997) Repair of DNA loops involves DNA-mismatch and nucleotide-excision repair proteins. Nature 387: 929–931. 46. Livingstone-Zatchej M, Marcionelli R, Moller K, de Pril R, Thoma F (2003) Repair of UV lesions in silenced chromatin provides in vivo evidence for a compact chromatin structure. J Biol Chem 278: 37471–37479. 26. Li F, Dong J, Pan X, Oum JH, Boeke JD, et al. (2008) Microarray-based genetic screen defines SAW1, a gene required for Rad1/Rad10-dependent processing of recombination intermediates. Mol Cell 30: 325–335. 47. Waters R, Teng Y, Yu Y, Yu S, Reed SH (2009) Tilting at windmills? The nucleotide excision repair of chromosomal DNA. DNA Repair (Amst) 8: 146– 152. 27. Mazon G, Lam AF, Ho CK, Kupiec M, Symington LS (2012) The Rad1-Rad10 nuclease promotes chromosome translocations between dispersed repeats. Nat Struct Mol Biol 19: 964–971. 48. UV-Induced Recombination in Yeast Teng Y, Li S, Waters R, Reed SH (1997) Excision repair at the level of the nucleotide in the Saccharomyces cerevisiae MFA2 gene: mapping of where enhanced repair in the transcribed strand begins or ends and identification of only a partial rad16 requisite for repairing upstream control sequences. J Mol Biol 267: 324–337. 28. Barbera MA, Petes TD (2006) Selection and analysis of spontaneous reciprocal mitotic cross-overs in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 103: 12819–12824. 29. St Charles J, Petes TD (2013) High-resolution mapping of spontaneous mitotic recombination hotspots on the 1.1 Mb arm of yeast chromosome IV. PLoS Genet 9: e1003434. 49. Donnianni RA, Symington LS (2013) Break-induced replication occurs by conservative DNA synthesis. Proc Natl Acad Sci U S A 110: 13475–13480. 50. McMurray MA, Gottschling DE (2003) An age-induced switch to a hyper- recombinational state. Science 301: 1908–1911. 30. Mitchel K, Zhang H, Welz-Voegele C, Jinks-Robertson S (2010) Molecular structures of crossover and noncrossover intermediates during gap repair in yeast: implications for recombination. Mol Cell 38: 211–222. 51. Ho CK, Mazon G, Lam AF, Symington LS (2010) Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast. Mol Cell 40: 988–1000. 31. Argueso JL, Westmoreland J, Mieczkowski PA, Gawel M, Petes TD, et al. (2008) Double-strand breaks associated with repetitive DNA can reshape the genome. Proc Natl Acad Sci U S A 105: 11845–11850. 52. Garvik B, Carson M, Hartwell L (1995) Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint. Mol Cell Biol 15: 6128–6138. 32. Hochberg Y, Benjamini Y (1990) More powerful procedures for multiple significance testing. Stat Med 9: 811–818. g g 33. Mitchell DL, Jen J, Cleaver JE (1992) Sequence specificity of cyclobutane pyrimidine dimers in DNA treated with solar (ultraviolet B) radiation. Nucleic Acids Res 20: 225–229. 53. Hackett JA, Greider CW (2003) End resection initiates genomic instability in the absence of telomerase. Mol Cell Biol 23: 8450–8461. 54. Petes TD, Malone RE, Symington LS (1991) 8 Recombination in Yeast. In: Broach JR, Jones EW and Pringle JR, editors. The Molecular and Cellular Biology of the Yeast Saccharomyces. Cold Spring Harbor: Cold Spring Harbor Press. Pp. 407–521. 34. References Genetics 21: 625–730. 18. Covo S, Westmoreland JW, Gordenin DA, Resnick MA (2010) Cohesin Is limiting for the suppression of DNA damage-induced recombination between homologous chromosomes. PLoS Genet 6: e1001006. 9. Esposito MS (1978) Evidence that spontaneous mitotic recombination occurs at the two-strand stage. Proc Natl Acad Sci U S A 75: 4436–4440. 9. Esposito MS (1978) Evidence that spontaneous mitotic recombination occurs at the two-strand stage. Proc Natl Acad Sci U S A 75: 4436–4440. 19. Esposito RE (1968) Genetic recombination in synchronized cultures of Saccharomyces cerevisiae. Genetics 59: 191–210. 10. Fabre F (1978) Induced intragenic recombination in yeast can occur during the G1 mitotic phase. Nature 272: 795–798. 10. Fabre F (1978) Induced intragenic recombination in yeast can occur during the G1 mitotic phase. Nature 272: 795–798. October 2013 | Volume 9 | Issue 10 | e1003894 PLOS Genetics | www.plosgenetics.org 20 UV-Induced Recombination in Yeast Casper AM, Mieczkowski PA, Gawel M, Petes TD (2008) Low levels of DNA polymerase alpha induce mitotic and meiotic instability in the ribosomal DNA gene cluster of Saccharomyces cerevisiae. PLoS Genet 4: e1000105. gene cluster of Saccharomyces cerevisiae. PLoS Genet 4: e1000105 35. Gangloff S, Zou H, Rothstein R (1996) Gene conversion plays the major role in controlling the stability of large tandem repeats in yeast. EMBO J 15: 1715– 1725. 55. Hoang ML, Tan FJ, Lai DC, Celniker SE, Hoskins RA, et al. (2010) Competitive repair by naturally dispersed repetitive DNA during non-allelic homologous recombination. PLoS Genet 6: e1001228. homologous recombination. PLoS Genet 6: e1001228. 36. Szostak JW, Wu R (1980) Unequal crossing over in the ribosomal DNA of Saccharomyces cerevisiae. Nature 284: 426–430. 56. Kupiec M, Steinlauf R (1997) Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae. Mutat Res 384: 33–44. 37. Zamb TJ, Petes TD (1982) Analysis of the junction between ribosomal RNA genes and single-copy chromosomal sequences in the yeast Saccharomyces cerevisiae. Cell 28: 355–364. 57. Parket A, Kupiec M (1992) Ectopic recombination between Ty elements in Saccharomyces cerevisiae is not induced by DNA damage. Mol Cell Biol 12: 4441–4448. 38. Galli A, Schiestl RH (1995) On the mechanism of UV and gamma-ray-induced intrachromosomal recombination in yeast cells synchronized in different stages of the cell cycle. Mol Gen Genet 248: 301–310. 58. Guthrie C, Fink GR (2002) Guide to yeast genetics and molecular biology. San Diego: Academic Press. 59. Altman DG (1991) Practical statistics for medical research. London: Chapman and Hall. y 39. Smith CE, Llorente B, Symington LS (2007) Template switching during break- induced replication. Nature 447: 102–105. 60. Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW (1983) The double-strand- break repair model for recombination. Cell 33: 25–35. p 40. Aguilera A, Chavez S, Malagon F (2000) Mitotic recombination in yeast: elements controlling its incidence. Yeast 16: 731–754. October 2013 | Volume 9 | Issue 10 | e1003894 21 PLOS Genetics | www.plosgenetics.org
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Basal and longitudinal changes in serum levels of TSH in morbid obese patients experiencing failure or success of dietary treatment
Eating and weight disorders
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* Luca Chiovato luca.chiovato@icsmaugeri.it Abstract Purpose  The relationship between thyroid function and obesity is a widely investigated one. The impact of thyroid hormones in determining the outcome of dietary/lifestyle interventions remains to be fully elucidated. The aim of this study was to compare basal and post dietary-intervention circulating thyroid-function parameters, lipid profile and fasting-glucose in euthyroid obese patients according to a success or failure of a dietary intervention program. Methods  In a retrospective longitudinal case–control study we enrolled 50 euthyroid obese patients who experienced a success in dietary intervention, as defined by a BMI reduction of at least 5% from baseline (Success Group) and 50 sex and age-matched euthyroid obese patients who experienced failure in dietary intervention as defined by either stable or increased body weight throughout the follow-up (Failure Group). Serum thyroid function parameters and metabolic profile at baseline and at the end of follow-up were collected. Results  At baseline, the two groups showed similar BMI, total-cholesterol, HDL-cholesterol and fasting-blood-glucose, but patients in Success Group had a significantly higher TSH as compared with Failure Group (2.20 ± 0.97 vs 1.66 ± 0.73, respec- tively, p < 0.001). Throughout a mean follow-up of 21.4 months TSH significantly decreased in Success Group (2.20 ± 0.97 vs 2.06 ± 0.98; p = 0.029) and increased in Failure Group (1.63 ± 0.72 vs 2.01 ± 0.99; p < 0.001). Multiple regression analysis showed that the outcome of the dietary intervention was significantly and independently related to baseline BMI (0.925; 0.861–0.993), age (0.957; 0.922–0.993), TSH (0.531; 0.290–0.973) and TSH-changes (1.011; 1.000–1.022) during follow-up. Conclusions  Baseline serum TSH level is related to the final outcome of a dietary intervention program in obese patients. Level of evidence III  Evidence obtained from a retrospective cohort or case–control analytic studies. Keywords  Obesity · Thyroid · Weight loss · Diet Basal and longitudinal changes in serum levels of TSH in morbid obese patients experiencing failure or success of dietary treatment Laura Croce1,2,3 · Cristina Pallavicini1 · Silvia Crotti1 · Francesca Coperchini1 · Linda Minnelli2 Luca Chiovato1,2   · Mario Rotondi1,2 Received: 15 July 2020 / Accepted: 30 September 2020 / Published online: 17 October 2020 © The Author(s) 2020 1 Unit of Internal Medicine and Endocrinology, Laboratory for Endocrine Disruptors, Department of Internal Medicine and Therapeutics, Istituti Clinici Scientifici Maugeri IRCCS, University of Pavia, Via S. Maugeri 4, 27100 Pavia, Italy Keywords  Obesity · Thyroid · Weight loss · Diet Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity (2021) 26:1949–1955 https://doi.org/10.1007/s40519-020-01043-x Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity (2021) 26:1949–1955 https://doi.org/10.1007/s40519-020-01043-x Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity (2021) 26:1949–1955 https://doi.org/10.1007/s40519-020-01043-x ORIGINAL ARTICLE * Luca Chiovato luca.chiovato@icsmaugeri.it Introduction [1, 2]. This observation together with the notion that T3 is involved in the regulation of basal energy expenditure, lead to the hypothesis that small differences in thyroid function, even in the presence of euthyroidism, could influence body weight status in the long term [2, 3]. Thus, thyroid status has been regarded as one of the factors involved in the pathogen- esis of the obesity epidemics. Although this hypothesis is a long-standing one, in the last years a huge body of evidence supported an opposite theory, namely that these slight thy- roid alterations could be a consequence rather than a cause of obesity [4]. Indeed, in a specifically designed study from our group, it was shown that morbid obese patients display a high rate of isolated hyperthyrotropinemia not associated with the typical features of autoimmune hypothyroidism. In particular, the hyperthyrotropinemia of obese patients was characterized by: (1) low prevalence of positive tests A moderate elevation of TSH concentrations, often associ- ated with triiodothyronine (T3) values in or slightly above the upper normal range, is frequently found in obese humans 2 Department of Internal Medicine and Therapeutics, University of Pavia, 27100 Pavia, Italy 3 PhD Course in Experimental Medicine, University of Pavia, 27100 Pavia, Italy (0123 1 3456789) 3 1950 Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity (2021) 26:1949–1955 for thyroid antibodies; (2) lack of female gender prevalence; and (3) similar free T4 (FT4)/free T3 (FT3) ratio as com- pared to obese patients with normal serum levels of TSH [5]. In addition, in a study comparing the circulating lipid pro- file between morbid obese patients and non-obese patients with raised serum levels of TSH, despite similar serum lev- els of TSH, FT4 and FT3, morbid-obese patients displayed significantly lower mean levels of total cholesterol, further supporting the concept that these latter might not be truly hypothyroid [6]. The length of the follow-up time was calculated for each patient starting from the first measurement of TSH which was performed at diagnosis (T0) to the last TSH measure- ment (T1). For all patients, the TSH value measured at the beginning and at the end of the dietistic approach was collected. Serum FT4, total cholesterol, HDL cholesterol, fasting glucose val- ues at the beginning and at the end of the dietary interven- tion program were collected. Dietary and behavioral intervention Eating habits were investigated, through an accurate dietary history collection, using a specially prepared form, referred to 3 typical days (2 weekdays and 1 holiday), the remote food history, or dietary history, provided to understand the setting of the diet at home for individual patients. Based on the results of this enquiry the average calorie intake and the bromatological composition of the patient’s usual diet was calculated. Patients also filled in a questionnaire on food consumption frequencies to identify the most frequent errors and provide solutions to better balance their diet. A leaflet on “Healthy and correct nutrition” was delivered, containing the basics of proper nutrition and regular physical activity was given to each patients. This booklet was designed to give an educational aid for continuity at home. The aim of our study was to compare basal and post die- tary intervention circulating thyroid function parameters, lipid profile and fasting glucose in obese patients according to a success or failure of a dietary intervention program. Introduction All patients signed an informed consent concerning the future use of their clinic data for research purposes and data collected remained strictly confidential and anonymous, according to the ethical rules of our Hospital institutions and to the Declaration of Helsinki. Formal approval by the ethical committee was not required in accordance with the Italian regulation for non-interventional (observational) ret- rospective studies concerning human beings (AIFA Guide- lines for Observational Studies, see www.AIFA.gov). It seems worth highlighting that, according to the NHANES III survey, the prevalence of positive tests for thyroid antibodies was similar between patients with mor- bid obesity and normal serum TSH as compared with the general population [7]. Hyperthyreotropinemia related to obesity has been shown to be reversible after weight loss, either obtained with diet [8–11] or bariatric surgery [12, 13]. In particular, Reinehr et al. [9] reported that in obese children with similar levels of TSH, undergoing an inten- sive program of dietary and lifestyle intervention, a signifi- cant decrease in TSH levels was observed in those children experiencing weight loss but not in those with stable body weight. 3 Statistical analysis Statistical analysis was performed using SPSS software ver- sion 20 (SPSS, Inc., Evanston, IL, USA). Between-group comparisons were performed by Student’s t test for unpaired data and by Mann–Whitney U test according to a normal or a nonparametric distribution of the variable tested. Paired samples comparisons were performed by Paired Samples t test and by Wilcoxon Rank test according to a normal or a nonparametric distribution of the variable tested. Correla- tion between two variables was ascertained by Pearson and Spearman’s correlation tests, as appropriate. Frequencies among groups were compared by χ2 test with Fisher’s cor- rection, when appropriate. A multivariate logistic analysis was constructed with the outcome of dietary intervention as the dependent variable and basal BMI, age, sex, basal TSH, percentage of variation of TSH as covariates. A p value < 0.05 was considered statistically significant. Table 1   baseline biochemical and anthropometrical characteristics of the two study groups Bold indicates p value under 0.05 All data are reported as mean ± standard deviation as well as median(minimum–maximum). Independent samples T test was used unless stated otherwise *Mann–Whitney test Success group Failure group p value Age (years) 50.0 ± 13.8 45.6 ± 13.6 0.111 50.0 (19.0–80.0) 47.0 (18.0–75.0) Sex (M/F) 22/28 22/28 1.000 BMI at baseline (kg/m2) 47.7 ± 7.8 45.1 ± 5.8 0.080 47.2 (35.6–67.9) 43.3 (39.1–62.9) TSH (µU/ml) 2.20 ± 0.97 1.66 ± 0.73  < 0.001* 1.93 (0.75–4.57) 1.61 (0.46–3.28) FT4 (ng/dl) 1.08 ± 0.15 1.07 ± 0.17 0.639 1.03 (0.69–1.45) 1.11 (0.72–1.48) Total cholesterol (mg/dl) 185.3 ± 34.0 187.2 ± 32.2 0.773 185.0 (109.0–271.0) 181.0 (119.0–277.0) HDL (mg/dl) 45.6 ± 14.0 48.8 ± 13.7 0.243 45.0 (21.0–86.0) 48.0 (27.0–91.0) Fasting blood glucose (mg/dl) 114.3 ± 38.5 123.9 ± 55.4 0.317 98.5 (72.0–267.0) 103.0 (77.0–325.0) Bold indicates p value under 0.05 All data are reported as mean ± standard deviation as well as median(minimum–maximum). Patients and methods In this study, data were retrospectively collected from mor- bid obese patients recruited in the Unit of Internal Medicine and Endocrinology at ICS Maugeri Hospital (Pavia, Italy) who had undertaken a Dietary and Behavioural interven- tion program. Patients were enrolled only if they had experi- enced a successful intervention program as defined by a BMI reduction of at least 5% from baseline to the last evaluation. A total number of 50 patients (22 males and 28 females) were selected. The control group encompassed an equal number of sex and age-matched patients (22 males and 28 females) in whom failure of the dietary-behavioral interven- tion program (as defined by either stable or increased body weight throughout the follow-up) was observed. The energy needs were estimated by calculating the basal metabolic rate with the Harris-Benedict formula, multiplied by the appropriate level of physical activity. Using the nutritional analysis program “Reasoned Diet 3” (supplied by the Clinic of Dietetics and Clinical Nutrition of the Maugeri Scientific Clinical Institutes), a personalized diet was subsequently calculated for each patient. All diets were low caloric diets with an average energy deficit of 700- 800 kcal compared to the estimated expenditure. The dietary scheme was designed respecting the requirements dictated by the LARN (Reference intake levels of nutrients and energy), including 45–55% of car- bohidrates (with a content of simple sugars < 15%, that latter could be further restricted, in relation to the patient’s clinical condition, i..e diabetes mellitus, insulin resistance, hypertriglyceridemia, etc.) and 25–35% of lipids (with saturated fats < 10%, monounsaturated 10–15% and poly- unsaturated 8–10%). The daily protein intake was 0.8–1 g/ Common inclusion criteria were: (1) BMI ≥ 35 kg/m2; (2) Regular dietistic follow-up for at least 6 months; 3) avail- ability of TSH measurement at the beginning and at the end of the dietistic follow-up; 4) age ≥ 18 years old. Exclusion criteria for both groups were: (1) current or previous history of thyroid disease or any alteration of thy- roid function parameters; (2) current or past use of thyroid function-modifying drugs; (3) previous bariatric surgery. 1 3 3 1951 Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity (2021) 26:1949–1955 kg of desirable weight (desirable weight means a weight corresponding to a BMI of 22.5 kg/m2). Table 1   baseline biochemical and anthropometrical characteristics of the two study groups Results The diet was structured around 3 main meals, possibly adding 1 or 2 snacks throughout the day, depending on the patient’s needs, respecting the Mediterranean diet model. The study group encompassed 50 patients in the Success Group and 50 patients in the Failure Group. The anthropo- metric and biochemical characteristics of the two groups are described in Table 1. The two groups were similar in terms of age (50.01 ± 13.8 in Success Group vs 45.6 ± 13.6 in Failure Group, p = 0.111) and baseline BMI (47.6 ± 7.8 in Success Group vs 45.1 ± 5.8 in Failure Group, p = 0.080). At T0, patients from Success Group had a significantly higher TSH as compared with Failure Group patients (TSH 2.20 ± 0.9 for Success Group vs 1.66 ± 0.73 for Failure Group, p < 0.001) (Fig. 1). Other biochemical parameters including total cholesterol, HDL-cholesterol, and fasting blood glucose levels were similar between the two groups at baseline. 38 patients (17 in the Success Group and 21 in the Failure group, p = 0.412) were diabetic. Among these 38 patients, 10 patients were in therapy with insulin (2 in the Success group, 8 in the Failure group, p = 0.136), 30 were in therapy with metformin (14 in the Success group, 16 in the failure group, p = 0.709), 11 patients were in therapy with a sulfonylurea (2 in the Success Group, 9 in the Failure Group, p = 0.070), 5 patients were in therapy with pioglitazone (0 in the Success Group, 5 in the Failure Group, p = 0.053), 9 patients were in therapy with a GLP-1 analogue (3 in the success Group, 6 in the Failure Group, p = 0.476), 2 patients were in therapy with DPP-IV inhibitors (0 in the success group, 2 in the Failure Group, p = 0.492) and 5 patients were All data are reported as mean ± standard deviation as well as median(minimum–maximum). Independent samples T test was used unless stated otherwise Statistical analysis No significant modifications in serum FT4 levels were observed in both groups (1.08 ± 0.15 at T0 vs 1.11 ± 0.18 at T1 in Success Group, p = 0.220; 1.07 ± 0.17 at T0 vs 1.09 ± 0.24 at T1 in Failure Group, p = 0.528). Fig. 1   Distribution of basal TSH values in the two groups of patients. TSH basal values were significantly higher in the Success Group compared with the Failure Group [1.93 (0.75–4.57) µU/ml in Success group vs 1.61 (0.46–3.28) µU/ml in Failure group, p < 0.001]. Data are expressed as median and 25th and 75th percentiles in boxes and 5th and 95th percentiles as whiskers The mean follow-up time was similar between the two groups (20.5 ± 22.0  months for Success Group vs 22.3 ± 19.9 months for Failure Group; p = 0.656). To evaluate factors potentially associated with the dif- ferent outcome of dietary intervention program, a logistic regression model was constructed entering intervention out- come (success/failure) as a dependent variable and baseline BMI, sex, age, baseline TSH, TSH percentage of variation throughout the follow-up as covariates. As shown in Table 3, baseline BMI, age, levels of TSH at T0 and TSH changes (expressed as percentages of the TSH at T0) were indepen- dently and significantly related to the outcome of the dietary intervention. Fig. 1   Distribution of basal TSH values in the two groups of patients. TSH basal values were significantly higher in the Success Group compared with the Failure Group [1.93 (0.75–4.57) µU/ml in Success group vs 1.61 (0.46–3.28) µU/ml in Failure group, p < 0.001]. Statistical analysis Independent samples T test was used unless stated otherwise *Mann–Whitney test Success group Failure group p value Age (years) 50.0 ± 13.8 45.6 ± 13.6 0.111 50.0 (19.0–80.0) 47.0 (18.0–75.0) Sex (M/F) 22/28 22/28 1.000 BMI at baseline (kg/m2) 47.7 ± 7.8 45.1 ± 5.8 0.080 47.2 (35.6–67.9) 43.3 (39.1–62.9) TSH (µU/ml) 2.20 ± 0.97 1.66 ± 0.73  < 0.001* 1.93 (0.75–4.57) 1.61 (0.46–3.28) FT4 (ng/dl) 1.08 ± 0.15 1.07 ± 0.17 0.639 1.03 (0.69–1.45) 1.11 (0.72–1.48) Total cholesterol (mg/dl) 185.3 ± 34.0 187.2 ± 32.2 0.773 185.0 (109.0–271.0) 181.0 (119.0–277.0) HDL (mg/dl) 45.6 ± 14.0 48.8 ± 13.7 0.243 45.0 (21.0–86.0) 48.0 (27.0–91.0) Fasting blood glucose (mg/dl) 114.3 ± 38.5 123.9 ± 55.4 0.317 98.5 (72.0–267.0) 103.0 (77.0–325.0) Table 1   baseline biochemical and anthropometrical characteristics of the two study groups 1 3 1952 Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity (2021) 26:1949–1955 in therapy with acarbose (1 in the success group, 4 in the Failure group, p = 0.335). at T1, p < 0.0001) whereas BMI was significantly increased in Failure Group (45.1 ± 5.8 at T0 vs 47.5 ± 5.9 kg/m2 at T1, p < 0.0001) throughout the study span.i The mean BMI variation and the parallel changes in total cholesterol, HDL-cholesterol, fasting blood glu- cose and TSH observed from baseline (T0) to the end of follow-up (T1) in the 2 groups are shown in Table 2. As expected, patients in Success Group experienced a signifi- cant decrease in BMI (47.6 ± 7.8 at T0 vs 40.7 ± 7.7 kg/m2 While a significant decrease in total cholesterol occurred in both groups, no changes in the circulating levels of HDL- cholesterol nor fasting blood glucose were observed from T0 to T1 in any of the groups. Noteworthy, the longitudinal behavior of the serum levels of TSH was strikingly differ- ent between the 2 groups. Indeed, while a slight although a significant decrease in serum TSH was observed from T0 to T1 (2.20 ± 0.9 vs 2.06 ± 0.9, respectively; p = 0.029) in Success Group, an opposite and still significant trend was observed in Failure Group (1.63 ± 0.7 in T0 vs 2.01 ± 0.9 in T1, p < 0.001). All data are reported as mean ± standard deviation as well as median(minimum–maximum). Paired samples T test was used unless stated other- wise Discussion The present study was specifically designed to compare changes in circulating thyroid function parameters in euthy- roid obese patients experiencing a different outcome of a dietary intervention program. Obese patients were specifi- cally recruited according to either a successful outcome (as defined by a BMI reduction of at least 5%) or failure of the interventional program (as defined by stable and/or increased BMI).f A further finding of the present study is that higher basal TSH levels were observed in patients from the Suc- cess Group as compared to those of the Failure Group. This finding was strengthened by the logistic regression analysis, showing that differences in basal TSH were significantly and independently related to the outcome of the dietary program. These data are in line with what reported in a recent study [18] showing that baseline TSH levels were significantly higher in children who experienced a substantial weight loss as compared to those experiencing failure after a 1 year dietary intervention program. The here reported differences in baseline TSH levels between the two groups may be regarded as slight but, it should be considered that the pres- ence of abnormal values of serum TSH constituted a man- datory exclusion criterion. The interplay between thyroid function and obesity is a widely investigated one. Accord- ing to previous studies, the concept arose that elevated TSH levels, commonly observed in obese patients, would not be always indicative of subclinical hypothyroidism [4, 5]. Furthermore, some authors proposed that this increase in TSH levels should be regarded as an adaptation process to increase REE and, in turn, energy expenditure [1]. The clini- cal implications of these findings would require specifically designed studies, however, previous studies reported that TSH levels can normalize in obese patients following weight loss [14–17] and thyroxine administration in obese patients The two groups did not differ in any anthropometric and biochemical parameter at baseline, with the exception of a higher level of TSH which characterized patients of Success Group. The longitudinal evaluation highlighted an opposite trend in TSH behavior according to a different outcome. Indeed, in patients experiencing weight loss, a significant reduction of the circulating TSH concentrations occurred while in patients experiencing failure of the dietary program, a significant increase in serum TSH levels was observed. Statistical analysis The here reported signifi- cant decrease in serum TSH, although of little magnitude, might not be free of relevant consequences, in that the TSH decrease was previously identified as a potential cause of the difficulties in maintaining weight loss. Previous studies [8, 18] demonstrated that the decrease in serum TSH during diet was positively correlated with weight regain after stopping the dietary regimen which appears in line with studies per- formed in adults and children reporting a decrease in energy expenditure in parallel with weight loss. Unfortunately, the design of the present study does not allow evaluating the issue of body weight regain. Table 3   Logistic regression analysis entering intervention outcome (success/failure) as dependent variable and baseline BMI, sex, age, baseline TSH Bold indicates p values under 0.05 TSH percentage of variation throughout the follow-up as covariates p value Exp (B) 95% CI for EXP (B) Lower Upper Age (years) 0.020 0.957 0.922 0.993 Basal BMI (kg/m2) 0.031 0.925 0.861 0.993 Baseline TSH (µU/ml) 0.041 0.531 0.290 0.973 Percentage of variation of TSH (%) 0.045 1.011 1.000 1.022 Sex (M/F) 0.840 1.099 0.440 2.743 Table 3   Logistic regression analysis entering intervention outcome (success/failure) as dependent variable and baseline BMI, sex, age, baseline TSH TSH percentage of variation throughout the follow-up as covariates Statistical analysis Data are expressed as median and 25th and 75th percentiles in boxes and 5th and 95th percentiles as whiskers Table 2   variation of BMI and biochemical parameters throughout the follow-up in the two study groups Bold indicates p values under 0.05 Success group Failure group T0 T1 p value T0 T1 p value BMI (kg/m2) 47.6 ± 7.8 40.7 ± 7.7  < 0.0001 45.1 ± 5.8 47.5 ± 5.9  < 0.0001 47.2 (35.6–67.9) 39.8 (27.7–57.3) 43.3 (39.1–62.9) 46.7 (39.5–67.2) TSH (µU/ml) 2.20 ± 0.97 2.06 ± 0.98 0.029* 1.63 ± 0.72 2.01 ± 0.99 0.001* 1.93 (0.75–4.57) 1.95 (0.24–4.38) 1.61 (0.46–3.28) 1.71 (0.74–5.52) FT4 (ng/dl) 1.08 ± 0.15 1.11 ± 0.18 0.220 1.07 ± 0.17 1.09 ± 0.24 0.528 1.03 (0.69–1.45) 1.11 (0.84–1.46) 1.11 (0.72–1.48) 1.08 (0.67–1.86) Total cholesterol (mg/dl) 185.4 ± 34.0 174.6 ± 31.8 0.007 187.3 ± 32.2 176.9 ± 32.3 0.011 185.0 (109.0–271.0) 180.0 (79.0–245.0) 181.0 (119.0–277.0) 175.0 (116.0–273.0) HDL (mg/dl) 45.6 ± 14.0 45.6 ± 11.4 0.967 48.87 ± 13.7 51.93 ± 14.8 0.051 45.0 (21.0–86.0) 46.5 (20.0–72.0) 48.0 (27.0–91.0) 52 (25.0–91.0) Fasting blood glucose (mg/dl) 114.3 ± 38.5 106.9 ± 25.1 0.059 123.9 ± 55.4 131.9 ± 68.1 0.388 98.5 (72.0–267.0) 97.5 (79.0–187.0) 103.0 (77.0–325.0) 106.0 (65.0–367.0) Table 2   variation of BMI and biochemical parameters throughout the follow-up in the two study groups BMI and biochemical parameters throughout the follow-up in the two study groups 1 Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity (2021) 26:1949–1955 1953 levels was found in those children who did not lose weight. It should be highlighted that, while in our study the patients who experienced a failure of the intervention program were characterized by a significant body weight gain, the children included in the study by Reinehr et al. showed stable body weight at the end of follow-up. Several studies showed that isolated hyperthyreotropinemia due to obesity can revert after weight loss either achieved by the dietary intervention [8–11, 14–16] or bariatric surgery [12, 13, 17]. The specific design of the present study (all patients enrolled displayed normal TSH levels), allows to observe that a significant reduction in serum TSH occurs also in obese patients with normal TSH levels who experience weight loss. A poten- tial limitation of the present study could be represented by the fact that adherence to the program was not evaluated through structured questionnaires. References 1. Reinehr T (2010) Obesity and thyroid function. Mol Cell Endo- crinol 316(2):165–171. https​://doi.org/10.1016/j.mce.2009.06.005 2. Santini F, Marzullo P, Rotondi M, Ceccarini G, Pagano L, Ippolito S et al (2014) Mechanisms in endocrinology: the crosstalk between thyroid gland and adipose tissue: signal integration in health and disease. Eur J Endocrinol 171(4):R137–R152. https​:// doi.org/10.1530/EJE-14-0067 Discussion The multivariate regression model confirmed that baseline levels of TSH, TSH changes during the follow-up, age and basal BMI were all independently and significantly related to the outcome of the dietary intervention.i The findings that both basal TSH levels and its changes throughout the follow-up were found to be related to the different outcome of the dietary intervention program might require to be discussed separately. The here reported direct relationship between changes in TSH and in BMI in obese patients is in line with pre- vious studies showing that obese patients display higher TSH levels as compared to normo-weight subjects. Reinehr et al. [9], previously reported a significant reduction of TSH levels only in children who obtained a significant weight loss after a 1 yr treatment with hypocaloric diet. At differ- ence with our findings, no substantial change in serum TSH 1 3 1 1954 Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity (2021) 26:1949–1955 What is already known on this subject? 3. Knudsen N, Laurberg P, Rasmussen LB, Bulow I, Perrild H, Ovesen L et al (2005) Small differences in thyroid function may be important for body mass index and the occurrence of obesity in the population. J Clin Endocrinol Metab 90(7):4019–4024. https​://doi.org/10.1210/jc.2004-2225 3. Knudsen N, Laurberg P, Rasmussen LB, Bulow I, Perrild H, Ovesen L et al (2005) Small differences in thyroid function may be important for body mass index and the occurrence of obesity in the population. J Clin Endocrinol Metab 90(7):4019–4024. https​://doi.org/10.1210/jc.2004-2225 Hyperthyreotropinemia is a frequent finding in obese sub- jects. Substantial variations in thyroid function parameters are observed after body weight loss due to dietary interven- tion or bariatric surgery. 4. Rotondi M, Magri F, Chiovato L (2011) Thyroid and obesity: not a one-way interaction. J Clin Endocrinol Metab 96(2):344– 346. https​://doi.org/10.1210/jc.2010-2515 4. Rotondi M, Magri F, Chiovato L (2011) Thyroid and obesity: not a one-way interaction. J Clin Endocrinol Metab 96(2):344– 346. https​://doi.org/10.1210/jc.2010-2515 Conflict of interest  The authors declares that they have no conflict of interest. The results of the present study are not sufficient to confirm this intriguing hypothesis, and future, prospective studies will be needed to confirm it. i In conclusion, the results of the present study demonstrate that significant differences in both baseline and longitudinal behavior of serum TSH characterize patients with a different response to a dietary intervention program. Future longitu- dinal prospective studies will be required to establish firm conclusion as to the effect of TSH changes on weight regain as well as on the physiopathologic mechanism involved in this association. Conflict of interest  The authors declares that they have no conflict of interest. Conflict of interest  The authors declares that they have no conflict of interest. Taken together the above concepts could explain the dif- ference in basal TSH serum levels in obese patients accord- ing to success or failure of the dietary intervention program. It is interesting noting that a significant reduction in total cholesterol levels was observed in both groups of obese patients independently of the success/failure of the program, which could indicate that an attempt to modify eating habits was made by all the involved patients regardless of the final outcome. Indeed, data in the literature indicate that although there is a direct correlation between weight loss and reduc- tion in total cholesterol levels [20], a low-fat diet can induce a reduction in total cholesterol even when bodyweight loss is limited [21]. Ethical approval  Formal approval by the ethical committee was not required in accordance with the Italian regulation for non-interventional (observational) retrospective studies concerning human beings(AIFA Guidelines for Observational Studies, see http://www.AIFA.gov). Informed consent  All patients signed an informed consent concern- ing the future use of their clinic data for research purposes and data collected remained strictly confidential and anonymous, according to the ethical rules of our Hospital institutions and to the Declaration of Helsinki. Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. The above concept could be potentially relevant in that it could suggest the hypothesis that success in a lifestyle inter- vention and/or in weight regain could not only be due to the compliance of obese patients, but also influenced by other factors, possibly including thyroid function. Compliance with ethical standards with moderately elevated TSH levels did not modify their body weight status [19]. What your study adds? 5. Rotondi M, Leporati P, La Manna A, Pirali B, Mondello T, Fonte R et al (2009) Raised serum TSH levels in patients with morbid obesity: is it enough to diagnose subclinical hypo- thyroidism? Eur J Endocrinol 60(3):403–408. https​://doi. org/10.1530/EJE-08-0734 Differences in baseline and longitudinal behavior of serum TSH levels characterize obese patients with success or fail- ure of a dietary intervention program. 6. Rotondi M, Leporati P, Rizza MI, Clerici A, Groppelli G, Pallavi- cini C et al (2014) Raised serum TSH in morbid-obese and non- obese patients: effect on the circulating lipid profile. Endocrine 45(1):92–97. https​://doi.org/10.1007/s1202​0-013-9928-8 Funding  Open access funding provided by Università degli Studi di Pavia within the CRUI-CARE Agreement. This paper was not sup- ported by any grant or funding. 7. Spencer CA, Hollowell JG, Kazarosyan M, Braverman LE (2007) National health and nutrition examination survey III thyroid-stim- ulating hormone (TSH)-thyroperoxidase antibody relationships demonstrate that TSH upper reference limits may be skewed by occult thyroid dysfunction. J Clin Endocrinol Metab 92(11):4236– 4240. https​://doi.org/10.1210/jc.2007-0287 1 3 1955 Eating and Weight Disorders - Studies on Anorexia, Bulimia and Obesity (2021) 26:1949–1955 8. Reinehr T, Andler W (2002) Thyroid hormones before and after weight loss in obesity. Arch Dis Child 87(4):320–323 8. Reinehr T, Andler W (2002) Thyroid hormones before and after weight loss in obesity. Arch Dis Child 87(4):320–323 16. Sari R, Balci MK, Altunbas H, Karayalcin U (2003) The effect of body weight and weight loss on thyroid volume and function in obese women. Clin Endocrinol (Oxf) 59(2):258–262. https​://doi. org/10.1046/j.1365-2265.2003.01836​.x g y 9. Reinehr T, de Sousa G, Andler W (2006) Hyperthyrotropinemia in obese children is reversible after weight loss and is not related to lipids. J Clin Endocrinol Metab 91(8):3088–3091. https​://doi. org/10.1210/jc.2006-0095 17. Moulin de Moraes CM, Mancini MC, de Melo ME, Figueiredo DA, Villares SM, Rascovski A et al (2005) Prevalence of subclini- cal hypothyroidism in a morbidly obese population and improve- ment after weight loss induced by Roux-en-Y gastric bypass. Obes Surg 15(9):1287–1291. https​://doi.org/10.1381/09608​92057​74512​ 537 10. Radetti G, Longhi S, Baiocchi M, Cassar W, Buzi F (2012) Changes in lifestyle improve body composition, thyroid function, and structure in obese children. J Endocrinol Invest 35(3):281– 285. https​://doi.org/10.3275/7763 11. Licenziati MR, Valerio G, Vetrani I, De Maria G, Liotta F, Radetti G (2019) Altered thyroid function and structure in children and adolescents who are overweight and obese: reversal after weight loss. J Clin Endocrinol Metab 104(7):2757–2765. Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. What your study adds? https​://doi. org/10.1210/jc.2018-02399​ 18. Wolters B, Lass N, Reinehr T (2013) TSH and free triiodothyro- nine concentrations are associated with weight loss in a lifestyle intervention and weight regain afterwards in obese children. Eur J Endocrinol 168(3):323–329. https​://doi.org/10.1530/EJE-12-0981 19. Krude H, Biebermann H, Schnabel D, Tansek MZ, Theunissen P, Mullis PE et al (2003) Obesity due to proopiomelanocortin defi- ciency: three new cases and treatment trials with thyroid hormone and ACTH4-10. J Clin Endocrinol Metab 88(10):4633–4640. https​://doi.org/10.1210/jc.2003-03050​2f g j 12. Raftopoulos Y, Gagné DJ, Papasavas P, Hayetian F, Maurer J, Bononi P et al (2004) Improvement of hypothyroidism after lapa- roscopic Roux-en-Y gastric bypass for morbid obesity. Obes Surg 14(4):509–513. https​://doi.org/10.1381/09608​92043​23013​514 13. Neves JS, Castro Oliveira S, Souteiro P, Pedro J, Magalhães D, Guerreiro V et al (2018) Effect of weight loss after bariatric sur- gery on thyroid-stimulating hormone levels in patients with mor- bid obesity and normal thyroid function. Obes Surg 28(1):97–103. https​://doi.org/10.1007/s1169​5-017-2792-5 20. Dattilo AM, Kris-Etherton PM (1992) Effects of weight reduction on blood lipids and lipoproteins: a meta-analysis. Am J Clin Nutr 56(2):320–328. https​://doi.org/10.1093/ajcn/56.2.320 21. Nordmann AJ, Nordmann A, Briel M, Keller U, Yancy WS, Brehm BJ et al (2006) Effects of low-carbohydrate vs low-fat diets on weight loss and cardiovascular risk factors: a meta-analysis of randomized controlled trials. Arch Intern Med 166(3):285–293. https​://doi.org/10.1001/archi​nte.166.3.285 p g 14. Kok P, Roelfsema F, Langendonk JG, Frölich M, Burggraaf J, Meinders AE et al (2005) High circulating thyrotropin levels in obese women are reduced after body weight loss induced by caloric restriction. J Clin Endocrinol Metab 90(8):4659–4663. https​://doi.org/10.1210/jc.2005-0920 Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 15. Kozłowska L, Rosołowska-Huszz D (2004) Leptin, thyrotropin, and thyroid hormones in obese/overweight women before and after two levels of energy deficit. Endocrine 24(2):147–153. https​ ://doi.org/10.1385/ENDO:24:2:147 1 3 1 3
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Horizontal Transfer of a Novel Soil Agarase Gene from Marine Bacteria to Soil Bacteria via Human Microbiota
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Horizontal Transfer of a Novel Soil Agarase Gene from Marine Bacteria to Soil Bacteria via Human Microbiota Tao Song1,*, Hui Xu1,*, Congchong Wei1, Tengfei Jiang1, Shishang Qin1, Weijia Zhang1, Yu Cao1,2, Chao Hu1, Fan Zhang1, Dairong Qiao1 & Yi Cao1 received: 22 February 2016 accepted: 08 September 2016 Published: 19 October 2016 Seaweed is receiving an increasing amount of attention as a “sea vegetable”. The microbiota of coastal populations may acquire seaweed associated enzymes through marine food. Several agarases have been found in non-marine environments; however, their origin is unknown. In this study, a hypothetical protein, Aga1, was identified as an agarase from an inland soil agar-degrading bacterium, Paenibacillus sp. SSG-1. Having low similarity to known glycoside hydrolases, Aga1 may be a distant member of the glycoside hydrolase family 86. Aga1 has good pH stability (pH 3–11) and is stable in the presence of various metal ions. Aga1 is an exo-type β-agarase that produces NA 4 (neoagarotetraose) and NA 6 (neoagarohexaose) as its main products. In addition, Aga1 may be a cell-surface-binding protein. The bioinformatic analysis showed aga1 may have been transfered together with its surrounding genes, from marine bacteria to soil bacteria via human microbiota. The use of seaweed as food and the disposal of human faeces or saliva were the most likely reasons for this gene transfer pathway. Notably, the results also indicated that microbes from inland humans may degrade agar and that these microbes may have acquired seaweed associated genes because of increased seaweed in diets. In 2012, the world’s annual production of seaweed reached 23.8 million tons, which is 3.5 times more than that pro- duced in the 1990s1. In recent decades, seaweed has become increasingly popular as food, not only in Asian countries, but also in western countries2–5. Compared to traditional crops, seaweed has many superior characteristics, such as being fertilizer-free and irrigation-free and having no land conflicts with traditional agricultural crops6,7. Considering its nutrient composition, including amino acids and fatty acids, seaweed is a promising food source. p g y p g Red seaweed, as an important marine plant, is widely used as food. Agar is the main component of red sea- weed, and it consists of 3-O-linked β​-D-galactopyranose and 4-O-linked α​-3,6-anhydro-L-galactopyranose linked to sulfate groups, methyl groups or pyruvic acid acetal8. Agarase is the enzyme that degrades agar into oligosaccharides or monosaccharides8,9. Based on cleavage patterns, agarases may be classified as α​-agarases, β​-agarases and β​-porphyranases10. According to the CAZy (carbohydrate-active enzymes) database, approxi- mately only 40 agarases have been characterized11. Some agarases belong to existing glycoside hydrolase (GH) families, such as GH1612,13 and GH5014–16. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 22 February 2016 accepted: 08 September 2016 Published: 19 October 2016 Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 Horizontal Transfer of a Novel Soil Agarase Gene from Marine Bacteria to Soil Bacteria via Human Microbiota Tao Song1,*, Hui Xu1,*, Congchong Wei1, Tengfei Jiang1, Shishang Qin1, Weijia Zhang1, Yu Cao1,2, Chao Hu1, Fan Zhang1, Dairong Qiao1 & Yi Cao1 Others have defined new families, such as GH8617,18, GH9619,20, GH11721,22 and GH11823,24. Compared with other well-studied glycoside hydrolases, such as cellulases or xyla- nases25, the number of agarases is small. Identifying and studying new agarases is essential. g y g y g g Until now, most reported agarases have come from marine environments13–17,19,23. Agar-degrading bacteria have also been found in different non-marine environments, such as soil26–28, plant endogenous environments29, rivers30 and even the human gut31,32. It is interesting that agarase exists in environments containing nearly no seaweed. It has been reported that coastal human microbiota may acquire glycoside hydrolases from the marine environment through food connections33. It is not known where non-marine source agarases come from, although marine environments or soil environments are possibilities. An outstanding question is how non-marine agarases came into existence. 1Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu, 610065, P. R. China. 2National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610065, P. R. China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to D.Q. (email: qiaodairong@scu.edu.cn) or Y.C. (email: geneium@scu. edu.cn) Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 1 www.nature.com/scientificreports/ Figure 1. (A) Structure of Aga1 from Paenibacillus sp. SSG-1. SLH regions represent the S-layer homology regions. (B) SDS-PAGE and Coomassie staining of purified Aga1. (C) Aga1’s enzymatic activity against different substrates. (D) Phylogenetic tree between aga1 and the characterized agarases. Numbers at nodes are levels of bootstrap support (%). Figure 1. (A) Structure of Aga1 from Paenibacillus sp. SSG-1. SLH regions represent the S-layer homology regions. (B) SDS-PAGE and Coomassie staining of purified Aga1. (C) Aga1’s enzymatic activity against different substrates. (D) Phylogenetic tree between aga1 and the characterized agarases. Numbers at nodes are levels of bootstrap support (%). In our previous work, we purified and characterized a natural agarase from the agar-degrading soil bacteria Paenibacillus sp. SSG-127. This natural agarase is a hypothetical protein in the genome of Paenibacillus sp. SSG-1. Furthermore, this hypothetical protein (Aga1) was synthesized, purified and characterized as agarase. Aga1 had low similarity to known agarases and was a distant member of GH86. A detailed biochemical characterization was conducted to determine the specific properties of Aga1. Bioinformatic analysis revealed that aga1 may be the result of horizontal gene transfer from a marine environment to a soil environment via human microbiota. Results l i Cloning and identification of agarase aga1. Using the matched peptide sequences of SSG1a, a BLAST search against the Paenibacillus sp. SSG-1 genome identified one hypothetical protein, which was subsequently designated Aga1. g g As demonstrated in Fig. 1A, Aga1 contains one signal peptide (1–55), a conserved region (330–613), and three S-layer homology (SLH) domains (1495–1536, 1554–1597 and 1624–1668).The signal peptide and SLH domains indicate that Aga1 is probably a secreted protein and may be located on the cell wall surface, which was supported by the subcellular location prediction results.h The recombinant protein Aga1, excluding the signal peptide and SLH domains, was inserted into the pET28a vector (His-tag fusion) and expressed in E. coli (DE3) as a soluble protein. SDS-PAGE showed the Aga1 protein had an appar- ent molecular mass of 165 kDa (Fig. 1B), which matched the calculated molecular mass of 166.3 kDa. Tandem mass analysis of the purified Aga1 confirmed that it was correctly expressed and purified (Supplemental Table S1). Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 2 www.nature.com/scientificreports/ Figure 2. (A) Effects of temperature on stability and activity of Aga1. (B) Effects of pH on stability and activity of Aga1. Data are mean ±​ SD of three independent experiments. (C) TLC analysis of the catalytic property against the oligosaccharide. (D) The TLC analysis of the end products at different time. NA2, NA4, NA6, NA8 represent the neoagarooctaose, neoagarotetraose, neoagarohexaose and neoagarooctaose, respectively. Figure 2. (A) Effects of temperature on stability and activity of Aga1. (B) Effects of pH on stability and activity of Aga1. Data are mean ±​ SD of three independent experiments. (C) TLC analysis of the catalytic property against the oligosaccharide. (D) The TLC analysis of the end products at different time. NA2, NA4, NA6, NA8 represent the neoagarooctaose, neoagarotetraose, neoagarohexaose and neoagarooctaose, respectively. As shown in Fig. 1C, Aga1 is an agarase that is active only in the presence of agarose. Aga1 had very low similarity (lower than 30%) to other glycoside hydrolases. The phylogenetic tree consisting of Aga1 and known agarases showed that Aga1 may be a distant member of GH86 (Fig. 1D). As shown in Fig. 1C, Aga1 is an agarase that is active only in the presence of agarose. Aga1 had very low similarity (lower than 30%) to other glycoside hydrolases. The phylogenetic tree consisting of Aga1 and known agarases showed that Aga1 may be a distant member of GH86 (Fig. 1D). Results l i Biochemical analysis of agarase. Aga1 maintained over 40% of its activity across a wide range of temper- atures (0–70 °C), and 50 °C was the optimal temperature for Aga1 (Fig. 2A). In addition, as shown in Fig. 2B, Aga1 showed strong stability over a wide pH range (pH 1–12). The decrease in Aga1 activity at pH 3.0 may be related to the pI of Aga1. Because the predicted pI of Aga1 was 4.5, it is possible that the real pI was close to pH 3.0, which caused this decrease. Meanwhile, most metal ions (1 mM) did not affect the activity of Aga1 (Supplemental Table S2). However, Cu2+ strongly inhibited its activity (37% activity). g y y y As shown in Fig. 2C, Aga1 could not hydrolyse neoagarobiose (NA 2), neoagarotetraose (NA 4) and neoaga- rohexaose (NA 6). Neoagarooctaose (NA 8) was the smallest oligosaccharide that Aga1 could hydrolyse. TLC analysis of the end product showed that Aga1 hydrolysed agarose into two main products, which should be NA 4 and NA 6 according to the standards (Fig. 2D). These two products were also subjected to HPLC, and the results showed that they had the same retention times as NA 4 and NA 6 (Fig. 3A). Moreover, as shown in Fig. 3B, the MALDI-TOF mass spectrometry results of the end products showed two main peaks, i.e., 653.2 m/z and 959 m/z, which corresponded to [M +​ Na]+​. These two peaks were attributed to NA 4 and NA 6, respectively. Taking these three results into consideration, the main hydrolysis products were NA 4 and NA 6. To analyse the cleavage pattern of Aga1, 13C NMR was conducted. As shown in Fig. 3C, resonances at approx- imately 97.04 ppm and 93.05 ppm corresponded to the β​ and α​ anomeric carbons, respectively, of the galactose residues. Resonance at 90.72 ppm, the typical signal of an α​-agarase, was not observed. Thus Aga1 is a β​-agarase. h TLC (Fig. 2D) and HPLC analysis (Supplemental Fig. S1) showed that the amounts of NA 4 and NA 6 increased as the hydrolysis time increased, and no other oligosaccharides were observed during hydrolysis. Endo-agarase decomposed agarose in a random way and produced oligosaccharides with different degrees of polymerization during hydrolysis. As only two products were observed in the hydrolysis procedure, Aga1 was determined to be an exo-type agarase. Inland aga1 may be from inland human symbionts. Results l i Control genes were protein-coding genes in Paenibacillus sp. SSG-1 and had same GC3s value with the genome. CAI: Codon Adaptation Index. CBI: codon bias index. Nc: effective number of codons. Fop: frequency of optimal codons. GC3s: GC of silent 3rd codon posit.GC: GC content of gene. Table 1. Correspondence analysis of codon usage of aga1, Paenibacillus sp. SSG-1 and control genes. Control genes were protein-coding genes in Paenibacillus sp. SSG-1 and had same GC3s value with the genome. CAI: Codon Adaptation Index. CBI: codon bias index. Nc: effective number of codons. Fop: frequency of optimal codons. GC3s: GC of silent 3rd codon posit.GC: GC content of gene. a foreign gene. Meanwhile, the GC content, GC3s content, and Codon Bias Index of aga1 were all significantly different from those of the genome (Table 1 and Supplemental Table S3). Additionally, the Relative Synonymous Codon Usage (RSCU) value of aga1 was clearly distinct from the genome’s value (Supplemental Table S4). Thus, these evidences strongly indicated that aga1 had been transfered horizontally from other microbes. a foreign gene. Meanwhile, the GC content, GC3s content, and Codon Bias Index of aga1 were all significantly different from those of the genome (Table 1 and Supplemental Table S3). Additionally, the Relative Synonymous Codon Usage (RSCU) value of aga1 was clearly distinct from the genome’s value (Supplemental Table S4). Thus, these evidences strongly indicated that aga1 had been transfered horizontally from other microbes. Given that aga1 was from an inland soil environment that was far from the sea and contained almost no seaweed, it was difficult for the enzymes to evolve into agarases without a substrate, and the distant geographic position of the enzyme made gene transfer nearly impossible. As showed in Fig. 4A, aga1 had a closer relationship with genes from symbiotic environments, other than those from marine environments. Meanwhile, we noticed that four aga1 like genes came from inland people’s microbiota; i.e., from the human gut or human mouth. Human faeces are usually used as fertilizer, and discarding saliva is also common. Given that aga1 was also from an inland location, it is reasonable to infer that aga1 may come from inland human microbiota. Horizontal gene transfer linkage. Analysis of Paenibacillus sp. SSG-1’s genome showed that aga1 was surrounded by other genes coding for agar, including α​-neoagarobiose hydrolase (NABH), galactosidase. 3,6-anhydro-L-galactose ( L-AnG) metabolic enzymes and sulfatase. Results l i To investigate the distribution of aga1, we searched for homologues of aga1 in NCBI none-redundant database. Interestingly, almost all of aga1’s similar genes came from other class or phylum. Among the 208 genomes of Paenibacillus sp., aga1 like gene only appeared in Paenibacillus sp. D14. This rare distribution of aga1’s similar gene in its own genus indicated that aga1 may be Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 3 www.nature.com/scientificreports/ Figure 3. (A) The HPLC result of the hydrolysis product produced by Aga1. (B) MALDI-TOF mass result of the end products. (C) The 13C NMR of the end products. G β​-D-galactopyranose, A 3,6-anhydro-a-L- aglactopyranose, r reducing end, nr non-reducing end, α​ α​ anomer, β​ β​ anomer. Figure 3. (A) The HPLC result of the hydrolysis product produced by Aga1. (B) MALDI-TOF mass result of the end products. (C) The 13C NMR of the end products. G β​-D-galactopyranose, A 3,6-anhydro-a-L- aglactopyranose, r reducing end, nr non-reducing end, α​ α​ anomer, β​ β​ anomer. CAI CBI Fop Nc GC GC3s Genome 0.267 0.127 0.489 47.43 0.536 0.633 aga1 0.251 -0.029 0.414 52.18 0.457 0.445 PA2609 0.235 0.092 0.468 49.23 0.561 0.634 PA1054 0.217 0.083 0.464 52.60 0.575 0.632 PA4965 0.246 0.013 0.425 57.25 0.511 0.630 PA4486 0.265 0.188 0.508 43.03 0.507 0.631 Table 1. Correspondence analysis of codon usage of aga1, Paenibacillus sp. SSG-1 and control genes. Control genes were protein-coding genes in Paenibacillus sp. SSG-1 and had same GC3s value with the genome. CAI: Codon Adaptation Index. CBI: codon bias index. Nc: effective number of codons. Fop: frequency of optimal codons. GC3s: GC of silent 3rd codon posit.GC: GC content of gene. CAI CBI Fop Nc GC GC3s Genome 0.267 0.127 0.489 47.43 0.536 0.633 aga1 0.251 -0.029 0.414 52.18 0.457 0.445 PA2609 0.235 0.092 0.468 49.23 0.561 0.634 PA1054 0.217 0.083 0.464 52.60 0.575 0.632 PA4965 0.246 0.013 0.425 57.25 0.511 0.630 PA4486 0.265 0.188 0.508 43.03 0.507 0.631 Table 1. Correspondence analysis of codon usage of aga1, Paenibacillus sp. SSG-1 and control genes. Control genes were protein-coding genes in Paenibacillus sp. SSG-1 and had same GC3s value with the genome. CAI: Codon Adaptation Index. CBI: codon bias index. Nc: effective number of codons. Fop: frequency of optimal codons. GC3s: GC of silent 3rd codon posit.GC: GC content of gene. Table 1. Correspondence analysis of codon usage of aga1, Paenibacillus sp. SSG-1 and control genes. Results l i All these genes were located in a region which had an atypical GC content value with the genome (Fig. 4B). Moreover, NABH, and galactosidase were also uncommon in Paenibacillus sp. Their closest homologues (>​70% identify) were found in other microbes, such as Clostridium sp. D5, Paenibacillus sp. D14. As showed in Fig. 4A, aga1 had a closer relationship with genes from human symbiotic environments. Thus, aga1 and its surrounding genes in Paenibacillus sp. SSG-1 may have been horizontally transfered from other microbes, such as human oral or gut symbionts. As discussed above, the most possible mode for this transfer was the disposal of human faeces or saliva. Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 4 www.nature.com/scientificreports/ Figure 4. (A) Maximum likelihood tree of Aga1 and similar proteins. Numbers at nodes are levels of bootstrap support calculated from 100 bootstrap replicates (%). (B) The GC content change around the aga1. The red arrow indicates the position of aga1. The blue line represents the GC content of the whole genome of Paenibacillus sp. SSG-1. (C) Schematics of clusters containing aga1 like genes in different species. Sequence related genes (higher than 30% identity) are linked. NABH: α​-neoagarobiose hydrolase; cycloisomerase: 3,6-anhydro-L-galactonate cycloisomerase; reductase: 2,5-diketo-3-deoxy-L-galactonate 5-reductase; dehydrogenase-1: 3,6-anhydro-L-galactose dehydrogenase; dehydrogenase-2: 2-keto-3-deoxy-L-galactonate 5-dehydrogenase. Figure 4. (A) Maximum likelihood tree of Aga1 and similar proteins. Numbers at nodes are levels of bootstrap support calculated from 100 bootstrap replicates (%). (B) The GC content change around the aga1. The red arrow indicates the position of aga1. The blue line represents the GC content of the whole genome of Paenibacillus sp. SSG-1. (C) Schematics of clusters containing aga1 like genes in different species. Sequence related genes (higher than 30% identity) are linked. NABH: α​-neoagarobiose hydrolase; cycloisomerase: 3,6-anhydro-L-galactonate cycloisomerase; reductase: 2,5-diketo-3-deoxy-L-galactonate 5-reductase; dehydrogenase-1: 3,6-anhydro-L-galactose dehydrogenase; dehydrogenase-2: 2-keto-3-deoxy-L-galactonate 5-dehydrogenase. Figure 4. (A) Maximum likelihood tree of Aga1 and similar proteins. Numbers at nodes are levels of bootstrap support calculated from 100 bootstrap replicates (%). (B) The GC content change around the aga1. The red arrow indicates the position of aga1. The blue line represents the GC content of the whole genome of Paenibacillus sp. SSG-1. (C) Schematics of clusters containing aga1 like genes in different species. Sequence related genes (higher than 30% identity) are linked. NABH: α​-neoagarobiose hydrolase; cycloisomerase: 3,6-anhydro-L-galactonate cycloisomerase; reductase: 2,5-diketo-3-deoxy-L-galactonate 5-reductase; dehydrogenase-1: 3,6-anhydro-L-galactose dehydrogenase; dehydrogenase-2: 2-keto-3-deoxy-L-galactonate 5-dehydrogenase. Results l i When the distribution of aga1 like gene, NABH and galactosidase genes from human symbionts, Paenibacillus sp. D14 and Clostridium sp. D5, were investigated, we also found that these genes were rare in their correspond- ing genus. Meanwhile, the homologue of these genes could be found in the marine bacterium Rhodopirellula sallentina SM41. Conserved gene pair was also the indicator of horizontal gene transfer34. As showed in Fig. 4C, conserved gene pairs could be observed among the clusters. Gene pair, encoding for dehydrogenase-2 and reduc- tase, was conserved between Paenibacillus sp. D14 (genes 2759 and 2758) and Rhodopirellula sallentina SM41 (genes 1698 and 1699). Gene pair, encoding for cycloisomerase and dehydrogenase-2, was conserved between Clostridium sp. D5 (genes 2446 and gene 2447) and Rhodopirellula sallentina SM41 (genes 1696 and 1697). Moreover, transposase genes were found around the gene cluster in Paenibacillus sp. D14 and integrase gene was also found in the downstream of cluster in Clostridium sp. D5 (data not shown). Both of them were associated with horizontal gene transfer35,36. Combining these evidences, aga1 like gene and surrounding genes in human symbiotic bacteria may have been horizontally transfered from marine bacteria. According to previous work of Hehemann et al.33, the most possible reason for gene transfer from marine to human microbiota may be seafood diet.i To further confirm this inferred pathway, phylogenetic trees of two other soil agarases were constructed. As shown in Fig. 5A,B, the same trend could be observed. Soil agarase showed a closer relationship to the agarases of symbiotic environments, such as the human gut or human mouth, than the agarases from marine environments. This evidence also indicated the same mode of gene dissemination; i.e., from marine to symbiotic environments to soil. Based on these results, we developed the hypothesis that soil agarases may be the result of horizontal gene transfer from a marine environment to a soil environment via human microbiota, and human symbiotic micro- biota and human faeces and saliva serving as the link between human microbiota and the soil environment (Fig. 5C). Inland human microbiota may use agar. Horizontal gene transfer linkage of marine-symbiont-soil was inferred. Agarases are abundant in marine environments and are found in inland soil environments. However, agarases have not been found in other inland populations, the missing link in the above chain.h g g Thus, we used 37 characterized agarases as queries to investigate their distribution in human symbiotic microbes. Figure 5. (A,B) Maximum likelihood tree of two soil agarases. Numbers at nodes are levels of bootstrap support calculated from 1000 bootstrap replicates. (C) The sketch map of the predicted gene transfer pathw Protein ID Strain Identity Source BACPLE_01670 Bacteroide splebeius DSM 17135 135/286 (47%) NS BACPLE_01689 Bacteroides plebeius DSM_17135 321/321 (100%) NS HMPREF0240_02413 Clostridium sp. D5 212/343 (61%) Inland HMPREF0240_02442 Clostridium sp. D5 155/348 (44%) Inland VE20221DRAFT_03240 Clostridiales bacteriumVE202-21 150/345 (43%) NS VE20221DRAFT_03208 Clostridiales bacterium VE202-21 198/338 (58%) NS VE20221DRAFT_03199 Clostridiales bacterium VE202-21 183/321 (57%) NS RSAG_01951 Ruminococcus sp. 5_1_39BFAA 205/348 (58%) NS RSAG_01951 Ruminococcus sp. 5_1_39BFAA 205/348 (58%) NS RSAG_01951 Ruminococcus sp. 5_1_39BFAA 194/347 (55%) NS POTG_02958 Paenibacillus sp. oral taxon 786 220/352 (62%) Inland POTG_02762 Paenibacillus sp. oral taxon 786 211/355 (59%) Inland Table 2. Distribution of the potential agarase genes in human microbiome. Protein IDs are from IMG database. NS: source is not specified. Table 2. Distribution of the potential agarase genes in human microbiome. Protein IDs are from IMG database. NS: source is not specified. Table 2. Distribution of the potential agarase genes in human microbiome. Protein IDs are from IMG database. NS: source is not specified. achieve positive results, possibly because of unsuitable cultural conditions. Given these results, the microbiota of inland people may also utilize agar. achieve positive results, possibly because of unsuitable cultural conditions. Given these results, the microbiota of inland people may also utilize agar. Results l i Several possible agarases could be found in the microbes of inland people (Table 2). Twenty faecal samples from inland people were used to test for the capacity to degrade agarose. Interestingly, 8 of 20 samples were found to degrade agarose partially (Fig. 6). The screening of agar-degrading bacteria on an agar plate did not Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 5 www.nature.com/scientificreports/ Figure 5. (A,B) Maximum likelihood tree of two soil agarases. Numbers at nodes are levels of bootstrap support calculated from 1000 bootstrap replicates. (C) The sketch map of the predicted gene transfer pathway. Discussion Until now, most studied agarases have come from marine environments, and very few studies have focused on agarases from terrestrial environments, such as soil. In our previous study, an agarase was purified from the soil agar-degrading bacterium Paenibacillus sp. SSG-127, whose genome was subsequently sequenced (data not pub- lished). Using the identified peptides (tandem mass result), we found that natural agarase matched a hypothetical protein in the genome. This hypothetical protein showed specific activity against agarose and was thus designated as Aga1. Aga1 showed very low similarity (lower than 30%) to characterized proteins and was thought to be a distant member of GH86. Aga1 is an exo-type β​-agarase, which hydrolyses agarose into NA 4 and NA 6 as end products. In contrast, most exo-agarases produce only one type of oligosaccharide instead of a mixture; for example, Aga50D37, Aga2138, and AgWH50A15 produce NA2 as an end product, and AgWH50C14 produces NA4 as an end product. The struc- ture of agarase greatly affects its end products39. Thus, the catalytic pattern of Aga1 may be different from those of known agarases. g Aga1 has S-layer homology (SLH) domains that are associated with anchoring to the cell wall surface, and the subcellular prediction results showed that Aga1 was located on the cell wall. Previously, most agarases were intracellular or extracellular40. The SLH domain was not found in other reported agarases, nor was a domain with Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 6 www.nature.com/scientificreports/ Figure 6. The TLC analysis of the hydrolysis product by inland human fecal sample. NA2, NA4, NA6: neoagarobiose, neoagarotetraose, neoagarohexaose. Figure 6. The TLC analysis of the hydrolysis product by inland human fecal sample. NA2, NA4, NA6: neoagarobiose, neoagarotetraose, neoagarohexaose. similar function. The cell-wall-binding enzymes were thought to be an efficient way to decompose polysaccha- rides into smaller components that were suitable for cells to absorb41. This was also found in the starch utilization system, which showed multiple functional proteins displayed on the surfaces of bacteria42. Thus, Aga1 was possi- bly located on the cell surface to degrade the agar, which has seldom been reported in agarases. similar function. The cell-wall-binding enzymes were thought to be an efficient way to decompose polysaccha- rides into smaller components that were suitable for cells to absorb41. This was also found in the starch utilization system, which showed multiple functional proteins displayed on the surfaces of bacteria42. Discussion Thus, Aga1 was possi- bly located on the cell surface to degrade the agar, which has seldom been reported in agarases. G h f l d d h d b h d Given the scarcity of agar in inland environments and the distance between these environments and geo- graphic locations with a sea environment, aga1 is unlikely to be the result of self-evolution or direct gene transfer from the sea. With the appearance of several agarases in inland symbiotic microbes, inland human microbiota are the most likely source. It has been reported that using human faeces as manure may cause antibiotic resistance gene transfer43. Additionally, saliva has also been shown to spread microbes in the environment. The “contami- nation” of soil with human faeces or saliva maybe the precondition for the gene transfer of agarase in an inland soil environment. An anomalous nucleotide composition indicated that aga1 was the result of horizontal gene transfer. In Paenibacillus sp. SSG-1, aga1 and other agar utilization genes, which encoding for NABH, galactosidase and L-AnG metabolic enzymes, were clustered. Meanwhile, gene clusters from different species were compared. The scarcity of agar utilization genes in their corresponding genus, the closest homologues between different microbes, the conserved gene pairs between different taxa and the appearance of transposase and integrase indi- cated aga1 may have been transfered together with other genes from marine environments to human microbiota, and to soil environments. This gene cluster transfer was also found in other human gut bacterium32. This gene cluster transfer is reasonable, because gene cluster transfer may enable microbes to utilize agar, while transfer of one agarase is not sufficient. fi All in all, soil agarase aga1 may be a result of horizontal gene transfer, from marine environment to soil envi- ronment via human microbiota. This gene transfer was also observed with two other soil agarases. In particular, Sco3481, the soil neoagarobiose hydrolase, was founded in microbes from terrestrial plants. This was consistent with the appearance of agar-degrading bacteria in plant associated environments29. Additionally, this evidence confirmed that human faeces and saliva affect not only soil but also plants in soil environments. Acquiring agarase may enable soil or plant associated microbes to use agar or agar-like polysaccharides. However, it is still unknown if this gene transfer provides an advantage. In this study, aga1 was from an inland bacterium. Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 Methods B i l Bacterial strains and culture medium. E. coli DH5α​ was used as the general gene-cloning host, and E. coli BL21 (DE3) was used as the host for protein expression. Unless otherwise noted, E. coli trains were cul- tured in Luria-Bertani (LB) medium with 100 μ​g/mL kanamycin. Paenibacillus sp. SSG-1 was cultured at 37 °C in LB medium. Strain SSG-1 had been deposited in the China Center Type Culture Collection (CCTCC) with the accession number CCTCC CB 2015001. Gene cloning. After overnight culturing, the cell pellet of Paenibacillus sp.SSG-1 was harvested, and the genomic DNA was extracted. The aga1 gene was amplified using high-fidelity PrimeSTAR Max DNA Polymerase (Takara, Japan). The PCR product was digested with XholI/Not I and then ligated into the pET28a vector, which was also digested with Xhol I /Not I. After transformation into E. coli BL21, the recombinant plasmid was sequenced to confirm the accuracy of PCR. The sequences of the primers are listed in Supplemental Table S5. Domain analysis of Aga1. Conserved domains of Aga1 were analysed using InterPro, and secondary struc- ture analysis was conducted at the PSIPRED site (http://bioinf.cs.ucl.ac.uk/psipred/). Cell-PLos 2.0 was used to predict the subcellular location of Aga1, and signal peptides were predicted using SignalP 3.0. Protein production and purification. Recombinant Aga1 was produced with an auto-induction method. E. coli BL21 harbouring the recombinant plasmid was cultured in LB medium containing 100 μ​g/mL kanamycin. After overnight culturing, the E. coli cells were inoculated into 2 L auto-induction-medium and then cultured at 28 °C for 48 h. After centrifugation, the cell pellet was collected and then suspended in 50 mL of 20 mM PB buffer with sonication. The supernatant was harvested. The protein was further purified using Ni-column (0.7 ×​ 2.5 cm; GE Healthcare). The elution fractions with agarase activity were collected and further analysed by SDS-PAGE. Unless otherwise noted, the protein purification procedure was conducted at 4 °C. Biochemical characterization of Aga1. To analyse the substrate specificity of Aga1, CMC-Na, pectin, carrageenan (mixture of κ​, λ​ and ι​), sodium alginate, arabic gum, neoagarooctaose, neoagarotetraose, neoaga- rohexaose and neoagarooctaose were tested.iii g To confirm whether Aga1 was correctly produced and purified, purified protein was obtained to conduct tandem mass spectrometry (MS) analysis. A local database was created using the protein data of Paenibacillus sp. SSG-1 and the Mascot search engine were used to identify the matched protein. Discussion It is known that microbiota in coastal humans may acquire agarase genes through seafood, which has not been reported in inland populations. If this gene transfer linkage exists, it is reasonable to infer that inland human microbiota have agarase genes. Using the 37 known agarases as queries, several possible agarases were found in human reference genomes, and some of them were from inland human microbes. In addition, a previous study indicated that agarases from GH86 and GH117 specifically appeared in the human digestive system. Moreover, these agarases were distributed in a North American population, as well as a Japanese population44. The lack of studies on agarases has made bioinformatic analysis difficult. As many agarases or seaweed associated genes may be annotated as hypothetical proteins or simple glycoside hydrolases, bioinformatic analysis are restricted. These data suggest that agarases may be distributed in human microbiota, including microbiota from inland people. In our study, 8 of 20 faecal samples from inland people were shown to have agar-degrading capacity. Given the difficulty in screening for agar-degrading bacteria due to unfavourable culture conditions, these findings are still encouraging. Additionally, a previous study showed that inland people’s microbiota could degrade agar-oligosaccharides and that an agar-degrading bacterium, B. uniformis L8, was isolated from inland human faecal samples31. These results suggest that microbiota from an inland population can degrade agar. 33 A previous study of Hehemann et al.33 showed that seaweed associated genes were horizontally transfered into Bacteroides plebeius of the Japanese population, which traditionally eats non-cooked seaweed. Seaweed food, known as “sea vegetables”, is a popular food, not only in coastal regions but also in inland areas2–4,45. Diet changes have always altered human microbiota46, and microbes living inside the body have employed gene transfer to gain Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 7 www.nature.com/scientificreports/ functions to adapt to changes44. Taking seaweed as food may explain how microbiota from inland people have acquired the agarases from marine bacteria. Meanwhile, agar-degrading bacteria have been reported to produce agar-oligosaccharides with biological functions, which may influence human microbiota47. Thus, using seafood as food may influence the balance of human microbiota, which has been shown to be important for human health. Further study is needed to determine whether eating seaweed can affect human microbiota. Conclusion In conclusion, we first cloned and characterized an exo-type β​-agarase; i.e., Aga1, from Paenibacillus sp. SSG-1. aga1 showed low similarity to known glycoside hydrolases and may be a distant member of the GH 86 family. aga1 gene may be the result of horizontal gene transfer from marine environments to humans to soil. Using seaweed as food and human faeces or saliva are the most likely linkages for this gene transfer pathway. Our results indicate that inland human microbiota also have the opportunity to acquire seaweed-associated genes from microbes that attach to the surface of seaweed foods. Methods B i l The DNS method was used to assay agarase activity, with D-galactose as the standard. The assay procedure was conducted as previously described. Enzyme activity (U) was defined as the amount of enzyme that liberated 1 μ​mol D-galactose per min- ute. The optimal pH and temperature and the stability at different pH values and temperatures were tested as previously described. Various metal ions and chemical reagents (1 mM) were added to the reaction solution to investigate their effects on agarase activity. All experiments were conducted in triplicate. Analysis of the degrading pattern of Aga1. To investigate the hydrolysis pattern of Aga1, 100 μ​g of purified enzyme was added to 50 mL of 0.5% substrate solution (0.5% agarose in deionized water). The reaction solution was incubated at 40 °C. Different samples were collected at fixed intervals. The collected samples were applied to silica G plates (Qingdao Haiyang Chemical Co., Ltd) using n-butyl alcohol:water:acetic acid =​ 2:1:1 as the developing solvent and then visualized using phenylamine/diphenylamine solution. High performance liquid chromatography (HPLC) was also used to detect the reaction (column HPX87-H Biorad 300 ×​ 7.8 mm). Bioinformatic analysis of Aga1 and related agarases. aga1 similar sequences were obtained from NCBI, Integrated Microbial Genomes (IMG) and the NIH Human Microbiome Project (HMP). Sequences were aligned with the Clustal W program and modified using Gblocks. The phylogenetic tree was constructed using a maximum likelihood method in PhyML. The nucleotide composition and codon usage analyses were conducted using the CodonW online service (http://mobyle.pasteur.fr/cgi-bin/portal.py#forms::CodonW). The species tree was constructed in MEGA 6.0 using a neighbour-joining method. Analysis of other two soil agarases was con- ducted using the same procedure. Agar degrading experiments using microbiomes from an inland population. This study was approved by the Ethics Committee of Sichuan University. 20 persons, who lived in Chengdu, were enrolled to collect stool specimens. Informed consent was obtained from all participants. All experimental procedures were Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 8 www.nature.com/scientificreports/ carried out in accordance with the Committee’s approved guidelines. The faecal samples were diluted (0.1 g samples were added to 10 mL of deionized water) and 50 μ​l of the diluted sample was added into 3 mL of the medium that contained agarose as a sole carbon source. The medium contained 0.1% NaCl, 0.1%K2HPO4, 0.1% (NH4)2SO4, 0.05% MgSO4, 0.01% CaCl2, 0.2% yeast extract and 0.2% agarose. Investigation of the distribution of agarase in human symbiotic microbes.  The protei Investigation of the distribution of agarase in human symbiotic microbes. The protein sequences of 37 characterized agarases were obtained from the CAZy database and were used as search queries. IMG online Blast service (https://img.jgi.doe.gov/cgi-bin/mer/main.cgi?section=​FindGenesBlast&page=​geneSearchBlast) was used to search for proteins with high similarity (over 40%). Similar proteins from human-related microbes were chosen,and information on geographic locations was also collected. Identification of agar utilization proteins. The agar’s utilization enzymes were studied in recent years, proteins in the database may be not annotated. Thus, experimental confirmed enzymes were used as the search queries and proteins with confident identity (higher than 30%) were deduced to have same function. The acces- sion numbers of the search sequences were listed in the Supplemental Table S6. 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FAO yearbook: fishery and aquaculture statistics 2012. Rome: Food and Agriculture Organization of the United Nations (2014). ( ) 2. Mouritsen, O. G. et al. On the human consumption of the red seaweed dulse (Palmaria palmata (L.) Weber & Mohr). J Appl Phyco 25, 1777–1791, doi: 10.1007/s10811-013-0014-7 (2013). 3. Fleurence, J. et al. What are the prospects for using seaweed in human nutrition and for marine animals raised through aquaculture Trends Food Sci Technol 27, 57–61, doi: 10.1016/j.tifs.2012.03.004 (2012). j 4. Edwards, M. D., Holdt, S. L. & Hynes, S. Algal eating habits of phycologists attending the ISAP Halifax Conference and me j 4. Edwards, M. D., Holdt, S. L. & Hynes, S. Algal eating habits of phycologists attending the ISAP Halifax Conference and members of the general public. J Appl Phycol 24, 627-633, doi: 10.1007/s10811-011-9760-6 (2011).i j 4. Edwards, M. D., Holdt, S. L. & Hynes, S. Algal eating habits of phycologists attending the ISAP Halifax Conference and membe the general public J Appl Phycol 24 627 633 doi 10 1007/s10811 011 9760 6 (2011) j 4. Edwards, M. D., Holdt, S. L. & Hynes, S. Algal eating habits of phycologists attending the ISAP Halifax Conference and members of the general public. J Appl Phycol 24, 627-633, doi: 10.1007/s10811-011-9760-6 (2011).i 4. Edwards, M. D., Holdt, S. L. & Hynes, S. Algal eating habits of phycologists attending the ISAP Halifax Conference a the general public. J Appl Phycol 24, 627-633, doi: 10.1007/s10811-011-9760-6 (2011).i y g g p y g g the general public. J Appl Phycol 24, 627-633, doi: 10.1007/s10811-011-9760-6 (2011).i g p pp y 5. Dawczynski, C., Schubert, R. & Jahreis, G. Amino acids, fatty acids, and dietary fibre in edible seaweed products. Food Chem 103 891–899, doi: 10.1016/j.foodchem.2006.09.041 (2007). , j ( ) 6. Wargacki, A. J. et al. An engineered microbial platform for direct biofuel production from brown macroalgae. Science 335, 308–313, doi: 10.1126/science.1214547 (2012).fi ( ) 7. Enquist-Newman, M. et al. Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform. Nature 505, 239–243, doi: 10.1038/nature12771 (2013). 8. Lahaye, M., Yaphe, W., Viet, M. T. P. & Rochas, C. 13 Cn. mr spectroscopic investigation of methylated and charged agaros oligosaccharides and polysaccharides. Carbohydr Res 190, 249–265 (1989). Methods B i l After cultivation at 37 °C for 5 days, 50 μ​l of supernatant was collected and subjected to TLC analysis. carried out in accordance with the Committee’s approved guidelines. The faecal samples were diluted (0.1 g samples were added to 10 mL of deionized water) and 50 μ​l of the diluted sample was added into 3 mL of the medium that contained agarose as a sole carbon source. The medium contained 0.1% NaCl, 0.1%K2HPO4, 0.1% (NH4)2SO4, 0.05% MgSO4, 0.01% CaCl2, 0.2% yeast extract and 0.2% agarose. After cultivation at 37 °C for 5 days, 50 μ​l of supernatant was collected and subjected to TLC analysis. www.nature.com/scientificreports/ Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559–563 (2014). . David, L. A. et al. Diet rapidly and reproducibly alters the human 6. David, L. A. et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559 563 (2014). 7. Holdt, S. L. & Kraan, S. Bioactive compounds in seaweed: functional food applications and legislation. J Appl Phycol 23, 543–597 doi: 10.1007/s10811-010-9632-5 (2011). Acknowledgements We thank Dr. Luo Dan for providing advice on gene cloning and appreciate Dr. Jingqiang Tang’s help with HPLC. We thank Dr. Jan-Hendrik Hehemann for advice on the classification of aga1. This work was supported by the National Twelfth Five-year Science and Technology support program (2014BAD02B02), the National Natural Science Foundation of China (31272659), the National Infrastructure of Natural Resources for Science and Technology Program of China (NIMR-2014-8) and the Sichuan Science and Technology Bureau (2014GXZ0005, 2017GXZ0005, 2012GZ0008, and 2015JPT0032). We thank Dr. Luo Dan for providing advice on gene cloning and appreciate Dr. Jingqiang Tangs help with HPLC. We thank Dr. Jan-Hendrik Hehemann for advice on the classification of aga1. This work was supported by the National Twelfth Five-year Science and Technology support program (2014BAD02B02), the National Natural Science Foundation of China (31272659), the National Infrastructure of Natural Resources for Science and We thank Dr. Luo Dan for providing advice on gene cloning and appreciate Dr. Jingqiang Tangs help with HPLC. We thank Dr. Jan-Hendrik Hehemann for advice on the classification of aga1. This work was supported by the National Twelfth Five-year Science and Technology support program (2014BAD02B02), the National Natural t Science Foundation of China (31272659), the National Infrastructure of Natural Resources for Science and Technology Program of China (NIMR-2014-8) and the Sichuan Science and Technology Bureau (2014GXZ0005, 2017GXZ0005, 2012GZ0008, and 2015JPT0032). Author Contributions T.S. and H.X. conducted most of the experiments, analysed the results and wrote most of the manuscript. C.W., T.J. and S.Q. conducted the bioinformatic analysis. W.Z., Y.C., C.H. and F.Z. purified proteins and analysed the data. D.Q. and Y.C. conceived the idea for this project and wrote the paper with T.S. and H.X. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 8. Li, X., Dong, X., Zhao, C., Zhen, C. & Feng, C. Isolation and some properties of cellulose-degrading Vibrio sp. LX-3 with agar liquefying ability from soil. World J Microbiol Biotechnol 129, 375–379 (2003). q y g y 9. Song, T. et al. Isolation and characterization of agar-degrading endophytic bacteria from plants. Curr Microbiol 70, 275–281 doi: 10.1007/s00284-014-0713-6 (2015).h 0. Agbo, J. A. & Moss, M. O. The isolation and characterization of agarolytic bacteria from a lowland river. J Gen Microbiol 115 355–368 (1979).l 1. Li, M. et al. Isolation and characterization of an agaro-oligosaccharide (AO)-hydrolyzing bacterium from the gut microflora o Chinese individuals. PLoS ONE 9, e91106, doi: 10.1371/journal.pone.0091106 (2014). j p 32. Hehemann, J. H. & Boraston, A. B. Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes. P Natl Acad Sci USA 109, 19786–19791 (2012). j p 2. Hehemann, J. H. & Boraston, A. B. Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active d f b l d ( ) enzyme updates from extrinsic microbes. P Natl Acad Sci USA 109, 3. Hehemann, J. H. et al. Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature 464 908–912, doi: 10.1038/nature08937 (2010).ii 4. Koonin, E. V., Makarova, K. S. & Aravind, L. Horizontal gene transfer in prokaryotes: quantification and classification1. Annu Rev Microbiol 55, 709–742 (2001).i 35. Canchaya, C., Fournous, G., Chibani-Chennoufi, S., Dillmann, M.-L. & Brüssow, H. Phage as agents of lateral gene transfer. Curr Opin Microbiol 6, 417–424 (2003). 6. Salyers, A. A., Shoemaker, N. B., Stevens, A. M. & Li, L.-Y. Conjugative transposons: an unusual and diverse set of integrated gene transfer elements. Microbiol Rev 59, 579–590 (1995). 37. Kim, H. T. et al. Overexpression and molecular characterization of Aga50D from Saccharophagus degradans 2-40 agarase producing neoagarobiose. Appl Microbiol Biotechnol 86, 227–234, doi: 10.1007/s00253-009-2256-5 (2010 7. Kim, H. T. et al. Overexpression and molecular characterization of Aga50D from Saccharophagus degradans 2-40: an exo-type beta r pr d in n r bi Appl Mi bi l Bi t h l 86 227 234 d i 10 1007/ 00253 009 2256 5 (2010) 37. Kim, H. T. et al. Overexpression and molecular characterization of Aga50D from Saccharophagus degradans 2-40: an exo-type agarase producing neoagarobiose. Appl Microbiol Biotechnol 86, 227–234, doi: 10.1007/s00253-009-2256-5 (2010).i 38. Li, J., Hu, Q., Li, Y. References Appl Microbiol Biotechnol 66, 266–275, doi: 10.1007/s00253-004-1757-5 (2004). hta, Y. et al. Cloning, expression, and characterization of a glycoside hydrolase family 86 beta agarase from a deep sea icrobulbifer-like isolate. Appl Microbiol Biotechnol 66, 266–275, doi: 10.1007/s00253-004-1757-5 (2004). pp 8. Ekborg, N. A. et al. Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40. App Environ Microbiol 72, 3396–3405, doi: 10.1128/aem.72.5.3396-3405.2006 (2006).ih 18. Ekborg, N. A. et al. Genomic and proteomic analyses of the agarolytic system expressed Environ Microbiol 72, 3396–3405, doi: 10.1128/aem.72.5.3396-3405.2006 (2006).ih 19. Ohta, Y. et al. Purification and characterization of a novel alpha-agarase from a Thalassomonas sp. Curr Microbiol 50, 212–216, doi: 10.1007/s00284-004-4435-z (2005).i 0. Potin, P., Richard, C., Rochas, C. & Kloareg, B. Purification and characterization of the α​‐agarase from Alteromonas agarlyticu (Cataldi) comb. nov., strain GJ1B. Eur J Biochem 214, 599–607 (1993).f 1. Rebuffet, E. et al. Discovery and structural characterization of a novel glycosidase family of marine origin. Environ Microbiol 13 1253–1270 (2011). 22. Ha, S. C. et al. Crystal structure of a key enzyme in the agarolytic pathway, alpha-neoagarobiose hydrolase from Saccharophagus degradans 2-40. Biochem Biophys Res Commun 412, 238–244, doi: 10.1016/j.bbrc.2011.07.073 (2011). g p y j 3. Ma, C. et al. Molecular Cloning and Characterization of a Novel β​-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24. J Bio Chem 282, 3747–3754, doi: 10.1074/jbc.M607888200 (2007). j 24. Dong, J., Hashikawa, S., Konishi, T., Tamaru, Y. & Araki, T. Cloning of the novel gene encoding beta-agarase C from a marine bacterium, Vibrio sp. strain PO-303, and characterization of the gene product. Appl Environ Microbiol 72, 6399–6401, doi: 72/9/6399 (2006). ( ) 25. Gilbert, H. J. & Hazlewood, G. P. Bacterial cellulases and xylanases. Microbiology 139, 187–194 (1993). y gy 26. Lakshmikanth, M., Manohar, S., Souche, Y. & Lalitha, J. Extracellular beta-agarase LSL-1 producing neoagarobiose from a n isolated agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1. World J Microbiol Biotechnol 22, 1087–1094, doi: 10.1 s11274-006-9147-z (2006).i 27. Song, T. et al. Purification and characterization of a novel beta-agarase of Paenibacillus sp. SSG-1 isolated from soil. 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Complex carbohydrate utilization by the healthy human microbiome. PLoS ONE 7, e2 (2012). 44. Cantarel, B. L., Vincent, L. & Bernard, H. Complex carbohydrate utilization by the healthy human microbiome. PLoS ONE 7, e28742 (2012). 45. Mchugh, D. J. A guide to the seaweed industry. FAO Fish Tech P 45. Mchugh, D. J. A guide to the seaweed industry. FAO Fish Tech Pap (2003). 46 David L A et al Diet rapidly and reproducibly alters the human gut microbiome Nature 505 559 563 (2014) g g y p 46. David, L. A. et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559–563 (2014). 47 H ldt S L & K S Bi ti d i d f ti l f d li ti d l i l ti J Appl Ph l 23 543 59 46. David, L. A. et al. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Song, T. et al. Horizontal Transfer of a Novel Soil Agarase Gene from Marine Bacteria to Soil Bacteria via Human Microbiota. Sci. Rep. 6, 34103; doi: 10.1038/srep34103 (2016). How to cite this article: Song, T. et al. Horizontal Transfer of a Novel Soil Agarase Gene from Marine Bacteria to Soil Bacteria via Human Microbiota. Sci. Rep. 6, 34103; doi: 10.1038/srep34103 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 6:34103 | DOI: 10.1038/srep34103 10
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Microstructural characterization and inductively coupled plasma-reactive ion etching resistance of Y2O3–Y4Al2O9 composite under CF4/Ar/O2 mixed gas conditions
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www.nature.com/scientificreports OPEN Microstructural characterization and inductively coupled plasma‑reactive ion etching resistance of ­Y2O3–Y4Al2O9 composite under ­CF4/Ar/O2 mixed gas conditions Ho Jin Ma 1*, Seonghyeon Kim 2, Ha‑Neul Kim 1, Mi‑Ju Kim 1, Jae‑Woong Ko 1, Jae‑Wook Lee 1, Jung‑Hyung Kim 2, Hyo‑Chang Lee 3,4 & Young‑Jo Park 1* In the semiconductor manufacturing process, when conducting inductively coupled plasma-reactive ion etching in challenging environments, both wafers and the ceramic components comprising the chamber’s interior can be influenced by plasma attack. When ceramic components are exposed to long-term plasma environments, the eroded components must be replaced. Furthermore, nonvolatile reactants can form and settle on semiconductor chips, acting as contaminants and reducing semiconductor production yield. Therefore, for semiconductor processing equipment parts to be utilized, it is necessary that they exhibit minimized generation of contaminant particles and not deviate significantly from the composition of conventionally used ­Al2O3 and ­Y2O3; part must also last long in various physicochemical etching environment. Herein, we investigate the plasma etching behavior of ­Y2O3–Y4Al2O9 (YAM) composites with a variety of mixing ratios under different gas fraction conditions. The investigation revealed that the etching rates and changes in surface roughness for these materials were significantly less than those of ­Y2O3 materials subjected to both chemical and physical etching. Microstructure analysis was conducted to demonstrate the minimization of crater formation. Mechanical properties of the composite were also analyzed. The results show that the composite can be commercialized as next-generation ceramic component in semiconductor processing equipment applications. Keywords Plasma etching, Y2O3-YAM composite, Microstructure, Etching resistance, Semiconductor manufacturing The transition of semiconductor-based devices from 2D to 3D-NAND memory, accompanied by increased miniaturization using advanced technologies, necessitates the incorporation of high aspect ratio and line width miniaturization. Consequently, there arises a need for a diversified plasma environment in the etching p ­ rocess1–3. Inductively coupled plasma-reactive ion etching (ICP-RIE) has proved a suitable method for achieving deeper and higher aspect ratio chip etching, as it achieves high plasma density at low pressures, thereby facilitating a heightened etching r­ ate4. Notably, during the etching process, the strong plasma affects both the Si wafers and the ceramic parts constituting the chamber interior, such as focus rings, confinement rings, life pins, and inner walls. These ceramic components have varying replacement cycles depending on where they are used and what materials they are made of. For instance, the confinement ring, which uses alumina, should be replaced every 1 Department of Engineering Ceramics, Korea Institute of Materials Science, Changwon 51508, Republic of Korea. 2Semiconductor Integrated Metrology Team, Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea. 3Department of Semiconductor Science, Engineering and Technology, Korea Aerospace University, Goyang 10540, Republic of Korea. 4School of Electronics and Information Engineering, Korea Aerospace University, Goyang 10540, Republic of Korea. *email: hojinma@kims.re.kr; yjpark87@kims.re.kr Scientific Reports | (2024) 14:7008 | https://doi.org/10.1038/s41598-024-57697-5 1 Vol.:(0123456789) www.nature.com/scientificreports/ 300 h; the focus ring every 6 ­months5. Longer replacement intervals can elevate semiconductor production yields by eliminating the need to shut down the chamber. Particulate contamination, originating from internal parts shaved off by physical ion sputtering and generated by chemical reactions between radicals and the surfaces of ceramics, can be produced during ICP-RIE process. Certain volatile reactants, like ­SiF4, are eliminated using a vacuum pump. On the other hand, others may become trapped within the sheath and have the potential to migrate towards the upper surface of the wafer once the plasma ceases to ­operate6,7. Given that this circumstance can result in reduced production yield of large-scale integrated circuit chips, it is imperative to carefully select and develop appropriate ceramic materials that do not generate c­ ontaminants8–10. Furthermore, for more sophisticated etching of semiconductor chips, processing is carried out in various gas environments such as ­CF4, ­CHF3, ­Cl2, etc.11–13. The fluorine and argon gas mixture has been studied and is applied for etching processes on Si-based ­semiconductors14,15. ­O2 gas can increase the yield of F atoms through its interaction with C ­ F4 gas, while also preventing polymerization on surfaces exposed to the plasma. Consequently, it helps in attaining the targeted etching selectivity and feature t­ opology14,16. The introduction of Ar elevates plasma density and enhances generation of ­F− active species. Given that physicochemical plasma etching processes operate concurrently, manipulation of the plasma gas fraction enables the realization of selective e­ tching17. For decades, research has been conducted on the plasma resistance properties of various ceramic bulk and coating materials in chamber during semiconductor etching in halogen plasma gas a­ tmosphere18,19. ­Y2O3 material has been the subject of interest and commonly utilized because of its outstanding chemical etching r­ esistance20–23. Through a reaction of Y ­ 2O3 surfaces with radicals, Y ­ 2O3 can form a thick and non-volatile fluorinated layer. This layer serves as effective protection for components against ions i­ mpingement24. However, in conditions in which physical etching predominates and there is a high bias voltage, the fluorinated layer on the surface of the ­Y2O3 matrix is prone to removal. This results in its conversion into contaminant particles. Also, cracks form at the interface of the matrix and the fluorinated m ­ aterial25. In addition, for sintered Y ­ 2O3 ceramics that become densified at high sintering temperature, large grain sizes can cause large craters after etching, resulting in rougher surface roughness and large contaminant p ­ articles26. The hardness is low, and so improvement of mechanical properties is essential for applications in ceramic p ­ arts27. We recently found that ­Y2O3–MgO nanocomposite material could improve the physicochemical plasma etching resistance under intense plasma c­ onditions28. In the temperature range at which the two composition do not react, each composition suppressed unintended grain coarsening via Zener pinning effect and minimized the development of large craters in the ­microstructure29,30. Research has also explored ­Y3Al5O12 (YAG) ceramic as a highly promising material resistant to plasma in etching c­ hambers31,32. However, for YAG, when encountering ­Y2O3, and depending on the mixing fraction, Y ­ AlO3 (YAP) phase with perovskite structure and Y ­ 4Al2O9 (YAM) phase with monoclinic structure can form as r­ eactants33,34. Therefore, the Y ­ 2O3-YAM composition is best suited to form composites with both compositions, without additional reaction. The YAM ceramic has a moderate thermal expansion coefficient (7.51 × ­10–6 ­K−1), high melting point (2020 °C), and low high temperature thermal conductivity (1.13 W/m/K)35,36. Therefore, it is anticipated that this material can be applied as a thermal barrier coating (TBC). Unlike YAG, little research has been done on the plasma etching resistance of YAM ceramics. When ceramic components used in semiconductor process equipment are completely changed from existing materials, it is difficult in practice to apply them due to the lack of reported processing stability. However, Y ­ 2O 3, ­Al2O3, and YAG materials are all being used or developed in chambers, so they are free of this problem. In addition, these composites can be used to overcome the shortcomings of conventional ­Y2O3 materials. In this study, we investigate the plasma etching characteristics of Y ­ 2O3-YAM composite across different volume fractions; material is intended for application in ceramic parts within etching equipment operating under high ICP power and bias voltage conditions. Firstly, dense ­Y2O3 and YAM composite sintered bodies with different volume ratios were fabricated via hot-press sintering. Next, the etching depths and surface roughness changes of ­Y2O3 and ­Y2O3-YAM composites were analyzed after physicochemical plasma etching with different mixture gas ratios. The mechanical properties of the composites and the surface microstructure after etching were analyzed. This investigation examined the capability of Y ­ 2O3-YAM composite to substitute for conventional materials to minimize production of contaminants and improve the production yield of semiconductors when ceramic components are subjected to intense plasma environments and diverse gas conditions. Methods Fabrication of ­Y2O3‑Y4Al2O9 nanocomposites ceramic Y2O3 (99.9%, Cenotec, Korea) and Y ­ 4Al2O9 (YAM, 99.9%, Syntech, Korea) were used as raw materials for the nanocomposites. They were mixed at 90:10, 70:30, 50:50, 30:70, and 10:90 volume ratios, and ball-milled with anhydrous alcohol (99.9%, Samchun, Korea) and zirconia grinding media with 5 mm diameter. The milling time was 12 h at 200 rpm. Specimens were named as shown in Table 1. After ball milling, slurries were dried and subjected to sieving through a 200-mesh sieve (75 µm ). Subsequently, the sieved powders were consolidated via hot-press sintering. The graphite sleeves and spacers used for sintering were coated with BN spray to reduce direct contact with the powders and minimize contamination of samples by carbon. The ­Y2O3-YAM pellets were sintered at 1500 °C with applied pressure of 40 MPa under vacuum condition. The dwell time was fixed at 2 h; this was followed by cooling. After the hot-pressing, annealing was conducted at 1200 °C for 20 h in a box furnace under air atmosphere. The post-annealing process was carried out due to remove residual carbon and oxygen vacancies, and to alleviate residual stress. To analyze the microstructure of the produced composites by SEM, they were subjected to thermal etching at 1150 °C for 2 h. Scientific Reports | Vol:.(1234567890) (2024) 14:7008 | https://doi.org/10.1038/s41598-024-57697-5 2 www.nature.com/scientificreports/ Sample name Y2O3 Y4Al2O9(YAM) (Vol%) Y2O3 (Vol%) 0 100 YAM1 10 90 YAM3 30 70 YAM5 50 50 YAM7 70 30 YAM9 90 10 Table 1.  Composition ratios of ­Y4Al2O9 (YAM) and ­Y2O3 ceramics. Plasma etching test To elucidate the plasma etching characteristics of the Y ­ 2O3-YAM composite across various mixed gas ratio environments, all samples were produced with uniform dimensions of 15 mm diameter and 1 mm thickness. Additionally, for comparative analysis of plasma etching behavior, commercial c-axis sapphire and ­Y2O3 polycrystalline ceramics were prepared. The Y ­ 2O3 samples were sourced from FineTech Co, Ltd. (Korea); the measured relative density of the Y ­ 2O3 ceramics was 99.4%. To accurately identify post-etching changes of Y ­ 2O3 and ­Y2O3-YAM polycrystalline ceramics, it was necessary to reduce the initial surface roughness to a level of 5 nm or less, so the surface was polished and chemical-mechanically planarized (CMP). The polished samples then underwent partial coverage with a shadow mask composed of a nickel–cobalt alloy, manufactured through the electroforming process. This shadow mask is mechanically flexible, reusable and full-surface contactable, so that it can be applied in actual plasma etching process. Therefore, this material was utilized as the shadow mask to create an environment similar to the actual process. The selectively exposed area had a length of 6 mm and a width of 1 mm. The thickness of the employed mask was 0.05 mm. Details of the plasma etching conditions and a simple schematic illustration of the plasma etching test chamber system are presented in Table 2. For the plasma test, a 13.56 MHz power supply was connected to a planar-type antenna. The input ICP power and RF bias voltage were 1.5 kW and 600 V, respectively. Under the specified discharge conditions, the plasma was sustained in inductive mode; this is a well-established approach in industrial ­semiconductors37. Plasma tests were executed within an RF-biased inductively coupled plasma (ICP) chamber, employing a gas mixture of ­CF4, ­O2 and ­Ar38. The plasma density measured by a microwave cut-off probe, was 9 × ­1010 ­cm-3. Exposure of all ceramic samples to the plasma environment was carried out for a duration of 1 h. The experimental pressure was 20 mTorr. To elucidate the physicochemical etching behavior of materials, the proportions of ­CF4:Ar:O2 gases were systemically adjusted at the following ratios: 40:10:10, 30:20:10, 20:30:10, and 10:40:10 sccm. In this study, the O ­ 2 flow rate was maintained at 10 sccm, with variation made solely to the C ­ F4 to Ar gas ratios to control the physical and chemical etching. The plasma gas composition ratios used in this experiment are shown in Table 3. Characterization Phase analysis of the sintered Y ­ 2O3-YAM composite ceramics was conducted utilizing an X-ray diffraction analysis equipment (XRD, D/Max 2500, Rigaku) with CuK α radiation, employing a scan rate of 5°/min within Conditions Samples Sapphire, ­Y2O3, ­Y2O3-YAM composite ICP power (frequency) 1.5 kW (13.56 MHz) Bias voltage (frequency) 600 V (2 MHz) Plasma gas CF4, Ar, ­O2 Pressure 20 mTorr Etching time 60 min Electrode distance 152 mm Table 2.  Details of plasma etching conditions. Gas conditions CF4 (sccm) Ar (sccm) O2 (sccm) 1 40 10 10 2 30 20 10 3 20 30 10 4 10 40 10 Table 3.  Mixed gas ratios for plasma etching test. Scientific Reports | (2024) 14:7008 | https://doi.org/10.1038/s41598-024-57697-5 3 Vol.:(0123456789) www.nature.com/scientificreports/ the range of 20° to 60°. The relative densities of all sintered samples were determined by the Archimedes method. The grain size was obtained by measuring the average line-intercept length of 150 grains. Average etching depths of sapphire, Y ­ 2O3, and Y ­ 2O3-YAM composites were assessed using a surface profiler (Tencor P-7 Stylus Profiler, KLA Co.) at three distinct positions for each specimen, employing scan length of 1 mm and scan rate of 200 Hz. Surface roughness and 3D topography of samples were further investigated through atomic force microscopy (AFM, XE-100, Park Systems), with the surface roughness (­ Ra) derived from measurements over 25 × 25 µm area. Microstructural images and EDS analyses of specimens were carried out using a field emission-scanning electron microscope (FE-SEM, JSM-7800F, JEOL). The Vickers hardness was measured using a Vickers hardness tester (HM200, Mitutoyo) with a 1 kg load applied to the unetched surface. Results and discussion Characterization of sintered ­Y2O3‑YAM nanocomposites Following the blending of Y ­ 2O3 and YAM powders at varying volume ratios, the phases of the resultant composite ceramics, consolidated through hot-pressing at 1500 °C, were identified by XRD analysis, as shown in Fig. 1a. Notably, with as little as 10% YAM powder by volume, a predominantly cubic ­Y2O3 (#86-1326) phase was identified, accompanied by a faint monoclinic YAM (#83-0935) phase. However, the intensity of the YAM phase peak relative to Y ­ 2O3 increased as the proportion of the YAM phase increased. When Y ­ 2O3 was more than twice as abundant as ­Al2O3, only YAM and ­Y2O3 phases existed as phase diagrams in the pseudo-binary system during sintering at 1500 °C39. Therefore, only YAM and ­Y2O3 phases were detected in the composites sintered in this study, while phases such as A ­ l2O3, ­Y3Al5O12 and ­YAlO3 were not identified. Figure 1b shows the measured and relative densities of the Y ­ 2O3-YAM composite sintered at 1500°C. YAM has a lower density (4.56 g/cm3) than that of Y ­ 2O3 (5.01 g/cm3), so when the volume fraction ranged from 10 to 90%, the measured density exhibited a gradual decline from 4.97 to 4.61 g/cm3. With increased percentage of YAM phase, despite a slight decrease in relative density from nearly 100% theoretical density to 99.4%, overall, well-densified specimens with minimal residual pores were obtained. In comparison to YAM, which typically necessitates sintering temperatures as high as 1800°C and high pressure, or Y ­ 2O3 single-composition ceramics, which require a temperature of 1600°C and applied pressure, the composite of the two compositions facilitated the production of high-density specimens at lower ­temperatures35,40,41. In addition, when compounding between ­Y2O3 and YAG compositions, densification was difficult due to reactant formation, including YAM and YAP phases; however, in this study, by compounding Y ­ 2O3 and YAM, we were able to solve this problem and achieve high density. The process also permitted examination of the plasma resistance properties among specimens of nearly equivalent density. SEM microstructural images of ­Y 2 O 3 -YAM composite with various volume ratios after consolidation at 1500°C for 2 h are represented in Fig. 2. Overall, dense specimens with few pores were achieved for all compositions after hot-pressing. The average grain sizes were 0.90 ± 0.53µm, 1.02 ± 0.44µm, 0.86 ± 0.41µm, 1.62 ± 1.02µm, and 0.97 ± 0.44µm from YAM1 to YAM9, in order, indicating an overall submicron meter size. In Fig. 2a–c, YAM1, 3, and 5 had a relatively fine grain size, with a small amount of nanopores at the triple pints, which could be the point of crater formation during plasma etching. On the other hand, the YAM7 specimen in Fig. 2d showed a relatively large grain size. It can be seen that due to the rapid grain coarsening during the sintering, pores are trapped within the grain without being able to escape. These intragranular pores are more difficult to eliminate than pores at grain boundaries or triple points. Figure 1.  (a) X-ray diffraction patterns of sintered ­Y2O3-YAM composite ceramics with different volume ratios. (b) Measured and relative density of sintered ­Y2O3-YAM nanocomposite ceramics with different mixed ratios. Scientific Reports | Vol:.(1234567890) (2024) 14:7008 | https://doi.org/10.1038/s41598-024-57697-5 4 www.nature.com/scientificreports/ Figure 2.  SEM microstructure images of Y ­ 2O3-YAM composites with different volume ratios after hot-pressing at 1500 °C with 40 MPa for 2 h; (a) YAM1, (b) YAM3, (c), YAM5, (d), YAM7, and (e) YAM9 composite ceramics. Plasma etching behavior of sintered ­Y2O3‑YAM nanocomposites Plasma etching testing was performed on reference materials including sapphire, ­Y2O3 ceramics, and ­Y2O3-YAM sintered composite materials, within an aggressive plasma environment. The simple scheme of plasma etching chamber is shown in Fig. 3. Figure 4 illustrates the resultant etching depths of the materials, delineating the impacts of varying plasma gas ratios of ­CF4 to Ar. Sapphire, the reference, showed a fast etching rate of more than 1000 nm/h under all conditions, and the etching depth decreased as the amount of C ­ F4 gas decreased because the effect of chemical etching weakened because of the low density of ­CF2 radicals in the gas ­mixture42. For ­Y2O3 ceramics, the etching depth increased slightly as the amount of Ar gas increased; it then decreased because the effect of physical etching increased when amount of Ar gas increased and the fluorinated layer generated on the surface of Y ­ 2O 3 was easily ­eliminated10. Overall, the ­Y2O3-YAM composites exhibited slower etching rates compared to the two reference materials, particularly when materials were subjected to C ­ F4:Ar:O2 ratios of 20:30:10 and 30:20:10, where simultaneous physicochemical etching was applied. In these conditions, all compositions had inductively coupled plasma-reactive ion etching resistance superior to that of Y ­ 2O3. The overall etching rates of ­Y2O3-YAM composites increased with increases in proportion of Ar in the plasma mixture gases. Moreover, the rise in etching rate was more pronounced for higher YAM compositions, notably in the case of the YAM9 composition, the highest YAM contents. As the C ­ F4:Ar:O2 ratios varied from 40:10:10 to 10:40:10, the etching depth of YAM9 exhibited a nearly threefold increase, increasing from 101 to 283 nm per hour, surpassing the value of ­Y2O3. Similarly, the YAM1 composition with 10% YAM content demonstrated an increase in etching depth from 96 to 138 nm. This phenomenon can be attributed to the lower boiling point (1275 °C) of A ­ lF3, which formed on the YAM composition surface through its reaction with F ­ − separated from C ­ F4 gas. This value is substantially lower than the boiling point of ­YF3 (2230 °C), making it challenging to produce ­AlF3 on the surface. Additionally, compared to ­YF3 (161 eV), the lower binding energy (77 eV) of ­AlF3 renders it more susceptible to removal by physical ion bombardment, thereby influencing the etching r­ ate43. To further analyze the plasma resistance properties of the composites, surface roughness changes before and after plasma etching, according to the variety of mixed gas ratios and compositions, were investigated. The results are presented in Fig. 5. In Fig. 5a, the ­Y2O3 single component ceramic demonstrates a substantial change in surface roughness following plasma etching across all gas composition environments. Especially, in the case of ­CF4:Ar:O2 gas with a ratio of 10:40:10, the surface roughness ( Ra ) increased significantly from 4.2 to 62.1 nm (14.8 times). The AFM 3D images in Fig. 5b,c reveal a smooth surface pre-etching, transforming at post-etching into one with large craters, each several micrometers in size. These craters emerged as a consequence of localized and intense impacts on residual pores in the microstructure of the ­Y2O3 sintered body by A ­ r+ ion sputtering, resulting in deterioration of the specimen ­surface5,6. The ceramic surface’s irregularities directly affect the release of sputtered neutral atoms and contaminants formation. Elevated irregularities contribute to an increased release of them, potentially giving rise to the generation of particles that are inadequately e­ vacuated8,44. Compared to ­Y2O3, all Y ­ 2O3-YAM composite had a lower roughness than Y ­ 2O3 after plasma exposure. AFM 3D images before and after etching for these compositions (Fig. 5d–g) distinctly show the discrepancy in plasma etching rates. In cases in which Y ­ 2O3 and YAM compositions were combined at 50:50 volume ratio, changes in surface roughness Scientific Reports | (2024) 14:7008 | https://doi.org/10.1038/s41598-024-57697-5 5 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 3.  Simple schematic illustration of plasma etching test chamber system. Figure 4.  Plasma etching rate of c-axis sapphire, Y ­ 2O3 polycrystalline ceramics and ­Y2O3-YAM composites with different volume ratios under a variety of mixed gas ratios between C ­ F4, Ar and ­O2 conditions. Scientific Reports | Vol:.(1234567890) (2024) 14:7008 | https://doi.org/10.1038/s41598-024-57697-5 6 www.nature.com/scientificreports/ Figure 5.  (a) Measured surface roughness ­(Ra) of ­Y2O3 and ­Y2O3-YAM composite ceramics at masked and plasma exposed regions with different volume ratios and mixed gas ratios of C ­ F4:Ar:O2. Representative 3D images of masked and exposed surface morphology were produced by atomic force microscopy (AFM): (b) masked and (c) etched ­Y2O3 polycrystalline ceramics; (d) masked and (e) exposed ­Y2O3-YAM (YAM1) composites; (f) masked and (g) exposed ­Y2O3-YAM (YAM9) composites. ­CF4:Ar:O2 gas ratio was 10:40:10. with plasma etching were minimal. When the C ­ F4:Ar:O2 gas mixture ratio was 10:40:10, the surface roughness change increased by a mere 1.9 times, from 2.9 to 5.4 nm. This aligns with previous findings on surface roughness change after plasma etching of nanocomposites, suggesting its efficacy in minimizing the formation of large craters. It is well-known that changes of surface roughness are significantly dependent on grain ­size26. Therefore, the positive effect in this study can be attributed to the inhibition of grain growth through a pinning effect and densification achieved by reducing the sintering t­ emperature25,45. Scientific Reports | (2024) 14:7008 | https://doi.org/10.1038/s41598-024-57697-5 7 Vol.:(0123456789) www.nature.com/scientificreports/ The SEM images presented in Fig. 6 show microstructures of Y ­ 2O3 and ­Y2O3-YAM composites after plasma etching test conducted with C ­ F4:Ar:O2 gas ratio of 40:10:10. In Fig. 6a, the Y ­ 2O3 polycrystalline ceramic exhibited the development of substantial craters, each several micrometers in size, after plasma etching, covering the entire specimen. As previously mentioned, the emergence of these craters is associated with pronounced etching of micropores within the specimen, particularly in open pores; the resultant large craters can induce noteworthy alterations in surface roughness. Contrastingly, in Fig. 6b–f, for the Y ­ 2O3-YAM composite, dark regions signify YAM composition, while light regions denote ­Y2O3 composition, revealing an evident discrepancy in etching rate based on composition. The YAM1 specimen, comprising 10% YAM by volume, underwent more profound etching over a small area, while YAM5, with the same proportion between two compositions, experienced deep etching across nearly half of the area. The lesser bonding energy of Al-O (512 kJ/mol) compared to that of Y–O (685 kJ/mol) underscores the significant role played by the reaction between Al-O bonding and fluorocarbon ­deposits46–48. This reaction results in the formation of A ­ lF3 layers, prone to removal via physical attack due to the vulnerability of fluorinated layers to ion sputtering. Consequently, an etching depth differential between the ­Y2O3 and YAM compositions a­ rose15,25,32. Craters within the Y ­ 2O3-YAM composite microstructure formed, but were very small in size, unlike the results of the ­Y2O3 mono-composition. Sizes of craters formed during plasma Figure 6.  SEM microstructure images of Y ­ 2O3 and ­Y2O3-YAM composites with different volume ratios after plasma etching under at 40:10:10 ­CF4:Ar:O2 gas ratio; (a) ­Y2O3 monolith ceramic, (b) YAM1, (c) YAM3, (d), YAM5, (e), YAM7, and (f) YAM9 composite ceramics. Scientific Reports | Vol:.(1234567890) (2024) 14:7008 | https://doi.org/10.1038/s41598-024-57697-5 8 www.nature.com/scientificreports/ etching is intricately linked to the ceramic grain size. Hence, amalgamation of varying YAM and Y ­ 2O3 compositions can reduce crater size and minimize changes in surface roughness if grain growth is effectually s­ uppressed49. For composition with little solidification at temperatures below the eutectic point, the pinning effect is much more effective at suppressing grain growth; it can dramatically reduce growth; resulting craters rarely form, and if formed, they are very fi ­ ne28. In light of these considerations, a strategic combination of compositions recognized for their robust plasma resistance properties holds promise in effectively diminishing crater size, thereby attenuating changes of surface roughness and reducing contaminant particle generation. SEM images of the microstructure after plasma etching are shown in Fig. 7; the ­CF4:Ar gas ratio was varied for the YAM5 specimen with a 50:50 volume mixture of ­Y2O3 and YAM ceramics. Overall, no significant differences in microstructure were seen with different plasma atmospheres, and there were no changes in composition of preferential etching. As the ratio of Ar gas increased, the number of craters formed on the surface with sizes of 1 µm or less increased, especially when the ratio of ­CF4:Ar:O2 was 10:40:10, meaning that there was a very high amount of Ar, as shown in Fig. 7d; in this case, the effect of physical etching increased and more craters were formed. As shown in Fig. 7c, when the ratio of C ­ F4:Ar:O2 gas was 20:30:10, a large number of irregular nanopores formed on certain ­Y2O3 ­grains15. SEM–EDS analysis of the surface of the YAM5 specimen after etching in C ­ F4:Ar:O2 gas mixture with ratio of 40:10:10 is shown in Fig. 8. Different compositions exhibited different levels of etching resistance, as shown in Figs. 6 and 8a; the composition with darker colored grains and etched faster by the plasma is YAM composition, and the EDS mapping results confirms that the darker grains are YAM composition, as shown in Fig. 8b. Other compositions of ­Y2O3 and F were detected throughout and no significant differences were found. In a recent study, bulk YAG ceramic was found to be etched slightly faster than bulk Y ­ 2O3 ceramic under C ­ F4:Ar:O2 plasma gas ratios of 40:10:10 and 10:40:1050. This study shows that YAM ceramics also etch faster microscopically than ­Y2O3 under the same conditions. Next, the mechanical properties of the ­Y2O3 and ­Y2O3-YAM composites were evaluated; results of Vickers hardness measurements are shown in Fig. 9. To serve as viable components in a practical semiconductor plasma etching apparatus, materials must have adequate mechanical properties alongside resistance to etching. The ­Y2O3 ceramic is acknowledged for its inherently low hardness, typically within a range of 7–8 GPa; a low hardness value of 6.9 GPa was obtained for the specimen in this s­ tudy51. The compromised mechanical properties of ­Y2O3 pose impediments to its sustained use of ceramic components, prompting frequent replacement cycles. Therefore, enhancing the mechanical properties without losing plasma etching resistance is imperative. The pure YAM composition has excellent hardness of about 11 G ­ Pa35. By compounding YAM in ­Y2O3, the Vickers Figure 7.  SEM microstructure images of Y ­ 2O3-YAM nanocomposite with 50:50 vol% after plasma etching under different C ­ F4:Ar:O2 gas ratios of: (a) 40:10:10, (b) 30:20:10, (c) 20:30:10, and (d) 10:40:10. Scientific Reports | (2024) 14:7008 | https://doi.org/10.1038/s41598-024-57697-5 9 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 8.  (a) SEM microstructure image and (b) element mapping result for ­Y2O3-YAM (YAM5) composites after plasma etching at 10:40:10 ­CF4:Ar:O2 gas ratio. Figure 9.  Vickers hardness of Y ­ 2O3 polycrystalline ceramics and sintered ­Y2O3-YAM composites with different volume ratios. hardness improved and, as the proportion of YAM increased from 10 to 90%, the Vickers hardness improved to 9.2 GPa. Consequently, the composite of Y ­ 2O3 and YAM did not deviate much from the Y ­ 2O3 and ­Al2O3 series of materials used as in-chamber materials for conventional semiconductor plasma etching processes, making it easy to apply in industry, while minimizing crater formation through grain growth inhibition. The excellent etching resistance, small surface roughness change, and enhanced mechanical properties are expected to enable the material to be used for a longer period and significantly improve the yield of semiconductor production, pushing it beyond the limitation of material components in plasma etching chambers that rely on conventional single composition and coating methods. Conclusion In conclusion, we studied the plasma etching characteristics of ­Y2O3-YAM composites and ­Y2O3 under harsh environment with different C ­ F4:Ar:O2 mixed gases ratios. Y ­ 2O3 polycrystalline ceramics showed fast etching rate, large change of surface roughness. In addition, there is formation of large craters at the surface after plasma etching. On the other hand, the ­Y2O3-YAM composite showed minimal surface roughness changes, while etching rate increased under the physical etching-dominant environment. Especially, under various gas conditions, composites with 50:50 volume fractions demonstrated superior physicochemical etching resistance compared to ­Y2O3 ceramics. The composites also effectively reduced the size of the craters produced on the Y ­ 2O3 surface after plasma attack. Based on these findings, Y ­ 2O3-YAM composites demonstrate remarkable inductively coupled plasma-reactive ion etching resistance in plasma etching conditions, accompanied by a notable decrease in the generation of contaminants. 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J., Kim, D. G. & Kim, D. K. Two-step sintering behavior of titanium-doped ­Y2O3 ceramics with monodispersed sub-micrometer powder. Ceram. Int. 45, 510–515 (2019). Acknowledgements This work was supported by the Nano & Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science and ICT (2020M3H4A310600321) and the Fundamental Research Program (PNK9500) of the Korea Institute of Materials Science (KIMS). Author contributions H.J.M.: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing, S.K.: Formal analysis, Data curation H-.K., M-.K., J-.K., J-.L., J.-K.: Formal analysis, H-.L.: Supervision, Investigation, Reviewing, Y-.P.: Conceptualization, Supervision, Investigation. Competing interests The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to H.J.M. or Y.-J.P. 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Tracking Human Mobility Using WiFi Signals
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Citation (APA): Sapiezynski, P., Stopczynski, A., Gatej, R., & Jørgensen, S. L. (2015). Tracking Human Mobility Using WiFi Signals. P L o S One, 10(7), Article e0130824. https://doi.org/10.1371/journal.pone.0130824 Tracking Human Mobility Using WiFi Signals Sapiezynski, Piotr; Stopczynski, Arkadiusz; Gatej, Radu ; Jørgensen, Sune Lehmann Published in: P L o S One Document Version Publisher's PDF, also known as Version of record General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Piotr Sapiezynski1*, Arkadiusz Stopczynski1,2, Radu Gatej3, Sune Lehmann1,4 Piotr Sapiezynski1*, Arkadiusz Stopczynski1,2, Radu Gatej3, Sune Lehmann1,4 1 Department of Applied Mathematics and Computer Science, Technical University of Denmark, Kongens Lyngby, Denmark, 2 Media Lab, Massachusetts Institute of Technology, Cambridge, MA, United States of America, 3 Department of Economics, University of Copenhagen, Copenhagen, Denmark, 4 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark * pisa@dtu.dk * pisa@dtu.dk a1111 OPEN ACCESS Citation: Sapiezynski P, Stopczynski A, Gatej R, Lehmann S (2015) Tracking Human Mobility Using WiFi Signals. PLoS ONE 10(7): e0130824. doi:10.1371/journal.pone.0130824 Citation: Sapiezynski P, Stopczynski A, Gatej R, Lehmann S (2015) Tracking Human Mobility Using WiFi Signals. PLoS ONE 10(7): e0130824. doi:10.1371/journal.pone.0130824 Academic Editor: Ye Wu, Beijing University of Posts and Telecommunications, CHINA Academic Editor: Ye Wu, Beijing University of Posts and Telecommunications, CHINA Academic Editor: Ye Wu, Beijing University of Posts and Telecommunications, CHINA Received: January 20, 2015 Accepted: May 26, 2015 Published: July 1, 2015 Received: January 20, 2015 Accepted: May 26, 2015 Published: July 1, 2015 Received: January 20, 2015 Accepted: May 26, 2015 Published: July 1, 2015 Abstract We study six months of human mobility data, including WiFi and GPS traces recorded with high temporal resolution, and find that time series of WiFi scans contain a strong latent loca- tion signal. In fact, due to inherent stability and low entropy of human mobility, it is possible to assign location to WiFi access points based on a very small number of GPS samples and then use these access points as location beacons. Using just one GPS observation per day per person allows us to estimate the location of, and subsequently use, WiFi access points to account for 80% of mobility across a population. These results reveal a great opportunity for using ubiquitous WiFi routers for high-resolution outdoor positioning, but also significant privacy implications of such side-channel location tracking. Link back to DTU Orbit Link back to DTU Orbit Citation (APA): Sapiezynski, P., Stopczynski, A., Gatej, R., & Jørgensen, S. L. (2015). Tracking Human Mobility Using WiFi Signals. P L o S One, 10(7), Article e0130824. https://doi.org/10.1371/journal.pone.0130824 this document breaches copyright please contact us providing details, and we will remove access to the work immediate ur claim. RESEARCH ARTICLE Introduction Copyright: © 2015 Sapiezynski 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. Due to the ubiquity of mobile devices, the collection of large-scale, longitudinal data about human mobility is now commonplace [1]. High-resolution mobility of individuals and entire social systems can be captured through a multitude of sensors available on modern smart- phones, including GPS and sensing of nearby WiFi APs (access points or routers) and cell tow- ers. Similarly, mobility data may be collected from systems designed to enable communication and connectivity, such as mobile phone networks or WiFi systems (e.g. at airports or on com- pany campuses) [2, 3]. Additionally, large companies such as Google, Apple, Microsoft, or Sky- hook, combine WiFi access points with GPS data to improve positioning [4], a practice known as ‘wardriving’. While widely used, the exact utility and mechanics of wardriving are largely unknown, with only narrow and non-systematic studies reported in the literature [5, 6]. As a consequence, it is generally not known how WiFi networks can be used for sensing mobility on a societal scale; this knowledge is proprietary to large companies. The dataset Out of the 130+ participants of the study [20], we selected 63 for which at least 50% of the expected data points are available. The methods of collection, anonymization, and storage of data were approved by the Danish Data Protection Agency, and complies both with local and EU regulations. Written informed consent was obtained via electronic means, where all invited participants read and digitally signed the form with their university credentials. The median period of WiFi scans for these users was 16 seconds, and the median period of GPS sampling was 10 minutes. The data spans a period of 200 days from October 1st, 2012 to April 27th, 2013. Tracking Human Mobility Using WiFi Signals assistants; for example Google Now [16] is a mobile application, which learns users’ habits to, among other services, conveniently provide directions to the next inferred location. Copenhagen. Please direct your queries to Sune Lehmann, the Principal Investigator of the study, at sljo@dtu.dk. Mobility traces are highly unique and identify individuals with high accuracy [17]. Sensitive features can be extracted from mobility data, including home and work locations, visited places, or personality traits [18]. Moreover, location data are considered the most sensitive of all the commonly discussed personal data collected from or via mobile phones [19]. Funding: This work was supported by Villum Foundation, http://villumfoundation.dk/ C12576AB0041F11B/0/ 4F7615B6F43A8EA5C1257AEF003D9930? OpenDocument, Young Investigator programme 2012, High Resolution Networks (SL) and University of Copenhagen, http://dsin.ku.dk/news/ucph_funds/, through the UCPH2016 Social Fabric grant (SL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This work was supported by Villum Foundation, http://villumfoundation.dk/ C12576AB0041F11B/0/ Here, we show that a time sequence of WiFi access points is effectively equal to location data. Specifically, having collected both GPS and WiFi data with high temporal resolution (median of 5 minutes for GPS and 16 seconds for WiFi) in a large study [20], we use six months of data for 63 participants to model how lowering the rate of location sampling influences our ability to infer mobility. The study participants are students with heterogeneous mobility pat- terns. They all attend lectures on campus located outside of the city center, but live in dormito- ries and apartments scattered across the metro area at various distances from the university. Competing Interests: The authors have declared that no competing interests exist. By mapping the WiFi data, we are able to quantify details of WiFi-based location tracking, which are usually not available to the general public. We find that the geo-positioning inferred from WiFi access points (APs or routers) could boost efficacy in other data collection contexts, such as research studies. In addition, our findings have significant privacy implications, indi- cating that for practical purposes WiFi data should be considered location data. As we argue in the following sections, this finding is not recognized in current practices of data collection and handling. Data Availability Statement: Data are from Data Availability Statement: Data are from Copenhagen Networks study (http://journals.plos.org/ plosone/article?id=10.1371/journal.pone.0095978). Due to privacy consideration regarding subjects in our dataset, including European Union regulations and Danish Data Protection Agency rules, we cannot make our data publicly available. The data contains detailed information on mobility and daily habits of 63 individuals at a high spatio-temporal resolution. We understand and appreciate the need for transparency in research and are ready to make the data available to researchers who meet the criteria for access to confidential data, sign a confidentiality agreement, and agree to work under our supervision in In the scientific realm, the mobility patterns of entire social systems are important for modeling spreading of epidemics on multiple scales: metropolitan networks [7–9] and global air traffic networks [10, 11]; traffic forecasting [12]; understanding fundamental laws govern- ing our lives, such as regularity [13], stability [14], and predictability [15]. Predictability and stability of human mobility are also exploited by commercial applications such as intelligent PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 1 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 Tracking Human Mobility Using WiFi Signals Fig 1. The time coverage provided by the routers with known position depends on who collects the corresponding location data and when it happens. In each subplot the orange line describes the scenario where each individual collects data about themselves and does not share it with others; the blue line corresponds to a system in which the location of routers discovered by one person is made known to other users; the green line presents a situation where each individual can use the common pool of known routers but does not discover access points herself. a. Stability of location. Learning the location of APs seen during the first seven (left panel) or 28 (right panel) days, leads to performance gradually decreasing with time in the personal case (orange line). The histograms of time coverage distribution for day 190 show that this decline is driven by a growing number of people who spend only *10% of time in the locations they visited in the beginning of the observation. The global approach (blue line) does not show this tendency, which indicates that people rotate between common locations rather than moving to entirely new places. b, c. Representativeness of randomly selected locations. Random subsampling with an average period of 24 hours (less than 1% of all available location estimations) is sufficient to find the most important locations in which people spend more than 80% of their time; using an average period of 4 hours (2.5% percent of all available location data) results in *85% coverage. The personal database does not expire since the location is sampled throughout the experiment, not only in the beginning. d. Limited number of important locations. Although querying commercial services for WiFi geolocation is costly, knowing the location of only the 20 most prevalent routers per person in the dataset results in an average coverage of *90%. Since people’s mobility overlaps, there is a benefit of using a global database rather than treating all mobility disjointly. doi:10.1371/journal.pone.0130824.g001 by the routers with known position depends on who collects the corresponding location data and when it Fig 1. The time coverage provided by the routers with known position depends on who collects the corresponding location data and when it happens. In each subplot the orange line describes the scenario where each individual collects data about themselves and does not share it with others; the blue line corresponds to a system in which the location of routers discovered by one person is made known to other users; the green line presents a situation where each individual can use the common pool of known routers but does not discover access points herself. a. Stability of location. Learning the location of APs seen during the first seven (left panel) or 28 (right panel) days, leads to performance gradually decreasing with time in the personal case (orange line). The histograms of time coverage distribution for day 190 show that this decline is driven by a growing number of people who spend only *10% of time in the locations they visited in the beginning of the observation. The global approach (blue line) does not show this tendency, which indicates that people rotate between common locations rather than moving to entirely new places. b, c. Representativeness of randomly selected locations. Random subsampling with an average period of 24 hours (less than 1% of all available location estimations) is sufficient to find the most important locations in which people spend more than 80% of their time; using an average period of 4 hours (2.5% percent of all available location data) results in *85% coverage. The personal database does not expire since the location is sampled throughout the experiment, not only in the beginning. d. Limited number of important locations. Although querying commercial services for WiFi geolocation is costly, knowing the location of only the 20 most prevalent routers per person in the dataset results in an average coverage of *90%. Since people’s mobility overlaps, there is a benefit of using a global database rather than treating all mobility disjointly. doi:10.1371/journal.pone.0130824.g001 subsampling scenario we select a set fraction of available GPS location estimations, each paired with a WiFi scan. Each GPS estimation provides information on the position of all routers seen in the paired scan. This scenario can be realized after the data collection is finished, as the loca- tion estimations are used to locate the WiFi scans which happened both before and after said esti- mations. The results are presented in Fig 1b. Top routers. We select the top routers in a greedy fashion after the data collection is finished. Known routers and coverage In the article we use a simple model of locating the WiFi routers. We consider an access point as known if it occurred in a WiFi scan within one second of a GPS location estimation. The shortcomings of this approach and possible remedies are described in more detail in S1 File. We define time coverage as a fraction of ten-minute bins containing WiFi data in which at least one known router was scanned. For example, let us assume that the user has data in 100 out of 144 timebins during a day, and in 80 of these timebins there is a known router visible. Therefore, that user’s coverage for that day is 80%. The average time coverage for a day is the mean coverage of all users who had any WiFi information in that day. This way our results are independent from missing data caused by imperfections in data collection system deployed in the study. In Fig 1 we present three different approaches to sampling, which we describe here in detail. Initial-period sampling. As presented in Fig 1a, we learn the location of the routers sequentially. With each GPS location estimation accompanied with a WiFi scan, we add the visible access points to the list of known routers. The learning curve can be observed for the first seven days (Fig 1a, left panel) or the first 28 days (Fig 1a, right panel). Random subsampling. In the random PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 2 / 11 Results Ubiquitously available WiFi access points can be used as location beacons, identifying locations based on BSSID (basic service set identifier, uniquely identifying every router) broadcast by APs. These locations are not intrinsically geographical, as the APs do not have geographical coordinates attached. However, since the placement of APs tends to remain fixed, mapping an AP to a location where it was seen once is sufficient to associate all the subsequent scans from the user device with geographical coordinates. See S1 File for details on inferring the geographi- cal locations of routers, as well as identifying (and discarding data from) mobile access points. WiFi networks are ubiquitous. In our population, 92% of all WiFi scans detect at least one access point, and 33% detect more than 10 APs, as shown in Fig 2c. In densely-populated areas, an average of 25 APs are visible in every scan, with population density explaining 50% of the variance of the number of APs, as shown in Fig 2b. WiFi scans containing at least one visible AP can be used for discovering the location of the user, with a typical spatial resolution on the order of tens of meters. We investigate three approaches to using access points as location beacons, all of which enable WiFi-based location tracking even with limited resources: (1) recovering APs’ locations from mobility traces collected during an initial training period (exploiting the long-term stabil- ity of human mobility), (2) recovering APs’ locations from randomly sampled GPS updates (exploiting low entropy of human mobility, see S1 File for distinction between stability and low entropy), and (3) using only the most frequently observed APs for which location can be feasi- bly obtained from external databases. The task is to efficiently assign geographical coordinates (latitude and longitude) to particular APs, so they can be used as beacons for tracking user’s location. In the following sections, we refer to time coverage as the fraction of ten-minute time- bins, in which at least one router with a known location is observed. We sort the routers in descending order by the subsampling scenario we select a set fraction of available GPS location estimations, each paired with a WiFi scan. Each GPS estimation provides information on the position of all routers seen in the paired scan. This scenario can be realized after the data collection is finished, as the loca- tion estimations are used to locate the WiFi scans which happened both before and after said esti- mations. The results are presented in Fig 1b. Top routers. We select the top routers in a greedy fashion after the data collection is finished. We sort the routers in descending order by the 3 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 Tracking Human Mobility Using WiFi Signals number of user timebins they occur in. We choose the top one router, and then we select the routers which provide the biggest increase in the number of user timbebins covered. Due to high density of access points, each semantic place is described by presence of several routers, but loca- tion of only one of them has to be established to find the geographic position of the place. In this sampling method we do not rely on our own GPS data—top routers are found purely based on their occurrence in the WiFi scans, regardless of availability of GPS scans within the one second time delta. The results of such sampling are presented in Fig 1e. Data collection scenarios Each subplot in Fig 1 contains series coming from three different simulated collection scenar- ios. In the global scenario, there is a pool of WiFi routers locations estimations coming from all users, and a router is considered known if at least one person has found its location. This scenario simulates the function of such services as for example mobile Google Maps. In the personal scenario each user can only use their own data, a router can be known to them only if they found its location themselves. It simulates collecting data in a disjoint society, where each person frequents different locations. Finally, in the global with no personal data scenario, each user can exploit estimations created by everybody else, but without contributing their own data. Stability of human mobility allows for efficient WiFi-based positioning This decline is evi- dent both when a week (Fig 1a, left panel) and four weeks (right panel) are used for training, with the time coverage dropping *18 percentage points between days 60 and 160. The histo- grams above each plot show the distribution of time coverage in selected points in time (at 7, 80, 190 days respectively). The distribution for day 190 reveals that the expiry of the personal database validity is driven by individuals who significantly altered routines, with 40% of partic- ipants spending only around 10% of time in locations they have visited in the first week. In contrast, when the inferred locations of routers are shared among people (the global database case, represented by a blue line) the information does not expire and shows no decreasing trend during the observation period. This implies that rather than moving to entirely new PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 5 / 11 routers seen during the first seven days (corresponding to *3.5% of the observations, shown in Fig 1a, left panel) provides APs’ locations throughout the rest of the experiment sufficient for recovering *55% of users mobility until the Christmas break around days 75–90. When the location of routers seen by each person is inferred using only this person’s data (the per- sonal-only WiFi database case, shown using an orange line in Fig 1), the information expires with time: there is a stable decrease in time coverage after Christmas break. This decline is evi- dent both when a week (Fig 1a, left panel) and four weeks (right panel) are used for training, with the time coverage dropping *18 percentage points between days 60 and 160. The histo- grams above each plot show the distribution of time coverage in selected points in time (at 7, 80, 190 days respectively). The distribution for day 190 reveals that the expiry of the personal database validity is driven by individuals who significantly altered routines, with 40% of partic- ipants spending only around 10% of time in locations they have visited in the first week. In contrast, when the inferred locations of routers are shared among people (the global database case, represented by a blue line) the information does not expire and shows no decreasing trend during the observation period. PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 Stability of human mobility allows for efficient WiFi-based positioning Human mobility has been shown to remain stable over long periods of time [13]. We find that participants in our study have stable routines, with locations visited in the first one, two, three, and four weeks of the study still visited frequently six months later. Learning the locations of PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 4 / 11 Tracking Human Mobility Using WiFi Signals Fig 2. WiFi routers are located where people live. a: Map of Greater Copenhagen Area, divided into parishes with color indicating average number of routers discovered per scan; rectangle overlay indicates the city center. b: The number of access points visible in each scan is correlated with the population density (r2 = 0.5). Even in low population density areas there are several routers visible on average in each scan. Therefore, knowing the positions of only a subset of routers is enough for precise location sensing. c: Distribution of number of routers per scan. In our dataset 92% of scans contain at least one router. d i 10 1371/j l 0130824 002 Fig 2. WiFi routers are located where people live. a: Map of Greater Copenhagen Area, divided into parishes with color indicating average number of routers discovered per scan; rectangle overlay indicates the city center. b: The number of access points visible in each scan is correlated with the population density (r2 = 0.5). Even in low population density areas there are several routers visible on average in each scan. Therefore, knowing the positions of only a subset of routers is enough for precise location sensing. c: Distribution of number of routers per scan. In our dataset 92% of scans contain at least one router. doi:10.1371/journal.pone.0130824.g002 doi:10.1371/journal.pone.0130824.g002 routers seen during the first seven days (corresponding to *3.5% of the observations, shown in Fig 1a, left panel) provides APs’ locations throughout the rest of the experiment sufficient for recovering *55% of users mobility until the Christmas break around days 75–90. When the location of routers seen by each person is inferred using only this person’s data (the per- sonal-only WiFi database case, shown using an orange line in Fig 1), the information expires with time: there is a stable decrease in time coverage after Christmas break. Stability of human mobility allows for efficient WiFi-based positioning This implies that rather than moving to entirely new routers seen during the first seven days (corresponding to *3.5% of the observations, shown in Fig 1a, left panel) provides APs’ locations throughout the rest of the experiment sufficient for recovering *55% of users mobility until the Christmas break around days 75–90. When the location of routers seen by each person is inferred using only this person’s data (the per- sonal-only WiFi database case, shown using an orange line in Fig 1), the information expires with time: there is a stable decrease in time coverage after Christmas break. This decline is evi- dent both when a week (Fig 1a, left panel) and four weeks (right panel) are used for training, with the time coverage dropping *18 percentage points between days 60 and 160. The histo- grams above each plot show the distribution of time coverage in selected points in time (at 7, 80, 190 days respectively). The distribution for day 190 reveals that the expiry of the personal database validity is driven by individuals who significantly altered routines, with 40% of partic- ipants spending only around 10% of time in locations they have visited in the first week. In contrast, when the inferred locations of routers are shared among people (the global database case, represented by a blue line) the information does not expire and shows no decreasing trend during the observation period. This implies that rather than moving to entirely new PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 5 / 11 Tracking Human Mobility Using WiFi Signals locations, people begin to visit places that are new to them, but familiar to other participants. The histograms of time coverage distribution in both panels of Fig 1a reveal that the individuals are heterogeneous in their mobility. The coverage in most cases is highly affected in the non- personal case (where the person does not collect their own location information, but data from others is used, marked using green in the figures), but 20% of participants retain a coverage of above 80% throughout the observation period, see Fig 1a, left panel. People living and working close to each other (like students in a dormitory) share a major part of their mobility and thus location of the APs they encounter can be estimated using data collected by others. The demonstrated stability of human mobility patterns over long periods has real-life pri- vacy implications. Human mobility can be efficiently captured using infrequent location updates Sampling location randomly across time (Fig 1b), rather than through the initial period (Fig 1a) provides a higher time coverage, which is retained throughout the observation. With around one sample per day per person on average, the location can be inferred 80% of the time in case of global lookup base and 70% in personal case (see Fig 1c, at training fraction of 0.007). h h b f h d b f h l The histograms in Fig 1b confirm that distribution of coverage in the non-personal case is bimodal within our population: mobility of some individuals can effectively be modeled using data from people around them, while patterns of others are so distinct they require using self- collected information. The single-mode distribution of coverage in the personal case and the fact that the distribution is unchanged between day 7 and day 190 show the lack of temporal decline when sampling happens throughout the observation period. The GPS sensor on a mobile device constitutes a major battery drain when active [21], whereas the WiFi frequently scans for networks by default. Our results show that GPS-based location sampling rate can be significantly reduced in order to save battery, while retaining high resolution location information through WiFi scanning. Our analyses also point to another scenario where WiFi time series can result in leaks of personal information. Infrequent location data can be obtained from a person’s (often public) tweets, Facebook updates, or other social networking check-ins and then matched with their WiFi records to track their mobility. Stability of human mobility allows for efficient WiFi-based positioning Denying a mobile application access to location data, even after a short period, may not be enough to prevent it from tracking user’s mobility, as long as its access to WiFi scans is retained. PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 Single-user analysis To illustrate the ubiquity of WiFi access points and how effectively they can be used to infer mobility patterns, we present a small example dataset containing measured and inferred loca- tion information of one of the authors, collected over two days. During the 48 hours of obser- vation, the researcher’s phone was scanning for WiFi with a median period of 44 seconds, measuring on average 19.8 unique devices per scan, recording 3 822 unique access points. Only one scan during the 48 hours was empty, and one scan yielded 113 unique results. Fig 3a shows the corresponding GPS trace collected with a median sampling period of 5 minutes. When dividing the 48 hours of the test period into 10 minute bins, a raw GPS trace provides location estimation in 89% of these bins. Four stop locations are marked with blue circles and include home, two offices, and a food market visited by the researcher. Fig 3b shows the estimation of this trace based on the inferred locations of WiFi routers, see S1 File for detailed information on the location inference. The four stop locations are clearly visible, but the transitions have lower temporal resolution and errors in location estimations. This method provides location information in 97% of temporal bins. Using WiFi increases overall coverage, but might intro- duce errors in location estimation of routers which were only observed shortly, for example during transition periods. Fig 3c shows the estimation of this trace based on the locations of top 8 (0.2%) WiFi routers. The four important locations have been correctly identified, but information on transitions is lost. Information in 95% of temporal bins is available. Finally, Fig 3d shows a graphical representation of how much time the researcher spends in any one of the top eight locations during the observation time. Note that the first four locations account for an overwhelming fraction of the 48 hours. Knowing the physical position of the top routers and having access to WiFi information reveals the location of the user for the majority of the timebins. The details of trajectories become lost as we decrease the number of routers we use to estimate locations. With too few routers might not be possible to determine which of possible routes the subject chose or how long she took to travel through each segment of the trip. Overall human mobility can be effectively captured by top WiFi access points As previously suggested [15], people’s mobility has low entropy and thus a few most prevalent routers can work effectively as proxies for their location. Fig 1d shows that inferring the loca- tion of just 20 top routers per person on average (which, given the median count of 22 000 routers observed per person, corresponds to 0.1% of all routers seen) translates to knowing the location of individuals 90% of the time. Since our population consists of students, who attend classes in different lecture halls in various buildings across the campus, we expect that the num- ber of access points necessary to describe mobility of persons with a fixed work location can be even lower. There are persons in our study, for whom just four access points correspond to 90% of time coverage (see Fig D in S1 File for details). That the mobility of individuals in our sample overlaps is apparent in Fig 1d as the time cov- erage of three top routers in the personal case is the same as in the global coverage using the total of 80 routers (instead of 189 disjoint routers). 6 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 Tracking Human Mobility Using WiFi Signals As a consequence, a third party with access to records of WiFi scans and no access to loca- tion data, can effectively determine the location of each individual 90% of time by sending less than 20 queries to commercial services such as Google Geolocation API or Skyhook. Single-user analysis On the other hand, the high temporal resolution of the scans allows for very precise discovery of arrival and departure times and of time spent in transit. Such information has important implications for security and privacy, as it can be used to discover night-watch schedules, find times when the occupants are not home, or efficiently check work time of the employees. PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 Discussion Our world is becoming progressively more enclosed in infrastructures supporting communica- tion, mobility, payments, or advertising. Logs from mobile phone networks have originally been considered only for billing purposes and internal network optimization; today they con- stitute a global database of human mobility and communication networks [13]. Credit card records form high-resolution traces of our spending behaviors [22]. The omnipresent WiFi networks, intended primarily for communication, has now became a location tracking infra- structure, as described here. The pattern is clear: every new cell tower, merchant with credit card terminal, every new private or municipal WiFi network offer benefits to the connected society, but, at the same time, create opportunities and perils of unexpected tracking. Cities entirely covered by WiFi signal provide unprecedented connectivity to citizens and visitors alike; at the same time multiple parties have to incorporate this fact in their policies to limit PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 7 / 11 Tracking Human Mobility Using WiFi Signals Fig 3. 48 hours of location data of one of the authors, with the four visited locations visited marked in blue: home, two offices, and a food market. Even though the author’s phone has sensed 3 822 unique routers in this period, only a few are enough to describe the location more than 90% of time. a. traces recorded with GPS; b. traces reconstructed using all available data on WiFi routers locations—the transition traces are distorted, but all stop locations are visible and the location is known 97% of the time. c. with 8 top routers it is still possible to discover stop locations in which the author spent 95% of the time. In this scenario transitions are lost. d. timeseries showing when during 48 hours each of the top routers were seen. It can be assumed that AP 1 is home, as it’s seen every night, while AP 2 and AP 3 are offices, as they are seen during working hours. The last row shows the combined 95% of time coverage provided by the top 8 routers. doi:10.1371/journal.pone.0130824.g003 Fig 3. 48 hours of location data of one of the authors, with the four visited locations visited marked in blu Fig 3. 48 hours of location data of one of the authors, with the four visited locations visited marked in blue: home, two offices, and a food market. doi:10.1371/journal.pone.0130824.g003 Discussion PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 8 / 11 Tracking Human Mobility Using WiFi Signals In the results, we focused on outdoor positioning with spatial resolution corresponding to WiFi AP coverage: we assume that if at least one AP is discovered in a scan, we can assign the location of this AP to the user. This is a deliberately simple model, described in detail in S1 File, but we consider the resulting spatial resolution sufficient for many aspects of research, such as studying human mobility patterns. The spatial resolution of dozens of meters is higher than for example CDR data [13], which describes the location with the accuracy of hundreds of meters to a few kilometers. Incorporating WiFi routers as location beacons can aid research by drasti- cally increasing temporal resolution without additional cost in battery drain. Students live in multiple dormitories on and outside of campus, take multiple routes com- muting to the university, frequent different places in the city, travel across the country and beyond. While the students spend most of their time within a few dozens of kilometers from their homes, they also make international and intercontinental trips (see Figs B and C in S1 File for details). Such long distance trips are not normally captured in studies based on telecom operator data. Our population is densely-connected and in this respect it is biased, in the same sense as any population of people working in the same location. We do simulate a scenario in which the individuals do not form a connected group by analyzing the results for personal- only database. We expect the obtained results to generalize outside of our study. Our findings connect to an ongoing debate about the privacy of personal data [23]. Location data has been shown to be among the most sensitive categories of personal information [19]. Still, a record of WiFi scans is, in most contexts, not considered a location channel. In the Android ecosystem, which constitutes 85% of global smartphone market in Q2 2014 [24], the permission for applications to passively collect the results of WiFi scans is separate from the location permission; moreover, the Wi-Fi connection information (ACCESS_WIFI_STATE) permission is not considered ‘dangerous’ in the Android framework, whereas both high-accu- racy and coarse location permissions are tagged as such [25]. Discussion Even though the author’s phone has sensed 3 822 unique routers in this period, only a few are enough to describe the location more than 90% of time. a. traces recorded with GPS; b. traces reconstructed using all available data on WiFi routers locations—the transition traces are distorted, but all stop locations are visible and the location is known 97% of the time. c. with 8 top routers it is still possible to discover stop locations in which the author spent 95% of the time. In this scenario transitions are lost. d. timeseries showing when during 48 hours each of the top routers were seen. It can be assumed that AP 1 is home, as it’s seen every night, while AP 2 and AP 3 are offices, as they are seen during working hours. The last row shows the combined 95% of time coverage provided by the top 8 routers. doi:10.1371/journal.pone.0130824.g003 privacy abuse of such infrastructure. Understanding and quantifying the dynamics of privacy and utility of infrastructures is crucial for building connected and free society. Since the creation of comprehensive databases containing geolocation for APs is primarily carried out by large companies [4], one might assume that WiFi based location tracking by ‘small players’, such as research studies or mobile applications, is not feasible. As we have shown above, however, APs can be very efficiently geolocated in a way that covers a large majority of individuals’ mobility patterns. privacy abuse of such infrastructure. Understanding and quantifying the dynamics of privacy and utility of infrastructures is crucial for building connected and free society. Since the creation of comprehensive databases containing geolocation for APs is primarily carried out by large companies [4], one might assume that WiFi based location tracking by ‘small players’, such as research studies or mobile applications, is not feasible. As we have shown above, however, APs can be very efficiently geolocated in a way that covers a large majority of individuals’ mobility patterns. Since the creation of comprehensive databases containing geolocation for APs is primarily carried out by large companies [4], one might assume that WiFi based location tracking by ‘small players’, such as research studies or mobile applications, is not feasible. As we have shown above, however, APs can be very efficiently geolocated in a way that covers a large majority of individuals’ mobility patterns. PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 Discussion While it has been pointed out that Android WiFi permissions may allow for inference of sensitive personal information [26], the effect has not been quantified through real-world data. Here we have shown that inferring location with high temporal resolution can be efficiently achieved using only a small percentage of the WiFi APs seen by a device. This makes it possible for any application to collect scanned access points, report them back, and inexpensively convert these access points into users’ loca- tions. The impact is amplified by the fact that apps may passively obtain results of scans rou- tinely performed by Android system every 15–60 seconds. Such routine scans are even run when the user disables WiFi. See S1 File for additional analysis on data privacy in the Android ecosystem. Developers whose applications declare both location and WiFi permissions are able to use WiFi information to boost the temporal resolution of any collected location information. We have shown that even if the location permission is revoked by the user, or removed by the app developers, an initial collection of both GPS and WiFi data is sufficient to continue high-reso- lution tracking of the user mobility for subsequent months. Many top applications in the Play Store request Wi-Fi connection information but not explicit location permission. Examples from the top charts include prominent apps with more than 100 million users each, such as Candy Crush Saga, Pandora, and Angry Birds, among others. We are not suggesting that these or other applications collect WiFi data for location tracking. These apps, however, do have a de facto capability to track location, effectively circumventing Android permission model and general public understanding. Due to uniqueness of location traces, users can be easily identified across multiple datasets [17]. Our results indicate that any application can use WiFi permission to link users to other public and private identities, using data from Twitter or Facebook (based on geo-tagged tweets and posts), CDR data, geo-tagged payment transactions; in fact any geo-tagged data set. Such PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 9 / 11 Tracking Human Mobility Using WiFi Signals cross-linking is another argument why WiFi scans should be considered a highly sensitive type of data. In our dataset, 92% of WiFi scans have at least one visible AP. Supporting Information S1 File. Additional details on the properties of the data and the employed analysis methods. In this Supporting File we present an example method of inferring the locations of WiFi rout- ers, explain the interplay between the long term stability and low entropy of human mobility, provide a detailed description of the mobility properties of the participants (Figs B and C), show the distributions of time coverage of top routers (Fig D), and explain how Android per- mission model allows apps to access the WiFi information of the user. (PDF) Acknowledgments We thank Yves-Alexandre de Montjoye and Andrea Cuttone for useful discussions. Discussion Even in the most challenging scenario, when there are no globally shared locations and each individual frequents different places, top 20 WiFi access points per person can be efficiently converted into geolocations (using Google APIs or crowd-sourced data) and used as a stable location channel. These results should inform future thinking regarding the collection, use, and data security of WiFi scans. We recommend that WiFi records be treated as strictly as location data. Author Contributions Conceived and designed the experiments: SL AS PS. Performed the experiments: PS AS RG. Analyzed the data: PS RG. Wrote the paper: PS AS RG SL. Conceived and designed the experiments: SL AS PS. Performed the experiments: PS AS RG. A l d h d PS RG W h PS AS RG SL Conceived and designed the experiments: SL AS PS. Performed the experiments: PS AS RG. Analyzed the data: PS RG. Wrote the paper: PS AS RG SL. PLOS ONE | DOI:10.1371/journal.pone.0130824 July 1, 2015 References 1. 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https://openalex.org/W3136907960
https://elib.dlr.de/141662/1/rob.22016.pdf
English
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The MADMAX data set for visual‐inertial rover navigation on Mars
Journal of field robotics
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cc-by
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Received: 17 August 2020 | Revised: 3 December 2020 | Accepted: 21 January 2021 Received: 17 August 2020 | Revised: 3 December 2020 | Accepted: 21 January 2021 DOI: 10.1002/rob.22016 F I E L D R E P O R T Lukas Meyer1 | Michal Smíšek1 | Alejandro Fontan Villacampa1 | Laura Oliva Maza1 | Daniel Medina2 | Martin J. Schuster1 | Florian Steidle1 | Mallikarjuna Vayugundla1 | Marcus G. Müller1 | Bernhard Rebele3 | Armin Wedler1 | Rudolph Triebel1 1Department of Perception and Cognition, Institute of Robotics and Mechatronics, German Aerospace Center (DLR), Wessling, Germany Abstract Planetary rovers increasingly rely on vision‐based components for autonomous navigation and mapping. Developing and testing these components requires re- presentative optical conditions, which can be achieved by either field testing at planetary analog sites on Earth or using prerecorded data sets from such locations. However, the availability of representative data is scarce and field testing in pla- netary analog sites requires a substantial financial investment and logistical over- head, and it entails the risk of damaging complex robotic systems. To address these issues, we use our compact human‐portable DLR Sensor Unit for Planetary Ex- ploration Rovers (SUPER) in the Moroccan desert to show resource‐efficient field testing and make the resulting Morocco‐Acquired data set of Mars‐Analog eX- ploration (MADMAX) publicly accessible. The data set consists of 36 different na- vigation experiments, captured at eight Mars analog sites of widely varying environmental conditions. Its longest trajectory covers 1.5 km and the combined trajectory length is 9.2 km. The data set contains time‐stamped recordings from monochrome stereo cameras, a color camera, omnidirectional cameras in stereo configuration, and from an inertial measurement unit. Additionally, we provide the ground truth in position and orientation together with the associated uncertainties, obtained by a real‐time kinematic‐based algorithm that fuses the global navigation satellite system data of two body antennas. Finally, we run two state‐of‐the‐art navigation algorithms, ORB‐SLAM2 and VINS‐mono, on our data to evaluate their accuracy and to provide a baseline, which can be used as a performance reference of accuracy and robustness for other navigation algorithms. The data set can be accessed at https://rmc.dlr.de/morocco2018. The MADMAX data set for visual‐inertial rover navigation on Mars Lukas Meyer1 | Michal Smíšek1 | Alejandro Fontan Villacampa1 | Laura Oliva Maza1 | Daniel Medina2 | Martin J. Schuster1 | Florian Steidle1 | Mallikarjuna Vayugundla1 | Marcus G. Müller1 | Bernhard Rebele3 | Armin Wedler1 | Rudolph Triebel1 Lukas Meyer1 | Michal Smíšek1 | Alejandro Fontan Villacampa1 | Laura Oliva Maza1 | Daniel Medina2 | Martin J. Schuster1 | Florian Steidle1 | Mallikarjuna Vayugundla1 | Marcus G. Müller1 | Bernhard Rebele3 | Armin Wedler1 | Rudolph Triebel1 J Field Robotics. 2021;1–21. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Journal of Field Robotics published by Wiley Periodicals LLC Abstract 2Department of Nautical Systems, Institute of Communications and Navigation, German Aerospace Center (DLR), Neustrelitz, Germany 3Analysis and Control of Advanced Robotic Systems, Institute of Robotics and Mechatronics, German Aerospace Center (DLR), Wessling, Germany Correspondence Lukas Meyer, Department of Perception and Cognition, Institute of Robotics and Mechatronics, German Aerospace Center (DLR), Muenchener Strasse 20, 82234 Wessling, Germany. Email: lukas.meyer@dlr.de wileyonlinelibrary.com/journal/rob | 1 1.1 | Hand‐held field testing Testing of planetary rovers and their navigation algorithms can be per- formed with increasing complexity levels. Tests in laboratory environ- ments and in artificially created outdoor testbeds are a good initial way to validate navigation and mapping solutions. A multitude of rover na- vigation solutions have been tested in such scenarios, for example, • ExoMars Test Rover (ExoTeR) is used to test localization and mapping components both in an indoor laboratory environment and in an outdoor test‐facility of the European Space Agency (Hidalgo‐Carrió et al., 2018). • Rovers Minnie and Mana are used outdoors on artificially created terrain that resembles the features of Mars (Post et al., 2018). However, planetary analog sites on Earth are generally remote locations (see Preston et al., 2012, for an overview) that are difficult to access. Bringing a robotic system to such areas for onsite testing usually results in costly logistics, comes with high demand in op- erations personnel, and involves the risk of damaging the robotic system. Offsite testing using previously recorded data sets is a more cost‐efficient approach, but suffers from the scarcity of data sets available for this purpose. Furthermore, data sets allow software components to be run repeatedly to improve algorithms and test them more efficiently. Algorithms can also be compared with each other with respect to the accuracy, robustness, and computational performance. For a state‐of‐the‐art validation of planetary rovers in general, and especially for navigation solutions, field tests in analog en- vironments become necessary. These analog environments are usually remote regions like deserts or volcanoes that resemble the environments of different celestial bodies, typically the Moon or Mars. A detailed list of analog sites on Earth is provided by Preston et al. (2012). In the past, many different planetary rover prototypes were placed in such environments for system testing or testing of the full rover mission including scientific operations. The list of such endeavors known to us comprises: This paper addresses the onsite and offsite testing of vision‐ based navigation solutions in planetary analog scenarios by con- sidering three aspects: • The long‐distance rover traverses in the Atacama Desert in Chile, pioneered by Wettergreen et al. (1999) and Wettergreen et al. (2005) with teleoperation and partial autonomy for the rovers Nomad and Zoë, respectively. And more recently, the long‐range autonomous exploration tests by the Seeker rover (Woods et al., 2014) in the same area. K E Y W O R D S exploration, extreme environments, navigation, planetary robotics, SLAM This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Journal of Field Robotics published by Wiley Periodicals LLC wileyonlinelibrary.com/journal/rob | 1 1 | INTRODUCTION 2 | MEYER ET AL. 2 • We use MADMAX with two state‐of‐the‐art navigation algorithms, ORB‐SLAM2 (Mur‐Artal & Tardós, 2017) and VINS‐Mono (Qin et al., 2018), to evaluate their algorithmic performance and to evaluate the challenges of the data set. We show that the hand‐held MADMAX can be considered representative for planetary rover navigation by comparing the navigation results in terms of accuracy with results obtained from a planetary rover prototype. Additionally, we use the results to provide a navigation baseline for our Mars‐analog scenario that can be used by other navigation algorithms as a performance reference for accuracy and robustness. Planetary surfaces are mostly explored by mobile robotic platforms, because these environments are difficult and expensive to reach for humans and exhibit hazardous environmental conditions. Owing to the interrupted and high‐latency communication, as well as the lack of prior knowledge about the environment, such mobile robotic platforms have to operate autonomously to some extent. The quality of autonomous decision‐making by a mobile robot de- pends heavily on the navigation and mapping software solutions, as precise localization capabilities are crucial for experiments to be com- pleted successfully. The navigation and mapping solutions increasingly rely on vision‐based components, as camera systems are commonly used in space missions since they are robust in harsh environments and space‐ qualified systems already exist. Extraterrestrial vision‐based robot navi- gation needs to be able to operate under optical conditions that differ greatly from most locations on Earth. While a software simulation can bootstrap initial phases of component development and testing, to satisfy the robustness requirements it must usually be tested against inputs with representative optical conditions. The maturity and usability of such navigation solutions therefore depends on the availability of the sensor input representative of the targeted planetary environment for devel- opment and testing. These data are typically obtained on Earth in pla- netary analog locations with optical features similar to the targeted extraterrestrial bodies. 1.2 • Our ROBEX demo mission on top of the volcano Etna in Italy focusing on a full modular Moon‐analog mission (Wedler et al., 2017), done by the DLR in 2017. All these tests campaigns were either a full rover mission, or even a full scientific mission scenario. Generally, planetary rovers are highly in- tegrated mechatronic systems, which makes field testing complex, something we experienced ourselves during the ROBEX demo mission (Wedler et al., 2017). The complexity is due to the multitude of com- ponents used, which require the presence of many specialists and equipment at the test site. This results in costly logistics and high man- power requirements. Furthermore, many of these endeavors are used to test various system components of the rover, allocating only limited time for navigation tests and possibly running the risk of technical failures that may delay or endanger the field test. As we deployed SUPER in the Moroccan desert, we use it to record a comprehensive visual inertial navigation data set, which constitutes the second contribution of this paper. The small size and mobility of SUPER allows it to cover several different locations with varying terrains and record a variety of trajectories in a short time. In addition, we use the mobility of SUPER to record data in sites that are not yet accessible for the current generation of wheeled plane- tary rovers, owing to the harsh terrain. In total, we collect data from 36 experiments at eight different locations in three general areas, each with an individual geological character. The recorded sensor input consists of a monochrome stereo camera pair, a RGB color camera, two omnidirectional cameras mounted in a vertical stereo setup, and an IMU. We also compute the ground truth pose from a Real‐time kinematic (RTK) Global Navigation Satellite System (GNSS) with two antennas mounted on SUPER, which allows us to obtain a ground truth not only in the position but also in the orientation. Our paper's first contribution is to address the complexity of field tests by providing a simplified hand‐held rover navigation platform that focuses solely on the visual inertial navigation systems (VINS) compo- nents of a planetary rover. This is achieved by our SUPER hand‐held device, which resembles a planetary rover in terms of sensors but leaves out all other components, such as locomotion, scientific instruments, and representative communication concepts. 1.2 1.2 and teleoperation (Sonsalla et al., 2017) and locomotion cap- abilities (Cordes et al., 2018). Only a few publicly available vision‐based navigation and mapping data sets exists, which specifically target planetary robotics. One is the Kat- wijk Beach Planetary Rover Data set (Hewitt et al., 2018), where a pla- netary rover prototype performs several long‐range traverses on a beach using stereo cameras and Lidar. Another is the resulting data set from the previously mentioned experiments on Devon Island, where the pushcart platform was used to record a long trajectory of 10 km (Furgale et al., 2012). We recorded two long range navigation runs on the outskirts of the volcano Etna during the ROBEX campaign, using a lightweight pla- netary exploration rover prototype that used stereo vision, IMU data, and wheel odometry for navigation (Vayugundla et al., 2018). Lamarre et al. (2020) present recordings from a Mars‐analog outdoor laboratory run by the Canadian Space Agency that include data for visual inertial and omnidirectional camera‐based navigation. They also consider energy‐ budget‐aware navigation by providing solar irradiation data. Lacroix et al. (2019) present navigation data recorded by several cameras, fiber optic gyro, IMU, and Lidar from the two rovers Minnie and Mana. These were obtained in the Moroccan desert—in the same region as MADMAX. Fi- nally, the Planetary Data System (NASA, 2019) from NASA makes it possible to access data directly obtained from current and past Mars missions. • The Mojave Desert field test in the United States (Bakambu et al., 2012) with a focus on evaluating navigation algorithms in a Mars‐ analog environment, specifically considering the visual motion estimation and inertial measurement unit (IMU) enhanced wheel odometry. • The Mojave Desert field test in the United States (Bakambu et al., 2012) with a focus on evaluating navigation algorithms in a Mars‐ analog environment, specifically considering the visual motion estimation and inertial measurement unit (IMU) enhanced wheel odometry. • A field test in the Teide Volcano National Park on the island of Tenerife, Spain, the results of which are used, among others, by Geromichalos et al. (2020) to validate a Simultaneous Localization and Mapping (SLAM) solution. • Visual odometry with omnidirectional cameras is evaluated with data from a field test in the Atacama Desert by Corke et al. (2004). 1.1 | Hand‐held field testing • We discuss the use of hand‐held sensor devices that resemble the sensor setup of a planetary rover as a cost‐efficient, low‐risk al- ternative to onsite field testing. For this, we present the human‐ portable Sensor Unit for Planetary Exploration Rovers (SUPER) of the German Aerospace Center (DLR) and its application in the 2018 Mars‐analog field test in the Moroccan desert. • Tests for the ExoMars rover mission, such as the SAFER field test (Gunes‐Lasnet et al., 2014) in the Atacama Desert and the ExoFit rover tests (Motaghian et al., 2019) in southern Spain. • During this field test, we recorded the Morocco‐Acquired Data set of Mars‐Analog eXploration (MADMAX). It is a comprehensive collection of visual inertial navigation data representative of a Mars‐roving scenario, containing 36 trajectories with a combined length of 9.2 km. We describe the data set in detail and make it publicly available. • The MARS2013 mission in Morocco, where full Mars‐analog op- erations were tested, including scientists in the field, commu- nication infrastructure, and rovers (Groemer et al., 2014). • The Utah field trials in the United States testing the SherpaTT rover and Coyote III robot, with a the focus on multirobot systems | 3 MEYER ET AL. 3 1.3 | Navigation algorithm performance reference We use MADMAX with two state‐of‐the‐art SLAM‐based navigation algorithms, the visual odometry algorithm ORB‐SLAM2 (Mur‐Artal & Tardós, 2017), and the visual inertial odometry algorithm VINS‐Mono (Qin et al., 2018) to evaluate their performance in a Mars‐analog scenario as the final contribution of our paper. The variety of MADMAX enables us to test the navigation solutions for optimal scenarios but also challenging corner cases. We compare the navigation accuracy of this hand‐held data set to the results of navigation sequences from an additional SUPER system, this time attached to a rover. This second sensor unit was integrated with the planetary rover prototype illustrated in the background of Figure 1, and was used to record several navigation FIGURE 1 The two SUPER units in the Moroccan desert on the Rissani 1 location: One unit is mounted on the SherpaTT rover (Cordes et al., 2018) of the DFKI Robotics Innovation Center (background) and the other is used as human‐carried device (foreground). The data presented in this article were captured by the hand‐held device [Color figure can be viewed at wileyonlinelibrary.com] 1.2 As SUPER is not a full planetary rover prototype like the LRU, the representativeness for a rover op- erations scenario on a celestial body can be questioned. However, our analysis in Section 6.3 suggests that the hand‐held approach is re- presentative for navigation. We therefore consider this approach as the optimal trade‐off between costs and representativeness. A similar ap- proach is taken by Furgale et al. (2012), where a pushcart platform equipped with stereo cameras, a sun sensor, and inclinometers is used for a combined Mars and Moon‐analog navigation experiment on Devon Island, in the Arctic North of Canada. MADMAX lies in line with the Etna data set (Vayugundla et al., 2018) as we used closely related systems with similar sensors and an identical software infrastructure in both cases. With these two data sets, it becomes possible to evaluate VINS algorithms in Moon‐ and Mars‐analog scenarios at the same time, without having to adapt to a different system setup. We use SUPER in a Mars‐analog site and perform navigation experiments to show that this kind of sensor unit allows for resource‐efficient field testing thanks to three factors: its small size, the use of key hardware components only, and the fact that only two persons are needed to operate the device. In our case, the Mars‐ analog site is located in the north‐western region of the Sahara Desert, close to the city of Erfoud in the Drâa‐Tafilalet region of Morocco, as shown in Figure 2. The Morocco data from Lacroix et al. (2019) combined with MADMAX allow to evaluate vision based navigation algorithms in similar environments—sometimes even in the same location—on very different systems. One of the main differences is the use of a longer stereo baseline for their cameras (270 mm vs. our 90 mm) that makes it possible to consider more distant features for navigation, whereas our configuration focuses on nearby scenery and local mapping. Lacroix et al. (2019) additionally records Lidar data, whereas SUPER MEYER ET AL. 4 sequences. We apply identical evaluation methods in both cases to study potential differences on the navigation performance. In the end, this experiment allows us to emphasize that MADMAX can be considered as representative for planetary rover navigation. provides omnidirectional stereo images as secondary sensor data. The wheeled platforms of Minnie and Mana provide a rover‐like movement of the system. We exploit the higher mobility of SUPER to access rougher terrain to collect data. These data become relevant for next‐generation planetary rovers with improved locomotion capabilities. Lacroix et al. (2019) provide data where trajectories were traversed several times by the rovers; we instead cover more trajectories of varied character in each location. We provide both a five and a six degrees of freedom (DoF) ground truth compared to the three DoF ground truth that is normally included in planetary navigation data sets. Finally, along with our data set we include an evaluation using two state‐of‐the‐art navigation algorithms. In addition, the results from the state of the art can be used as a baseline for other navigation algorithms. Our approach is similar to that of Antonini et al. (2020), however in our case targeted at pla- netary robotics instead of indoor unmanned aerial vehicle operation. To the best of our knowledge, no such evaluation and publicly available state‐of‐the‐art performance reference for navigation is available for planetary rover navigation yet. The three mentioned data sets therefore allow the development and evaluation of robust navigation algorithms that are able to perform independently of system architecture or environment. 1.4 | Outline The article is structured as follows. In Section 2, we describe the field test scenario in the Moroccan desert, where we recorded MADMAX. We present our sensor suite SUPER in Section 3, outline the system specifications, provide details on the installed sensors, and describe the reference frame definitions. We introduce the experiment setup in Section 4 together with operational aspects of field testing, our approach for dual‐antenna RTK GNSS ground truth computation, and the sensor calibration. We present the resulting data set with its 36 trajectories in Section 5 and discuss the specific characteristics of the different experiment locations and of the individual trajectories. We provide a detailed overview of all sensor data that can be found in the data set and how the different sensor readings can be related to each other spatially and temporally. We also address challenges that come up in MADMAX, such as influences from the extreme en- vironment, complications that we faced during the testing, as well as lessons learned from the operation. Finally, in Section 6, we discuss the results of two state‐of‐the‐art navigation algorithms with MADMAX and provide a performance analysis of them. 2 | SCENARIO OVERVIEW AND LOCATION SUPER is designed according to the specifications by the European Space Robotics Technologies Research Cluster (SRC) program to test percep- tion algorithms for planetary exploration. It is conceptualized in a versatile way, such that it can be either carried by a human in the stand‐ alone fashion or integrated with a robotic system via mechanical, elec- trical, and data interfaces. Two SUPER units were used in both config- urations as shown in Figure 1. They were deployed in a 40‐week field test in the Moroccan desert, close to the city of Erfoud, located in the northern Sahara during November and December 2018. Many areas in this region resemble an ideal Mars‐analog in terms of optical conditions (Preston et al., 2012). The experiment site locations are marked in Figure 2 and impressions from the sites are shown in Figures 1 and 3. FIGURE 1 The two SUPER units in the Moroccan desert on the Rissani 1 location: One unit is mounted on the SherpaTT rover (Cordes et al., 2018) of the DFKI Robotics Innovation Center (background) and the other is used as human‐carried device (foreground). The data presented in this article were captured by the hand‐held device [Color figure can be viewed at wileyonlinelibrary.com] There, we utilized the first SUPER in a hand‐held approach to record MADMAX. The second SUPER was used in the same area for the final validation experiments of the SRC technology development roadmap MEYER ET AL. | 5 FIGURE 2 Overview of the experiment locations. All experiments were performed in the vicinity of the city of Erfoud in the northern Sahara region. Map of Morocco (left) by Eric Gaba, local map (right) by OpenStreetMap‐Contributors and OpenTopoMap, both distributed under a CC BY‐SA 3.0 license [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 2 Overview of the experiment locations. All experiments were performed in the vicinity of the city of Erfoud in the northern Sahara region. Map of Morocco (left) by Eric Gaba, local map (right) by OpenStreetMap‐Contributors and OpenTopoMap, both distributed under a CC BY‐SA 3.0 license [Color figure can be viewed at wileyonlinelibrary.com] The data presented in MADMAX is widely varied, as the small size and mobility of SUPER allows it to access several different lo- cations for experimentation in a relatively short time. 2 | SCENARIO OVERVIEW AND LOCATION The region around Erfoud offers a rich variety of terrains: from flat to hilly, from sandy and featureless through pebbly to rocky with features of high saliency, from horizon landmarks being virtually nonexistent to salient landmarks on the horizon, from easily traversable areas to slopes nontraversable by locomotion systems of current planetary robots—such as high‐inclination hillsides or sandy dune fields. PERASPERA with the projects InFuse and Facilitators as a provider of sensoric data for localization, environment mapping, environment re- construction, and visual tracking (Post et al., 2018).1 For this purpose, it was integrated with the SherpaTT rover (Cordes et al., 2018) as shown in the background of Figure 1. This paper focuses on the field‐testing with the hand‐held SUPER and publishes only the data from the hand‐held experiments. For information on all other experiments, see Brinkmann et al. (2019). Nevertheless, navigation data from the rover‐mounted SUPER is used to show that MADMAX is representative for planetary rover navigation, and the evaluation results are included here. Additional vision‐based navigation data resulting from InFuse and the Morocco Field Test is described in Lacroix et al. (2019), where the rovers Minnie and Mana perform navigation experiments in several de- sert areas around the city of Erfoud. As mentioned before, MADMAX, the data set presented here, and the data set captured by Minnie and Mana can be seen as complementary in nature. 6 | MEYER ET AL. MEYER ET AL. 6 FIGURE 4 The SUPER sensor setup with the most relevant reference frames (color‐scheme: X—red, Y—green, and Z—blue). The body frame (B), the IMU frame, the frames of the upwards (omni,U) and downwards (omni,D) facing omnidirectional cameras, the front‐facing left (cam,L), right (cam,R), color (cam,C) cameras, and the left GNSS antenna (L) are shown. IMU, inertial measuring unit [Color figure can be viewed at wileyonlinelibrary.com] Both systems were developed for the scenario of planetary ex- ploration, share the same software architecture with SUPER, and use similar algorithms. See Schuster et al. (2019), for details on the LRU system and Lutz et al. (2020) for a comprehensive description of ARDEA. The core sensor and processing components of SUPER are a copy of the components used on LRU. LRU's navigation, locomotion, and manipulation capabilities have been manifested in multiple campaigns, including the SpaceBotCamp challenge in 2015 (Schuster et al., 2019) and the ROBEX demo mission, a month‐long Moon‐ analog field test on Mt. Etna in Sicily in 2017 (Wedler et al., 2017). While such competitions and field tests provide good opportu- nities for system testing and data recording, they come at significant organizational and logistics costs. To minimize costs while maximiz- ing the scientific yield, the design of SUPER is focused solely on planetary rover sensors. The result is a device almost identical to LRU in perception, on‐board data processing, and power manage- ment capabilities, but one that omits other aspects such as active locomotion and manipulation. See Figure 4, for a detailed view. FIGURE 4 The SUPER sensor setup with the most relevant reference frames (color‐scheme: X—red, Y—green, and Z—blue). The body frame (B), the IMU frame, the frames of the upwards (omni,U) and downwards (omni,D) facing omnidirectional cameras, the front‐facing left (cam,L), right (cam,R), color (cam,C) cameras, and the left GNSS antenna (L) are shown. IMU, inertial measuring unit [Color figure can be viewed at wileyonlinelibrary.com] As it was already mentioned, SUPER can be used in two sce- narios as is shown in Figure 1: it can be either carried by a human or mounted on a robotic platform and integrated with that robot. De- pending on the scenario, SUPER is either powered by two batteries with a capacity of 208 Wh each and hot‐swap capabilities during stand‐alone operations, or can be powered by a carrier robotic platform. 6 | The batteries allow it to be operated for up to 5 h. It weighs 14 kg and has a core‐body size of × × 34 26 40 cm .2 Two on‐board computers are integrated, both Kontron mITX‐KBL boards with CoreTM i7‐7820EQ CPUs and one Xilinx Spartan‐6 LX75T FPGA for depth image computation. used by the carrier. Generally, during our hand‐held experiments, the stereo camera bench is located approximately 1.20 m above the ground. Furthermore, the hand‐held approach implies the absence of wheels, therefore no wheel odometry is available—a sensor input typically present in planetary robotic platforms. The data from SUPER is only targeted at VINS algorithms for navigation and map- ping, which can be developed independently from the wheel odo- metry or other sensor inputs. Wheel odometry is a challenging scientific topic by itself and was omitted for this field test. Interested readers can learn more about our investigation into this topic in Bussmann et al. (2018), where slip of the LRU was investigated on a Moon‐analog site. Optionally, two GNSS antennas can be mounted to the sides with a wingspan of 1.28 m. We make use of the two antennas to provide a five DoF ground truth that includes not only the position but also roll and yaw information. Together with the antennas on the body of SUPER, a base GNSS station is installed on each experiment location to eliminate atmospheric delays, thus allowing precise po- sitioning estimates. The computation of the ground truth is discussed in Section 4.3. SUPER is focused on the perception aspect of planetary robotic applications. To keep the system simple, actuators were excluded from the design. This design choice implies that SUPER does not possess an active pan‐tilt unit to change the camera orientation. The cameras point downwards at a fixed pitch of ∘ 28 relative to the body of SUPER. The camera orientation can be actively guided by the carrier— especially its heading angle—and is aligned with the or- ientation of the carrier. The height of the sensors depends on the height of the carrier and the adjustments made to the harness that is 1In the context of InFuse, SUPER is referred to as Hand‐held Central Rover Unit. 2SUPER's dimensions are the result of using identical components as the LRU. This is de- sired for comparability between the systems, however contributes to a nonoptimal design in terms of size and weight. 2SUPER's dimensions are the result of using identical components as the LRU. This is de- sired for comparability between the systems, however contributes to a nonoptimal design in terms of size and weight. 3 | SYSTEM OVERVIEW The design of SUPER is inspired by the DLR mobile robotic systems, lightweight rover unit (LRU), a four‐wheeled full‐body‐actuated pla- netary rover prototype, and ARDEA, a microaerial vehicle (MAV). FIGURE 3 Impressions from the experiment locations: Kess Kess with the locations D (top left) and E (top right) and Maadid with locations F (bottom left) and H (bottom right) [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 3 Impressions from the experiment locations: Kess Kess with the locations D (top left) and E (top right) and Maadid with locations F (bottom left) and H (bottom right) [Color figure can be viewed at wileyonlinelibrary.com] 6 | 1In the context of InFuse, SUPER is referred to as Hand‐held Central Rover Unit. 3.1 | SUPER as stereo and VINS An overview of the sensors of SUPER is given in Table 1. The pla- cement of the sensors together with the relevant coordinate frames is shown in Figure 4. SUPER is equipped with an optical bench carrying three cameras mounted in a row at the front of the device, with parallel optical axes. The left and right cameras are monochrome and set apart at a 90 mm baseline. These constitute the stereo camera bench, which is our primary navigation sensor. The color camera is mounted centrally between them. Data from the color camera can be used as an ad- ditional navigation source, or for finding landmarks and points of | 7 MEYER ET AL. TABLE 1 Main components of SUPER that were used for recording MADMAX Sensor Name Specifications Navigation cameras AlliedVision Mako G‐319 14 Hz, monochrome × 1032 772 px images, rectified, auto exposure RGB color camera AlliedVision Mako G‐319 4 Hz, color × 2064 1544 px images, rectified, auto exposure Camera lenses RICOH FL‐HC0614‐2M 6 mm, F/1.4 Omnicam cameras AlliedVision Mako G‐319 4–8 Hz, monochrome × 2064 1544 px images, auto exposure Omnicam lens Entaniya 280 Fisheye 1.07 mm, F/2.8, × ∘ ∘ 280 360 field of view IMU XSENS MTi‐10 100 Hz, three‐axis acceleration and three‐axis angular rates GNSS receiver Piksi Multi GNSS SwiftNav 1 Hz, GNSS Data GNSS antenna SwiftNav GPS500 Frequencies GPS L1/L2, GLONASS L1/L2 and BeiDou B1/B2/B3 Note: They are listed with detailed specifications and, if applicable, the sensor frequency. Abbreviations: GNSS, global navigation satellite system; IMU, inertial measuring unit. TABLE 1 Main components of SUPER that were used for recording MADMAX Note: They are listed with detailed specifications and, if applicable, the sensor frequency. Abbreviations: GNSS, global navigation satellite system; IMU, inertial measuring unit. interest. The three‐axis accelerations and angular velocities are measured by a MEMS IMU. the lower camera has its optical axis pointing downwards. Their exposure mode is set to automatic with a target mean histogram level of the image at 60% brightness for the upward‐facing camera, and at 30% brightness for the downward‐facing camera. The differ- ent target levels are set to compensate for the bright sky or the shadows and terrain, respectively. 3.1 | SUPER as stereo and VINS The exposure mode for all cameras is set to automatic with a target mean histogram level of the image at 40% brightness, which results on average exposure times of 0.01–0.001 s for the stereo camera pair and 0.02–0.005 s for the color camera. All cameras have a horizontal field of view of ∘ 61 each. This field of view, together with the stereo baseline of 90 mm, allows for a stereo overlap starting at a minimum distance of 75 mm. Impressions from the stereo setup of both cameras are shown in Figure 5. We include the recorded data streams for this sensor pair in MADMAX for all trajectories, together with the associated cali- bration information. The processing pipeline for image acquisition, image rectifica- tion, and depth image computation is identical to that of the LRU (Schuster et al., 2019). We use the Semi‐Global Matching algorithm (Hirschmüller, 2008) to compute the depth images online onboard. This depth image stream is considered to be an intermediary data product and is included in the MADMAX data set. Note that the depth image computation is adjusted to the relevant working dis- tance, that is, it considers a maximum disparity of 128 px, which relates to a minimum depth of 60 cm. Developing and testing omnidirectional navigation solutions on SUPER was beyond the scope of the Morocco 2018 campaign and for this publication. However, it is a promising field of research and will form the subject of for future work. We hereby invite interested readers to use this collection of data for the development of novel navigation means for planetary robots. There are multiple ways in which these omnidirectional cameras can contribute to the overall navigation solution. First, the relatively large baseline and a large overlap in the fields of view of both cameras (illustrated in Figure 5) makes it possible to formulate an omnidirectional stereo‐based visual odometry. Second, both cameras 4.1 Similar to our previous data sets recorded on Mt. Etna in 2017 (Vayugundla et al., 2018), we performed intrinsic and extrinsic cali- bration of navigation cameras using the calibration toolbox DLR CalDe and DLR CalLab (see Strobl & Hirzinger, 2006 and Strobl et al., 2020, for the software) and IMU‐to‐cameras calibration as in Fleps et al. (2011). Due to the added omnidirectional stereo bench, the intrinsic and extrinsic calibration data sets are captured tying the configuration of all five cameras tightly into one joint calibration setup. The frame definitions associated with the GNSS ground truth are illustrated in the two Figures 4 and 6a. The GNSS raw data describes the position of the left and right GNSS antenna frames d L and d R with respect to the world, that is, the topocentric frame d T. Note that GNSS data only provides positional information and no or- ientation, however we define the corresponding GNSS frames for the sake of completeness. Section 4.3 gives details regarding raw GNSS data processing to calculate the ground truth of the SUPER pose, that is, orientation and position of the central body frame d B with respect to the world frame d T. This body frame is located above the IMU frame, precisely in the middle of the two GNSS antennas and is used as a central reference, also for the navigation algorithms in Section 6. Intrinsic and extrinsic camera calibration is performed before the first experiments. We record images of the DLR CalDe calibra- tion pattern (Strobl & Hirzinger, 2011) using the stereo cameras, the color camera, and two omnidirectional cameras. The topocentric reference frame d T for each experiment is de- fined as the respective position of the GNSS base station. Finally, d B,start defines the starting point of each trajectory and denotes the position of the body frame at time t0, with t0 being the starting time of the respective experiment. All relevant transformations between these reference frames are included in MADMAX for each experiment. have the potential to see distant landmarks on the horizon and use them for absolute orientational localization. Third, a Sun‐tracking solution can be formulated for the upper camera for the absolute orientation. Fourth, the lower camera can use the optical flow of features on the ground for improved navigation. In addition, on a full rover system, the lower camera can be used as a tool for visual inspection of the locomotion system's health. These are a few sug- gested uses of the omnidirectional stereo data for navigation and hazard‐avoidance purposes. 3.2 | Omnidirectional navigation The configuration of SUPER is easy to modify thanks to its design, which provides mechanical, electrical, and data connections for adding extra components. We use this advantage to include addi- tional perception sensors in our experiments. Inspired by the wide field of view from ARDEA (Lutz et al., 2020), we add a long‐baseline omnidirectional camera stereo bench to the system. Omnidirectional cameras are a promising addition or alternative to vision‐based na- vigation for planetary exploration, thanks to their high field of view. They are becoming popular for navigation and are considered in the field of planetary robotics by, for example, Corke et al. (2004) and Lamarre et al. (2020). FIGURE 5 Omnidirectional stereo images from a plain experiment site with a hill range in the background. The area without stereo‐overlap (red) and exemplary feature correspondences (green) between the two images are shown. The image area occupied by the operator is blacked out [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 5 Omnidirectional stereo images from a plain experiment site with a hill range in the background. The area without stereo‐overlap (red) and exemplary feature correspondences (green) between the two images are shown. The image area occupied by the operator is blacked out [Color figure can be viewed at wileyonlinelibrary.com] Our cameras have a field of view of ∘ 360 horizontally and ∘ 280 vertically and they are mounted as vertical stereo setup, in a coaxial configuration with a baseline of 61 cm. They are oriented in such a way that the upper camera has the optical axis pointing upwards and MEYER ET AL. 8 3.3 | Reference frames In this section, we discuss our field experiment methodology. The variability of conditions and the limited availability of equipment complicate the data set acquisition during field tests significantly, as opposed to well‐defined laboratory environments. To structure the data acquisition process in such conditions and to facilitate follow‐up data evaluation, we use predefined procedures for the experiment and the ground truth during all experiments. The relevant reference frames of SUPER are annotated in Figure 4 and are listed in Table 2. The resulting transformations between the frames are included in the data set. The IMU data is referenced to the d IMU frame. The stereo cameras provide image data with respect to the frames of the monochrome cameras d cam,L and d cam,R. The color camera provides images with respect to d cam,C. Depth images are associated with the left camera frame d cam,L. The omnidirec- tional cameras are referenced to the frames d omni,U and d omni,D for the upward and downward facing cameras, respectively. 4.2 | Experiment procedures • a GNSS+inertial 6 DoF reference, sampled at 100 Hz. It is ob- tained by fusing the IMU and GNSS measurements. We apply predefined procedures to each experiment to ensure consistency. We use a static platform as a base for SUPER, where we start and finish each trajectory to ensure that SUPER is placed in the same position and orientation both times. This allows for well‐ defined trajectory evaluation criteria and could also be used as loop closures for SLAM. We recommend the use of the 5 DoF ground truth solution for an inertial‐independent evaluation of visual‐inertial algorithms. The 6 DoF ground truth shall be used in the remaining cases, especially for the evaluation of purely vision‐based algorithms. The ground truth estimation solves the navigation problem, for which the position, velocity and attitude of a moving (rigid) body are determined. The kinematic quantities relate two coordinate systems: (i) the frame whose motion is described, body frame d B; (ii) the frame with which that motion is respect to, denoted as topocentric frame d T. This study adopts the conventions recommended for ro- tation and reference frames from Barfoot (2017). Figure 6a provides an illustration of the aforementioned navigation frames. The platform is leveled horizontally and oriented to the east using a spirit level and a compass. This procedure provides a rough initial alignment of the navigation results with the GNSS ground truth and facilitates later processing of the data. The initial seconds (between 8 and 42 s) of each data acquisition run are recorded in a stand‐still configuration to obtain static sensor readings for sensor bias evaluation. At each location, neither the platform nor the GNSS base station move. Therefore all runs from that location have common start and end points in image and ground truth data. This allows for navigation and mapping overlaps between the different trajectories. The state estimate is expressed as a discrete‐time state‐space model. 4.1 The derived camera TABLE 2 List of frames used on SUPER Frame Description X‐axis Y‐axis Z‐axis d T Topocentric frame East North Up d B SUPER body frame Forward Left Up d B,start body frame at t0 Forward Left Up d L Left GNSS antenna Forward Left Up d R Right GNSS antenna Forward Left Up d IMU IMU frame Forward Left Up d cam,L Left stereo camera Image horizontal Image vertical Optical axis d cam,R Right stereo camera Image horizontal Image vertical Optical axis d cam,C Color camera Image horizontal Image vertical Optical axis d omni,U Omnicam up Image horizontal Image vertical Optical axis d omni,D Omnicam down Image horizontal Image vertical Optical axis Frame Description X‐axis Y‐axis Z‐axis d T Topocentric frame East North Up d B SUPER body frame Forward Left Up d B,start body frame at t0 Forward Left Up d L Left GNSS antenna Forward Left Up d R Right GNSS antenna Forward Left Up d IMU IMU frame Forward Left Up d cam,L Left stereo camera Image horizontal Image vertical Optical axis d cam,R Right stereo camera Image horizontal Image vertical Optical axis d cam,C Color camera Image horizontal Image vertical Optical axis d omni,U Omnicam up Image horizontal Image vertical Optical axis d omni,D Omnicam down Image horizontal Image vertical Optical axis MEYER ET AL. | 9 (a) (b) FIGURE 6 Illustration of the SUPER ground truth computation set‐up and the processing steps for the GNSS‐based precise positioning and attitude determination. The ground truth computation allows for optional use of the IMU. (a) Geometrical setup with relevant reference frames; and (b) processing pipeline. EKF, extended Kalman filtering; GNSS, global navigation satellite system; IMU, inertial measuring unit; RTK, real‐time kinematic [Color figure can be viewed at wileyonlinelibrary.com] (a) (b) (b) (a) FIGURE 6 Illustration of the SUPER ground truth computation set‐up and the processing steps for the GNSS‐based precise positioning and attitude determination. The ground truth computation allows for optional use of the IMU. (a) Geometrical setup with relevant reference frames; and (b) processing pipeline. EKF, extended Kalman filtering; GNSS, global navigation satellite system; IMU, inertial measuring unit; RTK, real‐time kinematic [Color figure can be viewed at wileyonlinelibrary.com] information of both antennas to compute the pose of the body frame d B of SUPER as outlined in this section. 4.2 | Experiment procedures Thus, at the time t, the state is described by  q = ⎡⎣ ⎤⎦ ∈ ∈ x p v q p v q , , , , , t t T t T T t T T T T T 3 3 (1) where the position p T and velocity v T are expressed in the topo- centric frame and the quaternion θ θ ≔ ∕ + ∕ q u cos( 2) sin( 2), where = + + u u i u j u k x y z is an unitary axis and θ is a rotation angle, follows the Hamilton convention and expresses the body‐to‐topocentric rotation. The estimated state is denoted as xˆt and the corresponding covariance matrix as Pˆt. 4.1 The data set contains the raw GNSS observations from the left, right, and base antennas as well as the computed ground truth solution. Two distinct ground reference solutions are made readily available: parameters of the stereo system are consequently used for image rectification and depth map computation during all Morocco ex- periments. The raw images of the calibration are included in the data set, together with the derived intrinsic and extrinsic camera para- meters for all cameras. • an inertial‐independent (GNSS‐only) 5 DoF solution, sampled ev- ery second, estimation workflow. The prediction step is based on a constant‐ velocity, constant‐attitude model for the GNSS‐only solution, while classical inertial integration is employed as prediction for the GNSS +IMU solution. The Rissani area provides rich geological features for navigation cameras—rocks, pebbles, and sand patches. The area is featured in the background of Figure 1. Several landmarks are visible in omnidirectional cameras, most dominantly the tabletop mountain. The terrain is mostly traversable for a typical planetary rover with occasional challenges. The area is generally barren with vegetation visible sparingly. Additionally, it is the main site of testing for Brinkmann et al. (2019) and one of the three acquisition sites of Lacroix et al. (2019). During the GNSS processing stage, the raw code and phase observations from left and right antennas, along with the base sta- tion are fed to the open source RTKlib (Takasu & Yasuda, 2009). First, the RTK module performs a least‐squares (LS) adjustment for the position solution p T and its covariance matrix ∑p (Teunissen & Montenbruck, 2017). The Baseline module estimates the inter‐ antenna baseline vector r T and its covariance matrix ∑r. The SD for horizontal and vertical positioning of such a setup is approximately 2 and 5 cm, respectively, for fixed solutions (Medina et al., 2021). A fixed solution denotes a position/baseline result for which the esti- mated integer carrier phase ambiguities are considered valid, which is generally the case for MADMAX. The Kess Kess area consists of a ridge formed by carbonate mounds (Preston et al., 2012, p. 74) with a wide flat terrain in its vicinity. This site has previously featured a 4‐week long Mars‐analog mission campaign, where many scientific experiments were conducted under simulated Martian surface conditions (Groemer et al., 2014). Our data from that site is the result of two distinct acquisition sessions. The first one at the location (D) was realized in front of the ridge. The area is flat, barren, and has many pebbles providing fea- tures of high saliency as seen in the top left image of Figure 3. For omnidirectional cameras, the nearby mound provides a plethora of landmark features. The resulting GNSS‐based attitude precision coarsely relates to the accuracy of the estimated baseline (i.e., the positioning error for each antenna) compared to the actual antenna baseline (Giorgi et al., 2012). The SUPER configuration has a baseline length of 1.28 m. 5 | DATA SET The focus of our Morocco experiments was to gather relevant planetary‐analog data for navigation and mapping that offers a variety, both in the type of the trajectory as well as in the type of the terrain. An overview of the locations (labeled A–H) is given in Table 3, alongside with a brief description of the terrain, images of the scene, and the corresponding plots of the GNSS ground truth trajectories. Compared to the estimated baseline length, the attitude precision results mostly below 0.5° for roll and yaw estimates for our data set. Generally, the GNSS accuracy is denoted in the corresponding SDs in the ground truth data. Our second Kess Kess data acquisition location (E) is situated between the mounds of the ridge—see the top right image of Figure 3. Here, the geological situation is different as a variety of rocks and stones was present and the landmark richness for omni- directional cameras was high. In terms of terrain steepness and the number of rocks, we consider the location to be virtually non- traversable for planetary rovers of current generation, thus we ex- ploit the high mobility of SUPER and its carrier to obtain the navigation data there. Since only two GNSS antennas were installed, attitude de- termination for the GNSS‐only ground truth becomes an ill‐posed problem. As a result, roll and yaw can be accurately estimated, while pitch is not observable, thus providing a ground truth in 5 DoF. We provide the MATLAB code for our two ground truth estimation approaches together with the data set online. Lastly, the Maadid area hosts three distinct data acquisition sessions in very different terrain. The first one (F) is captured in a rather flat location covered with pebbles, easy to traverse for a planetary rover, as seen in the bottom left image of Figure 3. The environment for the second session (G) consists of a mixture of composite rock formations embedded in a sandy area. It comes with many landmarks for omnidirectional cameras to work with, and a terrain difficult to traverse for a planetary rover. The third data acquisition session (H) is situated in an area mostly consisting of sand dunes—see the bottom right image of Figure 3. These provide low saliency of corner or line features, thus being challenging for most visual odometries. 4.3 | RTK GNSS ground truth One crucial aspect for field tests is the ground truth. In laboratory setups, the ground truth is usually obtained by high‐precision optical tracking systems. For outdoor scenarios, such tracking systems are rarely available and, instead, RTK GNSS constitutes the main in- formation source. Our GNSS setup is shown in Figure 6a together with the corresponding reference frames. We use the GNSS The navigation problem is addressed based on a standard mul- tiplicative (also known as error‐state) extended Kalman filtering (MEKF) formulation (Markley, 2003), with Figure 6b depicting the 10 MEYER ET AL. 5.1 | Planetary analog location description We made sure that all trajectories follow the same recording pro- cedures, to make the data set more consistent. As outlined in Section 4.2, all tracks are realized in such a ways that the initial and final pose are the same—with heading always to the east, and roll and pitch approximately zero—allowing for at least one loop closure within each. MADMAX contains 36 trajectories recorded during eight data ac- quisition sessions, each in a separate location, in three general areas: Gara Medouar, also known as Rissani 1 (locations A–C, see Preston et al., 2012, p. 73, for details), Kess Kess (locations D and E, see Preston et al., 2012, p. 74) east of the city of Erfoud, and Maadid (locations F–H) north of Erfoud. All locations are marked in the map of Figure 2. Table 3 features impressions from SUPER's color camera that show the geological character of each location. All runs which are obtained in one location have mutual identical starting and end poses, allowing for at least two overlaps between each pair of tracks for multirun mapping. Furthermore, all 3 Experiment overview: GNSS trajectories, color camera impression of the scenes, and additional information sorted by location. Camera decalibration is indicated with ries Scene Character ID Length [m] Type Flat area with stones, rock ridge at the end of the area. Low sun illumination. A0 133 homing A1 138 homing A2 134 homing A3 219 zig‐zag + homing A4 250 mapping A5 200 mapping A6 258 exploration Flat area with sand and pebbles and few big rocks, cliffs visible in the background. B0 1511 long range nav B1 193 homing B2 195 homing B3 195 homing B4 286 zig‐zag + homing B5 301 mapping B6 293 exploration B7 312 exploration Small flat and square area, half sandy and half stony. C0 341 zig‐zag C1 321 zig‐zag C2 378 zig‐zag Flat area with small stones and pebbles, hill formation in the background D0 141 circular homing D1 155 circular homing D2 134 circular homing D3 493 long range nav D4 422 exploration run Rough terrain inside a valley and big stones within the traversed path. E0 223 exploration E1 309 exploration E2 374 exploration Flat area with small pebbles, rough terrain at the end of the area. F0 121 homing F1 128 homing F2 121 homing (Continues) MEYER ET AL. | 11 MEYER ET AL. trajectories are recorded in such way that they overlap on several additional occasions to allow for combined mapping. The ground truth is provided in two ways, as the GNSS‐only 5 DoF ground truth, and in addition, as the 6 DoF fusion of GNSS and IMU data. Note that the timestamps of the postprocessed GNSS measurements are temporally synchronized with the other sensor data of SUPER. The raw GNSS data is provided in UTC time ac- cording to the RINEX specification, thus the temporal synchroniza- tion with the other sensors has to be taken into account. The temporal offset between GNSS time and the UNIX system time is listed in the metadata.yaml file for each experiment. Note that this time offset is different for each day, as SUPER was not con- nected to clock synchronization servers during the field campaign. For each experiment, we choose from predefined categories of trajectories that represent different aspects of navigation. Trajec- tories of the mapping type aim to cover an area with many overlaps within one run, trying to allow for dense terrain mapping. Zig‐zag trajectories also aim at dense mapping of an area. In this case, a structured grid pattern of motion is used, unlike the unstructured motions of the mapping trajectories. Long‐range‐navigation runs cover long distances and are targeted towards evaluating localization algorithms. We also record trajectories for homing algorithms that follow one path several times with a minor offset. Finally, exploration runs combine several of the characteristics of the aforementioned types in an unstructured manner. The different trajectory types at each location are listed in Table 3, where the characteristics and mapping overlap of each run can also be seen in the corresponding GNSS ground truth overview plots. Additionally, the metadata text file lists detailed information for each experiment, like precise location coordinates of the base sta- tion, the time stamps of the experiment start, and the start of data recording, respectively. One key information is the initial pose of SUPER with respect to the base station, that is, the transformation from d B,start to d T. In addition to the experiment data, we provide calibration data. This includes the intrinsic and extrinsic camera calibration as callab_camera_calibration*.txt together with the re- sulting camera parameters for the rectified images as {camera} _rect_info.txt. The transformations between the relevant coordinate frames from Table 2 are provided as well. 5.3 | Data set content The navigation results for ORB‐SLAM2 and VINS‐Mono from Section 6 are included as well. They are provided in two formats: The original result data with respect to the camera and IMU frames, respectively, and the data aligned with the GNSS ground truth. Both are text files with timestamp, position, and orientation quaternion for each pose. We provide sensor data from all sensors listed in Table 1. From the stereo cameras, we provide the rectified images of the left and right camera together with the resulting depth image. The color camera image stream includes rectified images as well. From the omnidirectional cameras, we provide the raw images. As the upper and bottom omni- directional cameras show the face and the legs of the human carrier, an area of ∘ 60 is blacked out in each image as shown in Figure 5. Finally, we provide the data set from the SUPER calibration. This includes raw images from each camera with the calibration pattern visible, the specification of the calibration pattern dimensions, as well as the calibration results in a text file. All images are named after the respective camera plus the timestamp of the UNIX time in nanoseconds, and indicate if the images are rectified, resulting in the following pattern: img_ {rect}_{camera}_{timestamp}.png. We use the common Portable Network Graphics data type for the images. The time- stamped IMU data are provided as comma‐separated values (.csv) files for each experiment. The stereo cameras are synchronized with the IMU, with the IMU being used to trigger the cameras. The two omnidirectional cameras provide synchronized image pairs. The other sensors run independently, however, all of SUPER's sensors use clock synchronization to provide precise timestamps in the sys- tem time. All data can be freely accessed online at https://rmc.dlr.de/ morocco2018. The website shows details on each experiment loca- tion and the experiments performed, plus one section for the cali- bration data. The data is available individually for each experiment, structured as shown in Figure 7 and is provided in a compressed format. This is a col- lection of transforms between a parent and a child frame, given as position and quaternion‐orientation in .csv files. The operator was instructed to move at a velocity, which is si- milar the movement speed of current or future planetary rovers and to keep this velocity constant. In the data set, the overall average velocity is at 29 cm/s, while the average velocities of the individual sequences range between 22 and 48 cm/s. F‐0, with an average ve- locity of 12 cm/s, is considered as special case. 5.1 | Planetary analog location description TABLE 3 Experiment overview: GNSS trajectories, color camera impression of the scenes, and additional information sorted by Trajectories Scene Character ID Flat area with stones, rock ridge at the end of the area. Low sun illumination. A0 A1 A2 A3 A4 A5 A6 Flat area with sand and pebbles and few big rocks, cliffs visible in the background. B0 B1 B2 B3 B4 B5 B6 B7 Small flat and square area, half sandy and half stony. C0 C1 C2 Flat area with small stones and pebbles, hill formation in the background D0 D1 D2 D3 D4 Rough terrain inside a valley and big stones within the traversed path. E0 E1 E2 Flat area with small pebbles, rough terrain at the end of the area. F0 F1 F2 Scene Trajectories Trajectories Scene Character ID Length [m] Type F3 172 zig‐zag + homing F4 167 mapping F5 141 mapping Navigation around big composite rock boulders with sandy surface in between. G0* 125 exploration G1* 115 exploration G2* 154 exploration Desert sand dunes. H0 90 exploration 12 | Trajectories Scene Character ID Length [m] Type F3 172 zig‐zag + homing F4 167 mapping F5 141 mapping Navigation around big composite rock boulders with sandy surface in between. G0* 125 exploration G1* 115 exploration G2* 154 exploration Desert sand dunes. H0 90 exploration 12 | MEYER ET AL. MEYER ET AL. 12 Trajectories Scene Character ID Length [m] Type F3 172 zig‐zag + homing F4 167 mapping F5 141 mapping Navigation around big composite rock boulders with sandy surface in between. G0* 125 exploration G1* 115 exploration G2* 154 exploration Desert sand dunes. H0 90 exploration Character MEYER ET AL. 13 • During some experiment runs, the SUPER antennas lost the sig- nals of several GNSS satellites for a few seconds. On such occa- sions, the precision was usually good enough to provide a satisfactory position estimate, but the orientation suffered significantly. The first issue to mention is the extrinsic calibration of cameras. Once the field tests had been completed, we evaluated the quality of the extrinsic stereo camera calibration by comparing the vertical displacement of sampled features within selected stereo pairs of each run. It turned out that the last runs of the Morocco campaign, labeled G runs, experienced a vertical feature offset bigger than 2 px, which we consider a sign of decalibration. As a result, the computed depth images for these runs are less accurate and contain several invalid regions. Furthermore, the accuracy of camera to IMU cali- bration is degraded in these runs. The cause for this calibration error is most likely an unexpected mechanical load during transport to the experiment site. Nevertheless, we publish the G runs so that the data can be used to test the robustness of algorithms against extrinsic decalibration. Indeed, the Section 6 shows that VINS‐Mono and ORB‐SLAM2 obtain accurate navigation results for the G runs. All other runs turned out to have accurate calibration. For future field tests, we recommend calibrating cameras, and IMU‐to‐camera, on a daily basis to ensure high data quality. The GNSS inaccuracies occurred in 11% of all measurement points, not counting the runs B7, C2, and F0, which were more strongly affected with rates exceeding 40%. The accuracy of the measurements is represented in the GNSS pose estimate SD for each timestamp. Any algorithm or any evaluation that also considers the associated uncertainties should not be affected by this issue. Most runs experience frame drops of only 5%–10% of the overall frame count. However, the F runs are strongly affected with a loss of 15%–19%. We attribute the issue to the network hardware used in SUPER, which was chosen due to its lightweight design. Reconfiguring the network settings made the issue less prominent, but the general problem still prevailed. Generally, no direct corre- lation was found between the number of frame drops and poor na- vigation results, which we discuss in Section 6 in detail. The individual losses per run are listed together with the data. To over- come frame drops in the future, a more robust network setting has to be considered, even though this would require more heavyweight components to be used. FIGURE 7 Example structure of the available data for an experiment from Location M with Run ID N. The data are provided in a compressed format Finally, our GNSS solution lost precision in its measurements occasionally (we consider position measurements with a SD of more than 0.06 m to be imprecise) for two reasons: FIGURE 7 Example structure of the available data for an experiment from Location M with Run ID N. The data are provided in a compressed format FIGURE 7 Example structure of the available data for an experiment from Location M with Run ID N. The data are provided in a compressed format • The RTK GNSS quality depends significantly on having a precise geo‐reference solution of the base station. During the G and H runs, we recorded the base station GNSS data for intervals that were too short to obtain a sufficiently precise GNSS base station solution, thus leading to poorer accuracy in the corresponding pose estimation of SUPER. For future experiments, a prolonged data recording for the GNSS base station should be considered in the experiment schedule. due to shadows in the field of view. The moving shadow of SUPER introduces the similar optical features in the recorded images like a planetary rover. Our analysis concluded that the human carrier did not introduce any additional undesired shadows into the image that might disturb navigation algorithms. All of these disturbances were desired to create challenging scenarios for planetary navigation al- gorithms as a robustness test. Due to the operator having moved slowly and exposure times in bright sunlight having been short, no significant motion blur was observed. 6 | EVALUATION WITH STATE‐OF‐THE‐ ART NAVIGATION ALGORITHMS We evaluated the data set using two state‐of‐the‐art SLAM‐based navigation algorithms. The algorithms provide us with a 6D‐pose of the SUPER system, which is subsequently compared with the GNSS ground truth. The motivation is to provide the navigation results and insights as a baseline against which other algorithms can be com- pared, and we invite interested researchers to do so. Throughout the field test, we experienced network problems that specifically affected the stereo cameras connected via gigabit Ethernet (GigE vision®). As a result, several frame drops occurred. These frame drops usually lasted for one to four consecutive frames (up to a quarter of a second) and seldom reached half a second, that is, up to eight consecutive missing frames. Our analysis shows that this still accounts for an inter‐frame overlap of 80%–90% and 70%–80%, respectively. 14 | 14 MEYER ET AL. 5.4 | Issues, challenges, and lessons learned Operations in extreme environments pose special challenges to the system, the operators, and the experiments. In our case, several challenges and technical issues were encountered, which we could partially account for. The raw GNSS data from the left, right, and base antennas are provided as .obs RINEX format (International GNSS Service, 2015) together with the satellite ephemeris (.nav) text files. The post‐ processed ground truth pose of the SUPER body center from Section 4.3 is provided in an additional .csv text file as position vector, rotation in quaternions, a linear velocity vector, together with the associated SDs and the timestamp in nanoseconds. Many recordings contain optical disturbances that make the data set challenging. One disturbance appeared in particular during afternoon experiments: lens flares due to a low sun position. Another disturbance was the strong over or underexposure of image regions map of the environment as well as the robot pose (position and orientation) from a stream of images. VO incrementally estimates the path of the camera/robot focusing mainly on local consistency, whereas SLAM obtains a globally consistent estimation of the cam- era/robot trajectory and map by recognizing previously mapped areas (loop closure). The extensive navigation sequences covered by MADMAX containing numerous loop closure opportunities are sui- table for testing both VO and SLAM algorithms. map of the environment as well as the robot pose (position and orientation) from a stream of images. VO incrementally estimates the path of the camera/robot focusing mainly on local consistency, whereas SLAM obtains a globally consistent estimation of the cam- era/robot trajectory and map by recognizing previously mapped areas (loop closure). The extensive navigation sequences covered by MADMAX containing numerous loop closure opportunities are sui- table for testing both VO and SLAM algorithms. As we mention above, we test MADMAX with two different state‐of‐the‐art SLAM baselines: ORB‐SLAM2 (Mur‐Artal & Tardós, 2017) and VINS‐Mono (Qin et al., 2018). ORB‐SLAM2 for stereo cameras is built on monocular feature‐based ORB‐SLAM (Mur‐Artal et al., 2015), a complete system for monocular cameras, including map reuse, loop closing, and relocalization capabilities. VINS‐Mono is a tightly coupled monocular visual inertial odometry that fuses IMU measurements and feature observations. Both systems work in real time on standard CPUs in a extensive variety of environments from small hand‐held sequences, to ground robots and drones. An open‐ source system integration is available for both algorithms and their performances have been validated on public data sets and real‐world experiments. VO and SLAM can work with just a single monocular camera. The cameras allow robust and accurate place recognition, and thanks to their small size, low cost, and easy hardware setup they are of great interest in the robotics community. However, there is a list of drawbacks limiting the use of monocular cameras in real‐world ro- botic applications. Monocular vision‐only systems cannot recover the metric scale of the scene, they also suffer from scale drift, and pure rotation movements cause VO systems to fail during exploration. In addition, the initial map required for system bootstrapping cannot be obtained from the first frame, so multiview or additional sensors are required to produce it. A wide variety of state‐of‐the‐art techniques is available to address these problems, especially in the field of sensor fusion. 6.2 | Evaluation results MADMAX is a large‐scale data set that provides suitable sequences to test stability and long‐term use of SLAM. Notice that results and evaluation shown in this section do not aim to compare performance specifically between ORB‐SLAM2 and VINS‐Mono but to evaluate general differences between stereo and visual inertial setups using the selected algorithms as respective examples. Their performance is also used to show the opportunities provided by the data set in the sense of navigation algorithm testing. Additionally, the evaluation aims to provide a navigation baseline for the respective category, which can be used to benchmark other algorithms. The SLAM algo- rithms compute 6D‐poses for every frame of the sequences that we compare with the GNSS ground truth. Next, we will list the details of our evaluation: The scene structure can be reliably obtained with stereo cam- eras through static triangulation with depths within a range of ~40 times the stereo baseline (Mur‐Artal & Tardós, 2017), that is, 3.6 m for our case. The possibility of having the structure of the scene instantly yields a true‐scale SLAM solution, solving among other problems the procedure of initialization from unknown initial states. A monocular VINS consists of a camera and an IMU. One ad- vantage of this setup is to observe the metric scale, as well as directly measuring roll and pitch angles. To estimate a valid scale, the robot has to experience a nonconstant acceleration, which is the case for most moving robots. Furthermore, cameras allow for accurate as- sessment of slow movements, whereas IMUs are well suited for observing of fast movements and rotation. In practice, an IMU is a valuable complement to the visual data, since compared to cameras, these sensors are independent of the environment; their high‐rate values are also cheap to process and have an accurate probabilistic model with little to no outliers. • Both systems have been tested using half‐resolution images from the monochrome cameras to achieve real‐time performance using our institute computers (Intel Xeon E5‐1630, 3.70 GHz, 16 GB RAM, CPU‐only computation). • We initialize VINS‐Mono with an estimation of the extrinsic cali- bration parameters from the initial calibration and let the system refine them online. • The association with the GNSS data has been performed by only considering GNSS measurement points with a SD lower than 0.06 m. Two common VO/SLAM configurations that overcome the challenges while also taking advantage of the features offered by MADMAX are: stereo and visual inertial. 6.1 Among the different motion estimation algorithms, visual odometry (VO) and SLAM are the processes of concurrently estimating the MEYER ET AL. 15 6.2.1 | Navigation robustness and the overall ATE is ∑ = − ∕ = p p N ATE ( ˜ ) t N t t 0 2 (3) (3) To evaluate the navigation robustness, the estimated percentage of accomplished trajectories is shown in Figure 9 for each sequence. It can be seen how the visual inertial navigation is more robust than stereo since it finishes most of the sequences, a significantly greater number than ORB‐SLAM2. It turned out that the frame drops in the recordings, as mentioned in Section 5.4, do not have a direct cor- relation with the navigation robustness of the algorithms. This is shown, for example, by the run F2 (also shown in Figure 8), which has one of the highest frame drop rates at 19% but VINS‐Mono and ORB‐SLAM2 complete the full trajectory with a very low error. It is due to the fact that the inter‐frame overlap of 70%–90% at such frame drop occurrences is sufficient for a continuous tracking for both algorithms. for a total of N trajectory segments. for a total of N trajectory segments. for a total of N trajectory segments. The RPE computes the RMSE of the difference of traveled dis- tances between the estimated trajectory and the ground truth. The traveled distance between two frames separated temporally by Δt is defined as = ∣∣ − ∣∣ +Δ p p dt t t t and the resulting RPE as ∑ = − ∕ = d d N RPE ( ˜ ) , t N t t 0 2 (4) (4) where we choose Δ = t 1 s. The step‐wise RPE is therefore where we choose Δ = t 1 s. The step‐wise RPE is therefore = ∥ − ∥ d d RPE ˜ . t t t (5) (5) Note that while on the one hand, ATE computes the absolute dif- ference between the two trajectories in meters, RPE evaluates the average pose drift in meters per second. On the other hand, both algorithms fail to navigate with A6, a run that has only a 5% rate of frame drops, as well as only a small number of consecutive dropped frames (3–4 dropped frames in a row). It turned out that critical events, that is, the combination of a low inter‐frame overlap of 70%–90% (e.g., due to frame drops) together with other disturbances can cause tracking and relocation failures. 6.2 | Evaluation results SLAM approaches are used to counteract the long‐term drift in the translation and orientation that can strongly affect the visual odometry navigation. SLAM systems can detect online when the rover returns to a mapped area (place recognition and loop closure modules) and correct the drift accumulated in the exploration (graph optimization and/or bundle adjustment). The relocalization of the camera after a tracking failure (due to lighting changes, aggressive movement or lack of a textured scene) produces a very robust and zero‐drift tracking method. For all these reasons, the place re- cognition and pose‐graph optimization are key modules that play an important role to operate in large environments as it is the case for MADMAX. • For evaluation, we use the absolute trajectory error (ATE) and the relative pose error (RPE), as proposed by Sturm et al. (2012). • We consider the fully optimized trajectories that use all data available at the end of each run. • We use ORB‐SLAM2 and VINS‐Mono with loop closing and re- localization capabilities enabled for each individual sequence, but without map reuse between the runs. The ATE calculates the root‐mean square error (RMSE) of all global positions pt along the frames of the estimated trajectory with respect to the GNSS ground truth correspondences p˜t, after both 16 MEYER ET AL. 16 curves have been aligned using the method from Horn (1987). The resulting error at timestep t is VINS‐Mono are illustrated in Figure 8 together with the GNSS ground truth. = ∥ − ∥ p p ATE ˜ t t t (2) (2) 6.2.1 | Navigation robustness Since, on occasion, one of the SLAM approaches may not be capable of calculating the complete trajectories, for a fair compar- ison, we use these metrics just when at least 75% of the trajectory traveled distance has been accomplished successfully. The exemplary results for four trajectories of SUPER computed by ORB‐SLAM2 and Such disturbances are the change of the exposure time, over and underexposure of parts of the image, lens flares, back light, or the FIGURE 8 Sample results of the navigation: The performance of ORB‐SLAM2 and VINS‐Mono with respect to the GNSS is shown for four experiments. Ideal results (F2), ORB‐SLAM2 loses track but relocates after recognizing a previously visited area (D2), successful navigation of both algorithms with the extrinsic decalibration (G0), and loss of ORB‐SLAM2 tracking (F3) are shown. GNSS, global navigation satellite system [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 8 Sample results of the navigation: The performance of ORB‐SLAM2 and VINS‐Mono with respect to the GNSS is shown for four experiments. Ideal results (F2), ORB‐SLAM2 loses track but relocates after recognizing a previously visited area (D2), successful navigation of both algorithms with the extrinsic decalibration (G0), and loss of ORB‐SLAM2 tracking (F3) are shown. GNSS, global navigation satellite system [Color figure can be viewed at wileyonlinelibrary.com] 17 17 MEYER ET AL. FIGURE 9 Percentage of each trajectory completed by ORB‐SLAM2 and VINS‐Mono. Only navigation results with more than 75% completion (red line) are considered in the error analysis [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 9 Percentage of each trajectory completed by ORB‐SLAM2 and VINS‐Mono. Only navigation results with more than 75% completion (red line) are considered in the error analysis [Color figure can be viewed at wileyonlinelibrary.com] being recorded under more challenging illumination conditions is reflected in the poor navigation outcomes in terms of the trajectory completion percentage. Sequences F3 and D2 in Figure 8 show the case of loss of tracking for ORB‐SLAM2, where relocation succeeds for the D2 run and fails for the F3 run. moving shadow of SUPER. Since both algorithms are feature‐based approaches, they deal with purely visual noise in a similar way. In our case, it turned out that ORB‐SLAM2 normally overcomes dis- turbances when the overlap between consecutive frames is greater than ~80%. Values below that threshold in combination with chal- lenging visual conditions cause tracking and relocation failures. 6.2.1 | Navigation robustness As might be expected, VINS‐Mono is more robust against tracking failures produced by visual effects. Since the higher IMU measure- ment rate allows for a continuous pose update between two con- secutive camera frames, VINS‐Mono is able to bridge the gaps and therefore keep the functioning of the tracking thread intact. 6.2.2 | Navigation accuracy Leaving aside the fact that the visual inertial algorithm manages to complete more sequences than its stereo‐based counterpart, it also performs slightly better in terms of ATE accuracy, as shown in Figure 10. Evaluating the 16 sequences in which both algorithms reach more than 75% completion, VINS‐mono outperforms ORB‐SLAM2 in 10 sequences, whereas ORB‐SLAM2 performs better in six runs. Nevertheless, ATEs for both systems are within the same range. In general, these critical events can occur at any time during a sequence, thus explaining the seemingly arbitrary difference in completed trajectory length for each experiment. Also note that the runs A3–A6 were recorded in the late afternoon (around 4 pm in December) with cameras facing the direction of the setting sun. Thus, (a) (b) (c) (d) (e) (f) (g) (h) FIGURE 10 Distribution of the step‐wise ATE of ORB‐SLAM2 and VINS‐Mono for each experiment. Only navigation results with a completion of the trajectory of at least 75% are considered. The whiskers on each vertical bar denote the minimum and maximum values of the error distribution for each run. The box denotes the first and third quartile of the data with the median as the dividing line in‐between. ATE, absolute trajectory error [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 10 Distribution of the step‐wise ATE of ORB‐SLAM2 and VINS‐Mono for each experiment. Only navigation results with a completion of the trajectory of at least 75% are considered. The whiskers on each vertical bar denote the minimum and maximum values of the error distribution for each run. The box denotes the first and third quartile of the data with the median as the dividing line in‐between. ATE, absolute trajectory error [Color figure can be viewed at wileyonlinelibrary.com] MEYER ET AL. 18 (a) (b) (c) (d) (e) (f) (g) (h) FIGURE 11 Distribution of the step‐wise RPE of ORB‐SLAM2 and VINS‐Mono for each experiment. Only navigation results with a completion of the trajectory of at least 75% are considered. The whiskers on each vertical bar denote the minimum and maximum values of the error distribution for each run. The box denotes the first and third quartile of the data with the median as the dividing line in‐between. RPE, relative pose error [Color figure can be viewed at wileyonlinelibrary.com] (a) (b) (c) (d) (e) (f) (g) (h) FIGURE 11 Distribution of the step‐wise RPE of ORB‐SLAM2 and VINS‐Mono for each experiment. 6.2.2 | Navigation accuracy Only navigation results with a completion of the trajectory of at least 75% are considered. The whiskers on each vertical bar denote the minimum and maximum values of the error distribution for each run. The box denotes the first and third quartile of the data with the median as the dividing line in‐between. RPE, relative pose error [Color figure can be viewed at wileyonlinelibrary.com] sequences F3 and D2 (Figure 8). The same figure also shows the G0 run, which is one of the three runs with extrinsic decalibration. It is clear that both algorithms cope with such decalibration and provide reliable navigation results. Apart from the obvious advantages of visual inertial SLAM versus stereo SLAM in terms of robustness for outdoor environments with long‐term trajectories, we have not been able to observe any major differences between the two state‐of‐the‐ art SLAM pipelines. On the other hand, ORB‐SLAM2 outperforms VINS‐mono in 15 out of the 16 sequences in terms of RPE as shown in Figure 11. Generally, it can be said that both algorithms provide accurate navigation results with minor differences with respect to ATE and RPE accuracy. We compare the performance of ORB‐SLAM2 and VINS‐Mono in MADMAX with respect to the EuRoC data set (Burri et al., 2016). The EuRoC data set contains sequences recorded by a MAV flying around different indoor environments, and both ORB‐SLAM2 and VINS‐Mono authors provide testing values from the resulting ATE for their systems. To remove the effect of different trajectory lengths on the resulting alignment error between EuRoC and MADMAX, we compute the median of the ATEs normalized with the length of each sequence in Table 4. The accuracy of EuRoC and MADMAX in terms of ATE lies at similar levels. 6.3 | Comparison of hand‐held and rover‐based navigation The box denotes the first and third quartile of the data with the median as the dividing line in‐between. ATE, absolute trajectory error; RPE, relative pose error [Color figure can be viewed at wileyonlinelibrary.com] FIGURE 12 Distribution of ATE and RPE of ORB‐SLAM2 and VINS‐Mono for the rover‐mounted SUPER sequences. Note that the RPE is scaled by a factor of 3 compared to the previous figure to account for the lower velocity. The whiskers on each vertical bar denote the minimum and maximum values of the error distribution for each run. The box denotes the first and third quartile of the data with the median as the dividing line in‐between. ATE, absolute trajectory error; RPE, relative pose error [Color figure can be viewed at wileyonlinelibrary.com] motion blur in neither of the two data sets, thus limiting the differ- ence solely to the type of motion. field test is the comprehensive Mars‐analog VINS data set MADMAX that we make publicly available. We make the claim that MADMAX can be considered a re- presentative data set for rover navigation if the ATE and RPE of the experiments match the errors of the rover‐mounted SUPER experi- ments. We consider the C runs and the D‐0 to D‐2 runs for a com- parison, as these were obtained in the same location or feature similar types of trajectories, respectively. Recall, that the RPE de- pends on the experiment velocity, as it is the distance error per time as stated in (5). We therefore expect it to be lower by a factor of three for the rover‐bound experiments. This data set includes recordings of monochrome stereo cameras, a color camera, two omnidirectional cameras in a vertical stereo setup, and an IMU. The experiments took place in several distinctive locations, and we outlined the variety and character of the different experiments. We discussed several operational aspects that turned out to be crucial for a successful data set recording, such as the ground truth computation of position and orientation from the GNSS data, procedures for data re- cording, and the calibration of five different cameras relative to each other, including the two omnidirectional cameras. Regarding the rover‐based navigation, ORB‐SLAM2 completes six sequences, except for run 3, whereas VINS‐mono completes all seven runs. Figure 12 shows the respectiv navigation results in terms of ATE and RPE. 6.3 | Comparison of hand‐held and rover‐based navigation Finally, we investigate how representative the hand‐held data is for planetary rover navigation, answering the question of whether human‐induced motions negatively affect the navigation algorithms. We take seven navigation sequences that were obtained by the rover‐mounted SUPER unit and test these using ORB‐SLAM2 and VINS‐mono. We apply identical evaluation methods to those in Section 6.2. Note that these data belong to the InFuse project (Post et al., 2018) and are therefore not included in MADMAX. Nevertheless, a degradation in performance occurs from EuRoC to MADMAX that cannot be attributed to longer trajectories. There are several potential explanations for this, for example, the chal- lenging visual content of the sequences or the different quality of the sensor calibration. Nevertheless, the resulting navigation results of both algorithms can be considered as accurate, see for example, the trajectories of run F2, which is shown in the Figure 8. Until tracking is lost, ORB‐SLAM2 also provides accurate navigation results for the The seven sequences are 26–54 m in length. The trajectories are mostly straight drives combined with wide curves and took place in, and close to, the area of the C runs. Therefore, both MADMAX and these seven rover based sequences experience close to identical environmental conditions. The average velocity of the rover‐ mounted SUPER is 12 cm/s, about a third of the velocity of the hand‐ held navigation (at 29 cm/s). The position of the stereo cameras is approximately 0.80 m above the ground. TABLE 4 Normalized absolute trajectory error (%) VINS‐Mono ORB‐SLAM2 ~ Length (m) EuRoC 0.16 0.27 80 MADMAX 0.27 0.35 175 Note: Comparison of the navigation performance of the EuRoC data set and MADMAX. TABLE 4 Normalized absolute trajectory error (%) The main difference in both data sets is therefore the different type of movement, where the human‐induced motions may influence the navigation in a negative way. Recall that we experienced no Note: Comparison of the navigation performance of the EuRoC data set and MADMAX. 19 19 MEYER ET AL. FIGURE 12 Distribution of ATE and RPE of ORB‐SLAM2 and VINS‐Mono for the rover‐mounted SUPER sequences. Note that the RPE is scaled by a factor of 3 compared to the previous figure to account for the lower velocity. The whiskers on each vertical bar denote the minimum and maximum values of the error distribution for each run. 6.3 | Comparison of hand‐held and rover‐based navigation Indeed, the ATE lies in the same range as the comparable runs from MADMAX, generally around 0.5 m with peaks at 2–3 m. The RPE is approximately one‐third compared to the hand‐held runs, which is ex- pected owing to the three‐fold difference in velocity. We therefore conclude that motions from the human‐based transportation do not negatively influence the navigation. This indicates that MADMAX con- sists of representative planetary rover navigation data, supporting our case in favor of hand‐held field testing. Finally, we showed that the recorded data can be used for na- vigation by applying the state‐of‐the‐art algorithms ORB‐SLAM2 and VINS‐Mono. We evaluated their performance for this planetary‐ analog setting, showed their mostly high accuracy, but also revealed corner cases were these algorithms fail. We compare the perfor- mance of the algorithms to a rover‐based data set and show that our hand‐held approach does not negatively influence the accuracy of the state of the art. It became apparent that MADMAX is a challenging data set for planetary navigation which can be used as robustness test and per- formance reference for new navigation approaches. We make the data publicly available and provide detailed information about it to facilitate the use of the recordings. REFERENCES International GNSS Service. (2015). RINEX: The receiver independent exchange format. https://igs.org/pub/data/format/rinex303.pdf Antonini, A., Guerra, W., Murali, V., Sayre‐McCord, T., & Karaman, S. (2020). The Blackbird UAV dataset. The International Journal of Robotics Research, 39(10–11), 1346–1364. Lacroix, S., De Maio, A., Labourey, Q., PaivaMendes, E., Narvor, P., Bissonette, V., Bazerque, C., Souvannavong, F., Viards, R., & Azkarate, M. (2019). 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They received funding from the European Union's Horizon 2020 re- search and innovation program under grant agreement No 730068 and 730014. We want to thank our colleague Klaus Strobl for his valuable ideas and feedback on the paper and our colleagues Bertram Willberg, Tim Bodenmüller, Bernhard Vodermayer, and many more for their help in preparing the field campaign. Open Access funding enabled and or- ganized by Projekt DEAL. Geromichalos, D., Azkarate, M., Tsardoulias, E., Gerdes, L., Petrou, L., & Perez Del Pulgar, C. (2020). Slam for autonomous planetary rovers with global localization. Journal of Field Robotics, 37(5), 830–847. Giorgi, G., Teunissen, P. J., & Gourlay, T. P. (2012). Instantaneous global navigation satellite system (GNSS)‐based attitude determination for maritime applications. IEEE Journal of oceanic engineering, 37(3), 348–362. ACKNOWLEDGMENTS In this paper, we presented a field testing approach for planetary robotics navigation and mapping algorithms and test data recording that fills the gap between laboratory tests and complex, full‐rover‐ system field tests. To do this, we deployed a compact hand‐held sensoric abstraction of a planetary rover—SUPER—in a Mars‐analog environment in the northern Sahara in Morocco. The result of the We want to thank all European Space Robotics Technologies Research Cluster participants in the Morocco 2018 field test for their help in the field, their valuable scientific input, and their general support. This study was funded by the DLR project MOdulares Robotisches EXplorationssystem (MOREX). This activity has been conducted jointly with the two European Commission Horizon 2020 Projects InFuse and Facilitators. MEYER ET AL. 20 DATA AVAILABILITY STATEMENT Groemer, G., Soucek, A., Frischauf, N., Luger, M., Stumptner, W., Ragonig, C., Sams, S., Bartenstein, T., Häuplik‐Meusburger, S., Petrova, P., Evetts, S., & Sivenesan, C. (2014). The MARS2013 Mars analog mission. Astrobiology, 14(5), 360–376. All data that is considered part of the publication is publicly available under the URL https://rmc.dlr.de/morocco2018. A registration form needs to be filled out before accessing the data. An exception to this is the evaluation in Section 6.3 of the paper, as these data belong to a consortium of project partners. Gunes‐Lasnet, S., Winnendael, M., Diaz, G., Schwenzer, S., Pullan, D., Kisdi, A., Josset, J.‐L., Ciarletti, V., Barnes, D., Griffiths, A., Paar, G., Allouis, E., Waugh, L., Woods, M., & Shaw, A. (2014). SAFER: The promising results of the Mars mission simulation in Atacama, Chile. Proceedings of the International Symposium on Artificial Intelligence, Robotics and Automation in Space. ESA. 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G., Bussmann, K., Büttner, S., Dömel, A., Hellerer, M., Lehner, H., Lehner, P., Porges, O., Reill, J., Riedel, S., Vayugundla, M., Vodermayer, B., Bodenmüller, T., Brand, C., Friedl, W., Grixa, I. L., Hirschmüller, H., Kassecker, M., … Wedler, A. (2019). Towards autonomous planetary exploration: The Lightweight Rover Unit (LRU), its success in the SpaceBotCamp challenge, and beyond. Journal of Intelligent and Robotic Systems, 93, 461–494. Wettergreen, D., Bapna, D., Maimone, M., & Thomas, G. (1999). Developing nomad for robotic exploration of the atacama desert. Robotics and Autonomous Systems, 26(2–3), 127–148. Wettergreen, D., Cabrol, N., Baskaran, V., Calderón, F., Heys, S., Jonak, D., Lüders, A., Pane, D., Smith, T., Teza, J., Tompkins, P., Villa, D., Williams, C., & Wagner, M. (2005). REFERENCES Cordes, F., Kirchner, F., & Babu, A. (2018). Design and field testing of a rover with an actively articulated suspension system in a Mars analog terrain. 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NASA planetary data system—Cartography and imaging sciences discipline node. https://pds-imaging.jpl.nasa.gov/ MEYER ET AL. 21 Second experiments in the robotic investigation of life in the Atacama Desert of Chile. Proceedings ISAIRAS 2005 Conference (Vol. 603, pp. 233–240). Sonsalla, R., Cordes, F., Christensen, L., Roehr, T., Stark, T., Planthaber, S., Maurus, M., Mallwitz, M., & Kirchner, E. (2017). Field testing of a Cooperative Multi‐Robot Sample Return Mission in Mars Analogue Environment. Proceedings of the Symposium on Advanced Space Technologies in Robotics and Automation. Woods, M., Shaw, A., Tidey, E., Pham, B. V., Simon, L., Mukherji, R., Maddison, B., Cross, G., Kisdi, A., Tubby, W., Visentin, G., & Chong, G. (2014). Seeker—autonomous long‐range rover navigation for remote exploration. Journal of Field Robotics, 31(6), 940–968. Strobl, K. H., & Hirzinger, G. (2006). Optimal Hand‐Eye Calibration. Proceedings of the IEEE/RSJ International Conference on Intelligent Robots and Systems. (pp. 4647–4653). Beijing, China. Strobl, K. H., & Hirzinger, G. (2011). More Accurate Pinhole Camera Calibration with Imperfect Planar Target. Proceedings of the IEEE International Conference on Computer Vision, Workshop on Challenges and Opportunities in Robot Perception (pp. 1068–1075). IEEE. How to cite this article: Meyer, L., Smíšek, M., Fontan Villacampa, A., Oliva Maza, L., Medina, D., Schuster, M. J., Steidle, F., Vayugundla, M., Müller, M. G., Rebele, B., Wedler, A., & Triebel, R. (2021). The MADMAX data set for visual‐inertial rover navigation on Mars. Journal of Field Robotics, 1–21. https://doi.org/10.1002/rob.22016 How to cite this article: Meyer, L., Smíšek, M., Fontan Villacampa, A., Oliva Maza, L., Medina, D., Schuster, M. J., Steidle, F., Vayugundla, M., Müller, M. G., Rebele, B., Wedler, A., & Triebel, R. (2021). The MADMAX data set for visual‐inertial rover navigation on Mars. Journal of Field Robotics, 1–21. https://doi.org/10.1002/rob.22016 Strobl, K. H., Sepp, W., Fuchs, S., Paredes, C., Smíšek, M., & Arbter, K. (2020). DLR CalDe and DLR CalLab. https://rmc.dlr.de/callab Sturm, J., Engelhard, N., Endres, F., Burgard, W., & Cremers, D. (2012). A benchmark for the evaluation of RGB‐D SLAM systems. Proceedings of the IEEE/RSJ International Conference on Intelligent Robots and Systems (pp. 573–580). IEEE.
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https://bmcinfectdis.biomedcentral.com/counter/pdf/10.1186/1471-2334-14-141
en
Extensive haemorrhagic necrosis of liver is an unpredictable fatal complication in dengue infection: a postmortem study
BMC infectious diseases
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cc-by
5,083
Kularatne et al. BMC Infectious Diseases 2014, 14:141 http://www.biomedcentral.com/1471-2334/14/141 RESEARCH ARTICLE Open Access Extensive haemorrhagic necrosis of liver is an unpredictable fatal complication in dengue infection: a postmortem study SAM Kularatne1, IVB Imbulpitiya2, RA Abeysekera2*, RN Waduge3, RPVJ Rajapakse4 and KGAD Weerakoon5 Abstract Background: Dengue infection carries a potential risk of death despite stringent management of plasma leak and haemorrhage. It appears that the extent of liver dysfunction determines the outcome. Methods: We present a postmortem study of five patients, died of dengue shock syndrome who had markedly elevated liver enzymes and irreparable circulatory failure. Results: All were females with a median age of 46 years (range 20–50 years). All had positive NS1 and IgM. Clinically, one patient developed severe degree of hepatic encephalopathy whilst three patients developed uncontrollable bleeding manifestations. Dengue virus was detected in three liver specimens by reverse transcription PCR. Histology of the liver revealed massive necrosis with haemorrhages in these patients with evidence of micro and macrovesicular steatosis with significant periportal inflammatory infiltrate. No significant ischaemic changes or necrosis was observed in the other organs. Conclusions: Severe haemorrhagic necrosis of the liver was the cause of death in these patients probably due to direct viral infection. Predilection for severe liver disease remains unknown. Therefore, it is prudent to think beyond plasma leak as the main pathology of dengue infection and attempts should be made to develop other treatment modalities to prevent and manage unforeseen fatal complications of dengue infection. Keywords: Dengue fever, Liver cell necrosis, Acute liver failure, Autopsy study Background Dengue fever is an arboviral infection transmitted by mosquitoes of the genus Aedes, which is widely distributed in tropical and subtropical regions of the globe affecting up to 100 million people per year with 2.5 billion people at risk [1]. In Sri Lanka, dengue fever epidemics have been occurring with increased magnitudes but the worst epidemic was reported in 2009 with 35008 cases and 346 deaths of which 6638 cases and 51 deaths reported in the Central Province of Sri Lanka [2]. Dengue infection is caused by a single stranded RNA virus in the family Flaviviridae, which consists of 4 serotypes (DEN 1–4). Infection with any of the dengue virus serotypes may be asymptomatic in the majority of cases, but in symptomatic cases the severity could vary from * Correspondence: rajithaasa55@gmail.com 2 Medical Unit, Teaching hospital, Peradeniya, Sri Lanka Full list of author information is available at the end of the article dengue fever (DF) to dengue haemorrhagic fever (DHF) including dengue shock syndrome (DSS) [1]. The virus can infect many organs including liver, described from 1950s [3,4]. Over the years the pathophysiology of dengue virus infection had been extensively studied [5]. Studies suggest that three main systems play an important role in the pathogenesis of DHF/DSS: the immune system, the liver and endothelial cell linings of blood vessels [6]. Increased permeability of microvasculature and plasma leak is supposed to be the main dysfunction that leads to DHF and DSS [6]. With this understanding many management guidelines have been developed totally based on fluid resuscitation, resulting in reduced mortality [7]. Despite these efforts and stringent management, there is still a small proportion of patients die due to severe form of dengue infection all over the world. Severe liver involvement is one of the risk factors identified in patients who die of dengue infection. In general, © 2014 Kularatne 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. Kularatne et al. BMC Infectious Diseases 2014, 14:141 http://www.biomedcentral.com/1471-2334/14/141 mild to moderate liver involvement with elevated liver enzymes is common in dengue infection [8] but, acute liver failure and hepatic encephalopathy are rare [9]. Many pathogenic mechanisms have been put forth to explain the liver involvement, but none has been fully conclusive. Therefore, further studies are needed to understand the exact mechanisms of liver damage. The Teaching Hospital Peradeniya (THP) in the hilly Central Province of Sri Lanka maintains a prospective registry of all adult dengue admissions to the hospital since year 2000. The patients are managed according to the guidelines of WHO and the National Guideline of Sri Lanka [7]. The attempts have been made to keep the deaths to the minimum by using stringent fluid management. Yet five patients succumbed to dengue haemorrhagic fever and shock in spite of fluid resuscitation, intensive care and adequate supportive therapy. They all had extensive liver involvement. This necropsy based study aims to describe the extent of liver damage in dengue infection with its impact on the outcome and to think beyond fluid management as the sole treatment. Page 2 of 10 member from the medical team. Sections from all organs were sent to Department of Pathology, Faculty of Medicine, University of Peradeniya for histopathological examination. Formalin fixed tissues were processed, embedded in paraffin, and were cut into sections. The sections were stained with hematoxylin and eosin for histological examination. Reticulin stain was done for liver. RNA extraction, reverse transcription and PCR amplification Fresh tissue samples from heart, lung, brain, kidney, pancreas, liver and spleen, and blood were collected at autopsies for molecular genetic analysis done at the Department of Veterinary Pathobiology of the Faculty of Veterinary Medicine and Animal Science, University of Peradeniya. RNA extraction was performed using RNA Mini Kit (Pure Link™ – ambion). RT- PCR was performed according to a previously described protocol [10]. Finally informed written consent from the next of kin for publication of these individual clinical details and the accompanying images was obtained. Methods Confirmation of the diagnosis Results All patients with fever admitted to the Professorial Medical Unit of THP were clinically assessed to identify dengue cases. The confirmation of the diagnosis was made using NS1 antigen in the first few days of fever and using serology later (dengue specific IgM and IgG ELISA). The clinical and laboratory data were recorded in a formatted data sheet and the regular daily assessments were recorded during the hospital stay. Depending on the severity of the infection, frequent monitoring of vital parameters were done and stringent fluid management was carried out. Autopsies were done in all deceased patients. All five cases included in this study presented during the period of year 2011 – 2013 and they qualified for the diagnosis of dengue infection as they had either positive NS1 antigen or positive serology or they had positive Reverse Transcription Polymerase Chain Reaction (RT-PCR) for dengue in tissues obtained at autopsy. Informed written consent for the autopsy study was taken from the next of kin and complete pathological postmortem was carried out in four cases whereas in one case, only core biopsies of liver, heart and kidneys were taken. This autopsy study is a part of dengue fever studies we are conducting, for which the ethical clearance has been obtained from the Ethics Committee, Faculty of Medicine, University of Peradeniya, Sri Lanka. Written consent for postmortem was obtained from next of kin of all deceased patients. Clinical presentation Histopathological study The autopsies were done by a pathologist with advanced experience in autopsy studies with the participation of a All five patients were females with an age range of 20 – 50 years (median, 46 years). Two patients were transferred from a regional primary care hospital, where as the other three patients directly admitted to THP. The two patients who were transferred were send due to persisting shock with fluid leak despite fluid resuscitation. On general appearance three of them were overweight and three had other co-morbid illnesses consisting of diabetes mellitus, hypertension and dyslipidaemia which were well controlled prior to this admission. The mean duration of fever on admission was 4 days (range 3–5 days) and by the time of death the total mean duration of the illness was 5 days (range 4–6). Three patients had diarrhoea and vomiting associated with fever, where as two patients had bleeding manifestations. All clinical features on admission are summarized in Table 1. Four patients had low volume pulse on admission and blood pressures were unrecordable in two of them. The other patient, despite having a slightly high blood pressure on admission (160/100 mmHg) developed a rapid reduction in blood pressure (110/60 mmHg) within one hour of admission. Four patients had evidence of plasma leakage with pleural effusions and ascites on admission. All five patients deteriorated despite proper management according to local guidelines [7]. Three patients developed uncontrollable profound bleeding manifestations and one patient developed grade 4 hepatic encephalopathy prior to death. Kularatne et al. BMC Infectious Diseases 2014, 14:141 http://www.biomedcentral.com/1471-2334/14/141 Page 3 of 10 Table 1 Clinical features of patients on admission Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Gender Female Female Female Female Female Age (years) 34 20 49 50 46 Appearance Average built Overweight Overweight Overweight Average built DM DM, HT DM, Dyslipidaemia Duration of fever on admission (Days) 3 4 5 3 5 Presenting complaints Fever Fever Fever Fever Fever - Headache - Myalgia - Myalgia - - Myalgia - Vomiting - Vomiting -Headache - Diarrhoea - Headache - PV bleeding - Diarrhoea - Menses - Vomiting Pulse – weak Pulse-100 bpm Pulse-100 bpm, low volume BP – 160/100 mmHg which dropped to 110/60 mmHg Pulse - weak Co-morbidities Cardiovascular system BP-unrecordable BP – 80/60 mmHg BP - unrecordable Evidence of plasma leak Ascites B/L Pleural effusion Right pleural effusion Clear lungs No ascites Right pleural effusion BP – 80/50 mmHg Ascites B/L Pleural effusion Progress Recurrent shock Recurrent shock - Hepatic encephalopathy Uncontrolled bleeding Bleeding 5 6 - Bleeding Duration of illness at death (Days) 4 5 6 DM-diabetes mellitus, HT-hypertension, BP-blood pressure in mmHg, bpm-beats per minute, B/L-bilateral. Laboratory investigations PCR analysis The Table 2 depicts the laboratory investigations during the hospital stay. It was not possible to do antibody titres in one patient but her NS1 antigen was positive. All five patients on admission had a very low platelet count of less than 20 × 109/L (range, 4 – 17 × 109/L). Severe degree of liver involvement was evident by the high aspartate aminotransferase (AST), alanine aminotransferase (ALT), prothrombin time and international normalized ratio ( PT/INR) and Bilirubin values in these patients. The PCR detected dengue infection in multiple organs (Table 3). Type specific PCR found them to be DEN 1 serotype in all positive cases. Three patients had positive dengue PCR in their liver specimens. Positive results were seen in lung, kidney, pancreas and spleen as well. Myocardium was affected in one patient and PCR was negative in all brain tissue. Table 2 Laboratory investigation findings Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Platelets (×109/L) 12 4 16 17 11 PCV % 40% 42 44.9 37 47.2 ALT (IU/L) 1473.9 400 AST (IU/L) 3340 1771 11040 715 1207 1.94 2.50 INR 2.77 2.51 1.96 Bilirubin (μmolL) 45.55 37.35 51.74 NS1 antigen Positive Positive Positive Positive Positive Dengue IgM Positive Positive Positive Positive Not done Macroscopic findings at autopsy (Table 4) Macroscopic examination of multiple organs was done in four patients. Patient number 2 had only histology of core biopsy of liver, kidney and heart. Macroscopically all other cases had enlarged and congested liver. Two specimens revealed subcapsular haemorrhages with multiple petechiae on the surface of the liver. All patients showed evidence of fluid leakage with pleural effusions and ascites with one patient additionally having a significant pericardial effusion. None of the organs showed macroscopic evidence of ischaemic changes or evidence of necrosis. Histopathological analysis (Figures 1, 2, 3, 4, 5, 6, 7 and 8) PCV – packed cell volume, ALT- alanine aminotransferase, AST – aspartate aminotransferase, INR- international normalized ratio. Histology of the liver was almost same in all five specimens which showed distortion of liver architecture, red cell extravasation with collapse in the reticulin frame work and disintegration of hepatocyte nuclei suggestive of massive liver cell necrosis. These changes were Kularatne et al. BMC Infectious Diseases 2014, 14:141 http://www.biomedcentral.com/1471-2334/14/141 Page 4 of 10 Table 3 Results of PCR analysis in tissue samples from different organs Patient1 Heart Lungs Brain Kidney Pancreas Spleen Liver (−) ve (+ ) Ve (−) ve (+) Ve (+) Ve (+) Ve (+) Ve Patient 2 Not done Not done Not done Not done Not done Not done (−) ve Patient 3 (−) ve (−) ve (−) ve (−) ve (−) ve (−) ve (−) ve Patient 4 (−) ve (+) Ve (−) ve (+) Ve (+) Ve (+ )Ve (+) Ve Patient 5 (+) Ve (+) Ve (−) ve (+) Ve (+) Ve (+) Ve (+) Ve (−) ve – negative. (+ )Ve - positive. predominantly seen in zone 1,2 and periportal regions. Micro and macrovesicular steatosis was also evident in these specimens. Acute inflammatory mononuclear cell infiltrates were predominantly seen in the periportal region with two specimens showing evidence of bridging necrosis with one specimen showing evidence of piecemeal necrosis. Other organs such as the kidneys, suprarenal glands, pancreas, lung and heart predominantly showed evidence of congestion. None showed evidence of ischaemic changes or necrosis. Lungs showed evidence of interstitial pneumonitis suggestive of viral infection in patient 5. Brain specimens were all histologically normal. Discussion Over a period of three years, a single tertiary care centre experienced five deaths due to dengue infection in Sri Lanka. Necropsies revealed extensive haemorrhagic liver necrosis as the main pathology behind these fatalities. Detection of viral genetic material by PCR indicated viral invasion of liver and causing direct liver damage. Simultaneously, infection was found in the other organs as well except brain. Clinically, these patients had unstable vital parameters and failed the resuscitation despite prompt fluid therapy and supportive care. Except for many fold of raised hepatic transaminases, severity of hepatic involvement was not apparent clinically. However, autopsy study unraveled the extent of hepatic necrosis in these patients. All these patients had erratic and unpredictable plasma leak, bleeding tendency and hypotension when compared to most of the cases of DHF which were amenable to management with meticulous fluid therapy. One limitation of the study was that we were unable to perform a complete autopsy study in one patient due to limitation of consent. Severe to fulminant hepatitis tends to occur more often in DHF or DSS compared to classic dengue infections [11]. Unlike conventional viral hepatitis, in dengue Table 4 Summary of macroscopic findings of organs Patient 1 Fluid accumulation Liver Patient 3 Patient 4 Patient 5 Ascitic – 1200 ml Blood stained Ascites – 1500 ml Blood stained PlE – 1000 ml Ascites –1800 ml PlE – 800 ml Ascites – 1000 ml PlE – 300 ml PeE – 100 ml PlE – 1500 ml Congested Enlarged Congested Congested Subcapsular haemorrage Subcapsular haemorrage Enlarged Congested Petechial haemorrage Gastrointestinal tract Eso. and stomach Stomach and mesentery Stomach and mesentery Stomach and SI - Congested - Petechiae - Petechiae - Haemorrhagic Area - Pin point haemorrage - Haemorrhagic spots - Haemorrhagic Spots Heart Normal Increased adiposity LVH Congested Epicardial haemorrhagic patches Brain Normal Normal Normal Normal Lung No haemorrhage Congested Trachea- Petichiae Interstital pneumonia Petichiae No haemorrhage Kidney & supra renals Congested Haemorrhage in both renal pelvices Petechial haemorrhage Supra renal glands – petechial haemorrhgaes PlE – pleural effusion, PeE-pericardial effusion, Eso- oesophagus, SI-small intestine, LVH-left ventricular hypertrophy. Congested Kularatne et al. BMC Infectious Diseases 2014, 14:141 http://www.biomedcentral.com/1471-2334/14/141 Page 5 of 10 Figure 1 (Case 1) – H & E section of liver showing extensive haemorrhagic necrosis of liver parenchyma. infections the level of AST is higher than that of ALT [12]. It has been suggested that this may be due to excess release of AST from damaged myocytes during dengue infections [12]. Significantly higher elevations of AST and ALT were observed in patients with episodes of bleeding [12]. The five cases discussed here showed clinical, biochemical as well as pathological evidence of severe liver involvement. The elevation of transaminases is usually less than five-fold greater than upper limit of normal [13] in DF where as in these patients they were more than 10 times higher with an elevated INR. The mechanism of hepatic involvement and hepatocyte damage in dengue fever is poorly understood. The histopathological findings of fulminant hepatitis associated with dengue haemorrhagic fever is often characterized by hepatocellular necrosis, typically localizing to zone 1 and 2 of the hepatic plate, a cellular inflammatory infiltrate and fatty changes. Councilman bodies are frequently present, and may represent necrosis around viral particles [14]. It is well established that most viral infections cause damage by an interplay of direct infection and concomitant host response, evidenced by up- Figure 2 (Case 1) – Reticulin stain of liver with collapsed reticulin framework. Kularatne et al. BMC Infectious Diseases 2014, 14:141 http://www.biomedcentral.com/1471-2334/14/141 Page 6 of 10 Figure 3 (Case 2) – H & E stain of Core biopsy of liver shows confluent necrosis. regulation of cytokine production, notably TNFα, IL-2, IL-6, IL-8 and other chemicals [15,16]. Some literature suggest that liver damage in DHF is mainly due to viral replication in hepatocytes, and cytokine related damage due to adverse consequence of dysregulated host immune responses against the virus [17]. A dengue virus-specific CD4+ and CD8+ T cells may cause cytolysis. The dengue virus targets both the hepatocyte as well as kupffer cell and following internalization induces tumor necrosis factor-related apoptotic damage. The virus enters the hepatocytes and kupffer cells by phagocytosis and receptor-mediated endocytosis, respectively [18]. The main feature is the early generation of a unique cytokine, human cytotoxic factor (hCF) that initiates a series of events leading to a shift from Th1-type response in mild illness to a Th2-type response resulting in severe DHF. The shift from Th1 to Th2 is regulated by the relative levels of interferon-gamma and interleukin (IL)-10 and between IL-12 and transforming growth factor-beta, which showed an inverse relationship in Figure 4 (Case 3) – H & E stain of liver showing extensive haemorrhagic necrosis with surviving hepatocytes reveal macro and micro vesicular fatty change. Kularatne et al. BMC Infectious Diseases 2014, 14:141 http://www.biomedcentral.com/1471-2334/14/141 Page 7 of 10 Figure 5 (Case 3) – H & E stain of liver showing lymphocytic infiltrate of the portal tract. patients with DF [19]. It is also noted that hepatosplanchnic venous pooling and/or dysfunction occur and correlate with the severity of circulatory derangement and shock in patients with DHF [20]. There was a report of a case of liver failure from dengue virus infection with reversal of portal venous blood flow which postulated that hepatic sinusoidal obstruction coupled with shock might be the underlying mechanism of liver failure in this disease [21]. The four DENV serotypes (1, 2, 3, and 4) have been cocirculating in Sri Lanka for more than 30 years with dengue epidemics occurring since 2009 due to DEN-1 [22]. All four serotypes have been associated with denguerelated fulminant hepatitis [23]. DEN-1 and DEN-3 seem Figure 6 (Case 4) – Macroscopy of liver showing subcapsular haemorrhage. to have more prominent liver affinity [24]. In addition, with another study, DEN-2 or DEN-3 viruses were recovered from the livers of 5 of 17 fatal cases [25]. We found DEN-1 as the serotype causing extensive hepatic necrosis in these patients. We have considered many factors as contributory causes for the development of acute liver cell necrosis in these patients. The main factors being direct viral effect on liver cells, adverse consequence of dysregulated host immune responses against the virus, prolonged shock and haemorrhage, metabolic acidosis, intake of drugs during the early stages of the illness such as acetaminophen and pre-existing liver diseases such as fatty liver. Despite all these factors contributing, presence of the viral antigen in the liver (positive PCR), prominent inflammatory cells in liver, prominent damage seen in the periportal as well as zone 1 and 2 and the absence of ischaemic or necrotic changes in any of the other organs indicate that the liver damage was mainly due to the dengue virus infection rather than the other contributory causes. One would expect to have predominant changes in zone 3 as well as a less prominent inflammatory cell response if it were mainly due to ischaemic necrosis as a result of prolong shock. Studies indicate that other predisposing factors such as race, diabetes and sickle cell anaemia [26] can contribute to the liver damage. Three of above study patients were diabetics which may have contributed for fatty changes in liver. Therefore it is vital in the management to avoid or control the above mentioned factors occurring or take rapid steps to correct them as soon as they are identified. Another observatioin was the significant steatosis in all liver samples which could be due to a result of the infection itself Kularatne et al. BMC Infectious Diseases 2014, 14:141 http://www.biomedcentral.com/1471-2334/14/141 Page 8 of 10 Figure 7 (Case 4) – Microscopy of liver H & E stain showing extensive necrosis. or the other possibility of premorbid fatty liver disease in these patients putting them at higher risk of developing liver cell necrosis should be strongly suspected. It was interesting to note that four out of the five patients presented with low blood pressure very early in the disease. It could be that this is the natural course of DSS with evidence of fluid leakage giving rise to hypovolaemic shock. At the same time with such early presentation (mean 4 days) with such severe disease, it is possible that the severe liver cell necrosis was the primary damage with subsequent low blood pressure as a consequence to that. The pathophysiology of hypotension in patients with fulminant hepatic failure was explained by Trewby et al. [27] where it was mentioned that the hypotension could be due to both a central as well as a peripheral effect. Peripheral vasodilatation may be a consequence of the appearance of vasoactive compounds in the circulation which may fail to be metabolised by the necrotic liver, or are released from it. Alternatively they postulated the presence of metabolic insult to the vasomotor center resulting in a low blood pressure. In one study mortality and morbidity associated with fulminant hepatitis were higher in those with circulatory failure, severe thrombocytopenia and spontaneous bleeding, Figure 8 (Case 5) – Microscopy of liver H & E stain showing distortion of hepatic architecture by a confluent haemorrhagic necrosis. Kularatne et al. BMC Infectious Diseases 2014, 14:141 http://www.biomedcentral.com/1471-2334/14/141 with deaths in these cases occurring in the first week of hospitalization which is also consistent with our study findings. In these instances, the fatality rate were as high as 12–44% [28,29]. There is no specific therapy for fulminant hepatitis in dengue except for supportive care. Better outcomes for DHF/DSS with fulminant hepatitis occurred in hospitals with more experience in managing such conditions, with case fatality rates as low as 0.2% [28]. In our cases, supportive care was instituted in the high dependency unit and intensive care given as early as possible. However, case studies of this nature intend to open up new research ideas to identifying the mechanisms of liver involvement in severe dengue infection and to discover therapeutic options. There is limited research that has been carried out to see whether treatment with drugs such as N-acetylcysteine (NAC) which is used in acute liver failure would be of benefit if given early in the course of the illness, to patients with liver involvement with dengue fever [30]. Conclusion Severe hepatic necrosis is an infrequent phenomenon seen with dengue haemorrhagic fever but when present carries a high mortality rate. It is therefore important to identify at risk individuals early and take steps to prevent them progressing into hepatic failure. It is also important to consider dengue haemorrhagic fever as one of the differential diagnosis for acute liver failure especially in endemic and epidemic areas of dengue. Even though, fluid management is the mainstay of treatment in dengue infection, attempts should be made to develop other treatment modalities to prevent and manage unforeseen fatal complications of dengue infection. Abbreviations DEN: Dengue virus serotype; DF: Dengue fever; DHF: Dengue haemorrhagic fever; DSS: Dengue shock syndrome; THP: Teaching hospital peradeniya; RT-PCR: Reverse transcription polymerase chain reaction; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; PT/INR: Prothrombin time and international normalized ratio. Competing interests The authors declare that they have no competing interests. Author contributions SAMK conceived the idea. SAMK, IVB, RAA, KGADW and VJR recorded clinical data. RNW did autopsies and histopathological studies. SAMK, KGADW, IVB and RAA drafted the manuscript. VJR did PCR analysis. All the authors read and approved the final version of the script. Author details 1 Department of Medicine, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka. 2Medical Unit, Teaching hospital, Peradeniya, Sri Lanka. 3 Department of Pathology, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka. 4Department of Veterinary Pathobiology, Faculty of Veternary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka. 5Department of Parasitology, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Saliyapura, Sri Lanka. Received: 22 November 2013 Accepted: 10 March 2014 Published: 14 March 2014 Page 9 of 10 References 1. WHO: Comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. World Health Organization; 2011. 2. Annual Health Bulletin. Sri Lanka: Ministry of Health; 2010. 3. Havens WP Jr: Hepatitis, yellow fever and dengue. Annu Rev Microbiol 1954, 8:289–310. 4. Weerakoon KG, Kularatne SA, Edussuriya DH, Kodikara SK, Gunatilake LP, Pinto VG, Seneviratne AB, Gunasena S: Histopathological diagnosis of myocarditis in a dengue outbreak in Sri Lanka, 2009. BMC Res Notes 2011, 4:268. 5. Malavige GN, Fernando N, Ogg G: Pathogenesis of dengue viral infections. Sri Lanka J Infect Dis 2011, 1:2–8. 6. de Marcedo FC, Nicol AF, Cooper LD, Yearsley M, Pires AR, Nuovo GJ: Histologic, viral and molecular correlates of dengue fever infection of the liver using highly sensitive immunohistochemistry. Diagn Mol Pathol 2006, 15:223–228. 7. Guidelines on management of dengue fever and dengue haemorrhagic fever in adults. Sri Lanka: Ministry of Health, National guidelines; 2012. 8. Trung DT, Thao LTT, Hien TT, Hung NT, Vinh NN, Hein PTD, Ching NT, Simmons C, Wills B: Liver involvement associated with dengue infection in adults in Vietnam. Am J Trop Med Hyg 2010, 83(4):774–780. 9. Kularatne SA, Gawarammana IB, Kumarasiri PR: Epidemiology, clinical features, laboratory investigations and early diagnosis of dengue fever in adults: a descriptive study in Sri Lanka. Southeast Asian J Trop Med Public Health 2005, 36:686–692. 10. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV: Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992, 30:545–551. 11. Larreal Y, Valero N, Estevez J, Reyes I, Maldonado M, Espina LM, Arias J, Melean E, Ariez G, Atencio R: Hepatic alterations in patients with dengue. Invest Clin 2005, 46(2):169–178. 12. Kuo CH, Tai DI, Chang-Chien CS, Lan CK, Chiou SS, Liaw YF: Liver biochemical tests and dengue fever. Am J Trop Med Hyg 1992, 47(3):265–270. 13. Nguyen TL, Nguyen T, Tieu NT: The impact of dengue hemorrhagic fever on liver function. Res Virol 1997, 148:273–277. 14. Huerre MR, Lan NT, Marianneau P, Hue NB, Khun H, Hung NT, Khen NT, Drouet MT, Huong VT, Ha DQ, Buisson Y, Deubel V: Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children. Virchows Arch 2001, 438:107–115. 15. Nuovo GJ: Histologic distribution of hepatitis A, B, C, D, E and G with the concomitant cytokine response in liver tissues. Diagn Mol Pathol 1998, 7:267–275. 16. Napoli J, Bishop GA, McGuinness PH, Painter DM, McCaughan GW: Progressive liver injury in chronic hepatitis C infection correlates with increased intrahepatic expression of Th1-associated cytokines. Hepatology 1996, 24:759–765. 17. Seneviratne SL, Malavige GN, de Silva HJ: Pathogenesis of liver involvement during dengueviral infections. Trans R Soc Trop Med Hyg 2006, 100:608–614. 18. Stalin V, Nayyar I, Anemon PP G, Shyam K: Fatal fulminant hepatic failure in a diabetic with primary dengue. J Trop Med 2010, 2010:413561. 19. Chaturvedi UC, Agarwal R, Elbishbishi EA, Mustafa AS: Cytokine cascade in dengue hemorrhagic fever: implications for pathogenesis. FEMS Immunol Med Microbiol 2000, 28(3):183–188. 20. Khongphatthanayothin A, Lertsapcharoen P, Supachokchaiwattana P, Satupan P, Thongchaiprasit K, Poovorawan Y: Hepatosplanchnic circulatory dysfunction in acute hepatic infection: the case of dengue hemorrhagic fever. Shock 2005, 24(5):407–411. 21. Khongphatthanayathin A, Mahayosnond A, Yong P: Possible cause of liver failure in patient with dengue shock syndrome. Emerg Infect Dis J 2013, 19(7):1161–1163. 22. Sirisena PDNN, Noordeen F: Evolution of dengue in Sri Lanka-changes in virus, vector and climate. Int J Infect Dis 2014, 19:6–12. 23. Fabre A, Couvelard A, Degott C, Lagorce-Pages C, Bruneel F, Bouvet E, Vachon F: Dengue virus induced hepatitis with chronic calcific changes. Gut 2001, 49(6):864–865. 24. Mourao MP, Lacerda MV, Bastos Mde S, Albuquerque BC, Alecrim WD: Dengue hemorrhagic fever and acute hepatitis: a case report. Braz J Infect Dis 2004, 8(6):461–464. 25. Rosen L, Khin MM UT: Recovery of virus from the liver of children with fatal dengue: reflections on the pathogenesis of the disease and its possible analogy with that of yellow fever. Res Virol 1989, 140(4):351–360. Kularatne et al. BMC Infectious Diseases 2014, 14:141 http://www.biomedcentral.com/1471-2334/14/141 Page 10 of 10 26. Morier L, Kouri G, Guzman G, Soler M: Antibody-dependent enhancement of dengue 2 virus in people of white descent in Cuba. Lancet 1987, 1(8540):1028–1029. 27. Trewby PN, Williams R: Pathophysiology of hypotension in patients with fulminant hepatic failure. Gut 1977, 18:1021–1026. 28. Rigau-Perez JG, Clark GG, Gubler DJ, Reiter P, Sanders EJ, Vorndam AV: Dengue and dengue haemorrhagic fever. Lancet 1998, 352(9132):971–977. 29. Poovorawan Y, Hutagalung Y, Chongsrisawat V, Boudville I, Bock HL: Dengue virus infection: a major cause of acute hepatic failure in Thai children. Ann Trop Paediatr 2006, 26(1):17–23. 30. Kumarasena RS, Senanayake M, Sivaraman K, de Silva A, Dassanayake AS, Premaratna R, Wijesiriwardena B, de Silva HJ: Intravenous N-acetylcysteine in dengue-associated acute liver failure. Hepatol Int 2010, 4(2):533–534. doi:10.1186/1471-2334-14-141 Cite this article as: Kularatne et al.: Extensive haemorrhagic necrosis of liver is an unpredictable fatal complication in dengue infection: a postmortem study. BMC Infectious Diseases 2014 14:141. 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SLANG ANTHROPONYMS IN THE UZBEK LANGUAGE
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EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center UIF = 8.1 | SJIF = 5.685 www.in-academy.uz SLANG ANTHROPONYMS IN THE UZBEK LANGUAGE Nazarova Nigora Djahangirovna Master student of the UzSWLU https://www.doi.org/10.5281/zenodo.7964940 SLANG ARTICLE INFO Received: 15th May 2023 Accepted: 23th May 2023 Online: 24th May 2023 KEY WORDS Slang, anthroponyms, names, associative field, motivation. EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center UIF = 8.1 | SJIF = 5.685 www.in-academy.uz Innovative Academy Research Support Center Innovative Academy Research Support Center UIF = 8.1 | SJIF = 5.685 www.in-academy.uz y pp | SJIF = 5.685 www.in-academy.uz Синмоқ- the original meaning - to break. Синдираман- Break or beat - used as a threat. Телефонингни бермасанг мен сени синдираман (If you don’t give me a cell, phone, I’ll break you). Урғочи- A very rude word. I think it's something between a bitch and a hen. The original meaning is female. Example: -Урғочинг борми? (Do you have a girlfriend?) Ўзича - generally speaking, means "in one's own way". But in slang, this word becomes a noun and is used to refer to an arrogant person who puts himself above other people. Very often used in relation to girls, they say she refused me, here it is! What does she think of herself!- Ўзича-е, ким деб ҳисоблайди, ўзини?! The language of communication between merchants and the relationship of individual social groups used to hide one's identity from others among small social groups. Definitions of this nature, similar in content, are also given in the works of E.D.Suleimenova, Yu.S. Maslova, N.V. Vasilyeva, V.I. Belikov. In general, it is obvious that jargons and slang in the Uzbek language are in many cases used in Russian originally. In the encyclopedia of the Uzbek language at the moment there are 160 words associated with theft in the youth speech, 85 words and phrases associated with deceit, concealment [1, 140]. It is known that slangs by their nature are words that are used among people of a narrow circle, united by common interests, occupations or position in society, and which do not always form a low style. For example, sometimes in newspapers and magazines there is often information related to the activities of police officers, such as the operation “Burgut”, “operation of the second direction”. Of course, these are also not understandable phenomena, and some slang, after completing their task, obviously ceases to exist in the language. So, for example, the names of military equipment or geographical names, after they become known and understood by the whole youth, cease to exist as slang or jargon. Some words "die" very quickly and are replaced by new ones; others live for centuries and sometimes get into the literary language from slang. ABSTRACT Slang may be used by members of a particular profession, such as musicians, surgeons, soldiers, or sailors, but it doesn't really have anything to do with reputation or rank. This suggests that slang is a unit that is simpler to comprehend than jargon. Jargon is a term used in a profession whose meaning can only be understood by those who work in that industry. Unlike slang and jargon, which are made up of randomly chosen mutated components of one or more natural languages of a very small and restricted professional or social group, argot is a special criminal language. Slang is often replenished with vocabulary borrowed from other professional groups. Unfortunately, there are a lot of words included in the student slang from the lexicon of drug addicts, which in turn partly indicates that this particular segment of the population is a behavioral guide for a certain part of the students. In addition, about 30% of the words in student slang are taboo vocabulary related to sex. Uzbek anthroponymy is based on modern scientific methods studies devoted to learning began to appear in the 60s of the last century. An anthroponym is not just a word that distinguishes an individual from among similar ones. The concepts fixed in anthroponyms are a reflection of the features of objects, properties and relations of reality. The history of names is closely connected with the history, culture, ideology of the society in which they are created. Personal proper names have a national flavor to a greater extent than common nouns. That is why anthroponyms are so actively used in proverbs, sayings, literary texts (the so-called "speaking" names and surnames), in criminal jargon and in modern youth slang. The main functions of the anthroponym are traditionally considered identification, nominative and differentiating, however, in slang, its secondary functions are activated: social, emotional, address, expressive, aesthetic and stylistic. Қотиб қолмоқ – to be drunk. The usual meaning of the word қотиб қолмоқ is to freeze, stiffen. This word does not need a direct addition and is used throughout Uzbekistan. Example: – Қотиб қолдингми? (Are you drunk?) Example: – Қотиб қолдингми? (Are you drunk?) Volume 3 Issue 5, Part 3 May 2023 ISSN 2181-2020 Volume 3 Issue 5, Part 3 May 2023 ISSN 2181-2020 Page 186 Innovative Academy Research Support Center UIF = 8.1 | SJIF = 5.685 www.in-academy.u Innovative Academy Research Support Center UIF = 8.1 | SJIF = 5.685 www.in-academy.uz “Aбитур” (applicant). The Russian word entrant has been transformed into a diminutive абитур “Aбитур” (applicant). The Russian word entrant has been transformed into a diminutive абитур. “Мусор- ахлат- rubbish - ballyhoo, fiddle-de-dee” are rare case when all three languages describe a word according to the same principle, a set of meaningless letters and sounds that mean nonsense, nonsense. “Тўнка” (greedy person). In many stories and works, greedy people are described as plain and fat, obviously implying that greedy people eat a lot and do not share with anyone. The Kazakh version was no exception, karyn means belly, bai means a rich person. “Тўнка” (greedy person). In many stories and works, greedy people are described as plain and fat, obviously implying that greedy people eat a lot and do not share with anyone. The Kazakh version was no exception, karyn means belly, bai means a rich person. “Онасининг қизи” (cowardly, spineless). The literal translation of this phrase can be confusing. It in this case is translated as inert, weak character, and sheshe - mother. “Онасининг қизи” (cowardly, spineless). The literal translation of this phrase can be confusing. It in this case is translated as inert, weak character, and sheshe - mother. “Шайтон сув” (alcohol, vodka). The word “шайтон” is an adjective meaning naughty, mischievous. “Сув” is translated as water, liquid. Vodka, like all alcohol, has a detrimental effect on a person, changes his character, and makes him intractable or aggressive. Slangisms of the Uzbek language are formed mainly on the basis of a noun; other parts of speech (adjectives, verbs, etc.) are not activated in the associative base of the carrier of Uzbek youth slang due to a rather limited vocabulary of the literary norm of the language for most slang users [3, 17]. During the survey, we found that the vast majority of slang lexemes belong to the semantic field "Education" (concepts and ideas of school life, the realities of the school environment). The core of education slang, used for many decades, consists of such words as: Камчатка, Чукотка (back desks), шпора (cheat sheet), доза (задание на дом, homework), etc. Our survey showed that the following slang lexemes are most regularly repeated in the speech of students in recent years: tin, cool, cool, cool, hangout, count up, nishtyak, chip, dude, dumb, sucks, fly away, stir up. EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center UIF = 8.1 | SJIF = 5.685 www.in-academy.uz The use of non-literary elements in speech - vulgarisms, swear words, jargon, slang - is typical for students of almost any era. However, the activity of non-normative word usage has never been as high as in the communication of modern schoolchildren, and the problem of speech purity has never been raised as acutely as in the last 5-10 years. What specific invective (cf. invectivaoratio "rough speech") and slang lexemes are most characteristic of the speech of students in recent years? If the set of traditionally used invectives is relatively stable and mainly limited by offensive assessments of the mental abilities and external behavior of the addressee (fool, idiot, psycho, etc.), then school/university and - more broadly - youth slang is characterized by much more significant diversity and variability [2, 20]. Unfortunately, it was not possible for us to provide a large number of examples of Kazakh slang. “Узун қулоқ” (rumors). Translating this slang literally, we get "long ear", which should mean that having "long ears" you can hear a lot of information. “Чурвақалар” (children). It is worth noting that this slang is quite outdated for youth slang, and now it is mainly used by older people in their speech. Volume 3 Issue 5, Part 3 May 2023 ISSN 2181-2020 Volume 3 Issue 5, Part 3 May 2023 Page 187 EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center References: 1. Азиатские языки. Энциклопедия. Алматы: 1998.- 178 с. 2. Никитина Т.Г. Так говорит молодежь. Словарь молодежного сленга. –М.,1998. 3. Юганов И., Юганова Ф. Русский жаргон 60–90-х годов: Опыт словаря. –М.,1994. Innovative Academy Research Support Center UIF = 8.1 | SJIF = 5.685 www.in-academy.u To the question "Why do you use slang in your speech?" the following responses were given (responses are in descending order). 1. It's fashionable. 1. It's fashionable. 2. Not to be different from others. 3. With the help of such words, you can emotionally convey what you feel. 4. One word says it all. 5. With the help of slang, you can transfer information to each other so that others (more often adults) do not understand what is being said. 5. With the help of slang, you can transfer information to each other so that others (more often adults) do not understand what is being said. 6. In our circle, this is customary. 6. In our circle, this is customary. Many students in Uzbekistan use English words as slang. More often there are such words as: тичэ (teacher), фэйс (face), сюпер (super), гирл (girl), etc. Uzbek words are not used as often as English ones: ҳуш келганский (welcome), бўғирсоқ (fat person), чёткий from Russian word (cool). Comparing the slang and idiomatic expressions of the English and Russian languages, we came to the conclusion that in both languages slang is very common and in each of them it makes up about 15-20% of the total vocabulary of an ordinary person. Having drawn analogies with the Uzbek language, we came to the conclusion that Uzbek slang is much different from both Russian and English. If Russian, like English, is full of slang expressions, e 5, Part 3 May 2023 ISSN 2181-2020 Page 188 Volume 3 Issue 5, Part 3 May 2023 ISSN 2181-2020 Page 188 EURASIAN JOURNAL OF ACADEMIC RESEARCH Innovative Academy Research Support Center Innovative Academy Research Support Center UIF = 8.1 | SJIF = 5.685 www.in-academy.uz then, in turn, there is little slang in the Uzbek language, and most of them are borrowed from i hb i l then, in turn, there is little slang in the Uzbek language, and most of them are borrowed from neighboring languages. According to experts, this situation can be explained by two reasons. One of the explanations is that the Uzbek language itself is more colloquial, that is, it does not abound in complex phrases and scientific terms of Uzbek origin, as, indeed, in all languages, since scientific terms are almost always international. Another reason for the small volume of slang is the very blurred boundaries in the Uzbek language between slang and ordinary or literary language. In part, this is quite understandable, because the Uzbek language, although it is old, most of the time existed in oral form, due to historical circumstances. From a linguistic point of view, relatively recently formed, with a mass of borrowed words, Uzbek writing is undergoing constant changes, acquiring a mass of new words. Due to all of the above reasons, it is very difficult to trace and identify slang and jargon. After analyzing the above circumstances, we came to the conclusion that slang, professional jargon and slang are present mainly in the "old" languages with a relatively long-established written language. Volume 3 Issue 5, Part 3 May 2023 ISSN 2181-2020 Volume 3 Issue 5, Part 3 May 2023 Page 189
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Extroverted financialization: how US finance shapes European banking
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Citation for published version (APA): Beck, M. (2022). Extroverted financialization: how US finance shapes European banking. REVIEW OF INTERNATIONAL POLITICAL ECONOMY, 29(5), 1723-1745. https://doi.org/10.1080/09692290.2021.1949375 Citation for published version (APA): Beck, M. (2022). Extroverted financialization: how US finance shapes European banking. REVIEW OF INTERNATIONAL POLITICAL ECONOMY, 29(5), 1723-1745. https://doi.org/10.1080/09692290.2021.1949375 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. Review of International Political Economy ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/rrip20 Extroverted financialization: how US finance shapes European banking Mareike Beck General rights General rights Copyright and moral rights for the publications made accessible in the Research Portal are retained by the authors and owners and it is a condition of accessing publications that users recognize and abide by the legal requirements associ ral rights for the publications made accessible in the Research Portal are retained by the authors and/or other copyright condition of accessing publications that users recognize and abide by the legal requirements associated with these right •Users may download and print one copy of any publication from the Research Portal for the purpose of private study or research. •You may not further distribute the material or use it for any profit-making activity or commercial gain •You may freely distribute the URL identifying the publication in the Research Portal •Users may download and print one copy of any publication from the Research Portal for the purpose of private study •You may not further distribute the material or use it for any profit-making activity or commercial gain y y p g y •You may freely distribute the URL identifying the publication in the Research Portal Take down policy If you believe that this document breaches copyright please contact librarypure@kcl.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. p y If you believe that this document breaches copyright please contact librarypure@kcl.ac.uk providing details, and we w the work immediately and investigate your claim. Download date: 24. Oct. 2024 Review of International Political Economy Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=rrip20 CONTACT Mareike Beck Mareike.beck@kcl.ac.uk Department of European and International Studies, King’s College London, London WC2R 2LS, UK.  2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 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. Mareike Beck To cite this article: Mareike Beck (2022) Extroverted financialization: how US finance shapes European banking, Review of International Political Economy, 29:5, 1723-1745, DOI: 10.1080/09692290.2021.1949375 To link to this article: https://doi.org/10.1080/09692290.2021.1949375 © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 09 Jul 2021. Submit your article to this journal Article views: 2233 View related articles View Crossmark data Citing articles: 5 View citing articles REVIEW OF INTERNATIONAL POLITICAL ECONOMY 2022, VOL. 29, NO. 5, 1723–1745 h //d / / KEYWORDS KEYWORDS Liability management; money markets; market-based banking; banks; extroverted financialization; comparative political economy Mareike Beck Mareike Beck Mareike Beck Department of European and International Studies, King’s College London, London, UK ABSTRACT This paper reconceptualizes the impact of US finance on European banking as a process of ‘extroverted financialisation’. This impact is commonly associated with the rise of ‘market-based banking’ (MBB). While MBB exposes how commercial banking has been deeply transformed by disintermediation and borrowing from wholesale markets, the concept struggles to capture the distinct imperatives of this process, and its uneven nature. By contrast, the concept of extroverted financializa- tion captures the problems European banks have faced while adapting to US-led financialization. More specifically, the concept portrays the financialization of European banking as an outcome of new funding practices, called liability manage- ment (LM), developed in US money markets from the 1960s onwards. I show how this put pressures on European lenders because it allowed US banks to leverage extensively. To catch up, European banks had to improve their access to liquid USD, which forced them to find a way into the Eurodollar markets and into the US money markets. To operate in these markets, they had to gradually implement the practices of LM. This process of extroversion made their own banking models highly fragile and dependent on US money market funding. Despite adopting LM, they could not reduce their structural disadvantages vis-a-vis US banks. Extroverted financialization: how US finance shapes European banking Mareike Beck  2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group ke Beck Mareike.beck@kcl.ac.uk Department of European and International Studies, ondon, London WC2R 2LS, UK. Introduction The 2008 financial crisis exposed how damaging global banking can be. International Political Economy (IPE) scholars previously conceptualized this as a distinct US financial problem because of its tendency towards short-term specula- tion. Recently, however, IPE and finance scholars started to analyze the hitherto neglected role of large European banks in causing the financial collapse (Bayoumi, 2017; Hardie & Thompson, 2020; McCauley, 2018; Tooze, 2018a). They demon- strated that large European banks have engaged in a cross-country trade, taking trillions of USD out of US money markets only to invest them back into the US. This was a crucial mechanism that produced the mortgage-backed security (MBS) M. BECK 1724 M. BECK bubble in the 2000s and its global meltdown (Fligstein & Habinek, 2014; McGuire & von Peter, 2009). Studies revealed that, contrary to common belief, European banks were important actors of financialization (Hardie et al., 2013)1. The European banks’ involvement in the global financial crisis (GFC) reflects a profound change in their practices because European political economies were pre- viously considered to be ‘less amenable to financial logics and markets’ (Schelkle & Bohle, 2020, p. 1). Prior to the 2008 financial crisis, European banks engaged in selling long-term financial products and industrial support as opposed to short- term speculative practices. For many, this transformation demonstrated that bank- ing in Europe has become more like US finance (Engelen, 2008; Maxfield et al., 2017)2, which most closely resembles the ideal-typical decentralized ‘free market’ (cf. Zysman, 1983). However, given the banks’ previously different, indeed oppos- ing, financial models, ‘the question that needs to be answered is how the Europeans were sucked into that boom as deeply as they were’ (Tooze, 2018b). A conceptual framework commonly used to understand these changes is mar- ket-based banking (MBB) (Hardie et al., 2013). Hardie and colleagues’ seminal account convincingly demonstrates that since the 2000s, European banks have changed their business models by marketizing their own assets and liabilities. That is to say, short-term financial practices significantly replaced long-term loans and customer deposits. This made banks’ balance sheets increasingly fragile because they could not withstand market shocks anymore. As a result, formerly strong European banks ‘have undermined their financial power in lending to NFCs’ (non- financial corporations) (Hardie et al., 2013, p. 700). p p From the perspective of MBB, competitive market forces have driven European banks to engage with short-term financial innovations. Introduction p p The second feature is the need for European banks to raise large volumes of liquid USD to catch up to US banks. As the European’s deposits were in CHF, FF or DM, and later in EUR, this was initially a structural constraint as European banks lacked access to USD deposits. This has therefore been a key driver for them to direct their strategies towards finding new sources of USD. As I demonstrate, this forced European banks to find a way into foreign USD markets. At first, they experimented with joint European banking strategies in the 1960s. However, to compete with the flexibility of LM, the European banks had to change their practi- ces and improve their individual access to USD, first in the Eurodollar markets in the 1970s, and later in the US. The third feature is the importance of US money markets. This is because they uniquely accommodate the liquidity requirements of LM as they provide a large and deep pool of USD. As I show, the European banks had to find ways to institu- tionalize their USD funding networks into US money markets from the 1980s onwards to be able to keep pace with US banks. To operate in those markets, the Europeans had to initiate larger transformations of their business models towards the practices of LM, which subsequently allowed them to leverage large volumes of USD in the 2000s. The fourth feature is the structural contradictions of financialized banking. The process of extroversion meant that European banks adopted business models that became highly dependent on US money markets. As a result, albeit unintentionally, European banks have contributed to institutionalizing global funding structures that have entrenched the original problem they tried to overcome: having to access large volumes of short-term USDs from an outside, disadvantaged position. I argue that these factors are key in framing the financialization of European banks as a unique process. The reliance on USD funding from foreign markets has continuously posed crucial, if evolving, disadvantages to European banks in con- trast to US banks. As I show, European banks initially lacked access to short-term USD funding. Subsequently, they had to adapt to a new way of banking – LM – that the longer-term logics of their traditional banking practices and the institu- tional frameworks of their home markets did not support. Introduction I argue, however, that it is questionable if the concept of MBB allows us to fully understand the pressures that US finance has posed for European banks. The emphasis on generic market forces as core imperative has prevented an analysis of concrete financial practices that the processes of financialization are rooted in, and the constraints and power relations they represent. As a result, the concept of MBB leaves us with a paradoxical inter- pretation of the role of European banks during financialization: they have become central actors but have lost the power to navigate financial markets. To understand the role of internationally active European banks, I suggest we exam- ine the uneven nature of the rise of US finance, and the constraints it exerts on the European lenders. From this perspective, the distinct characteristic of the transformation of European banking is not its market-based but its extroverted nature. With extrover- sion, I refer to the systematic attempts of European banks to route themselves into for- eign markets and to use a foreign currency. More specifically, the European banks’ paths of financialization have been shaped by trying to capture USD from the 1960s onwards and by subsequently attempting to anchor themselves in US money markets. This means that European banks have had to manage financial practices that were originally devel- oped for a different context. US banks, by contrast, have enjoyed the institutional privi- leges of their home markets. As a result, European banks have faced distinct constraints and disadvantages as they transitioned towards a US-form of finance. To capture this process, this paper develops the concept of extroverted financial- ization (EF). Relying on secondary sources and publicly available data, the concept combines four factors that represent novel imperatives for European banks during US-led financialization. The first feature is the centrality of liability management REVIEW OF INTERNATIONAL POLITICAL ECONOMY 1725 (LM) for the rise of US finance. LM is a new funding practice developed in US money markets from the 1960s onwards. It allowed financial actors to raise and manage high volumes of capital by dynamically buying and selling short-term securities in US money markets, as opposed to relying on deposits which accumu- late slowly and steadily (Beck & Knafo, 2020; Dutta, 2020). The rise of LM enabled US banks to outcompete European banks on international credit markets. Market-based banking and the imperatives of financialization The role of European banks during financialization has become a central issue in the comparative political economy (CPE) literature since the 2008 financial melt- down (Bell & Hindmoor, 2015; Braun & Deeg, 2020; Deeg, 2012; Hardie et al., 2013; Hardie & Thompson, 2020; Thompson, 2015). While European banks were long thought to operate outside US financial logics, the unprecedented losses of European banks during the GFC showed their deep and devastating connections with US financial markets. To account for this new type of banking that emerged during financialization, Hardie et al. (2013) have developed the influential concept of market-based banking (MBB). g MBB is a significant conceptual innovation because CPE scholars had relied on the traditional assumption that European banks would assume long-term commit- ment to the productive industry (‘patient capital’), instead of trading in short-term financial markets (Hardie et al., 2013; Schelkle & Bohle, 2020). According to this literature, European banks enjoyed institutional advantages that ensured they had a central role in lending to the corporate sector. Firstly, banks could rely on retail deposits for funding which accumulated steadily, if slowly, because retail customers tend to be loyal. Secondly, banks enjoyed insider knowledge of their corporate cli- ents because they held personal relationships with corporations through cross- shareholdings and seats on supervisory boards. This allowed banks to monitor the profitability of their long-term loans, which was important because harvesting of market data from large stock exchanges or rating agencies was not common prac- tice. Thirdly, and as a result, this constrained competition from other financial actors who did not have similar access or preferential treatment from regulators. European banks thus enjoyed an oligopolistic status as lenders. By contrast, US financial markets are seen as representing most closely the ideal-typical understanding of ‘free markets’. By that view, US markets have a very competitive nature as many financial agents compete over resources, rather than having a few large banks that monopolize corporate lending. As a result, decentral- ized market forces ensure that financial actors cannot impose their own conditions of lending because they face price constraints from competitors. This institutional comparison is most famously articulated by Zysman’s (1983) typology of bank- based and market-based financial systems. With the concept of MBB, Hardie et al. (2013) present the first framework that moves beyond this oppositional conceptualization of banks and financial markets. Introduction By contrast, US money markets, which did support LM, have had significant regulatory restrictions for European banks as late as the 1990s. In addition, European banks had to buy costly new institutions and personnel to transform their business models towards US money market funding. As a result, European banks became less resilient to market downturns, suffer from additional difficulties and costs to swap GBP or EUR into USD, and are dependent on money markets that institutionally privilege US banks. In short, while European banks have become important actors of financialization through their strategies of extroversion, their own success in adopting LM has not reduced their structural disadvantages vis-a-vis US banks. I develop the concept of extroverted financialization in two steps. Firstly, I crit- ically interrogate MBB. I demonstrate that the emphasis on markets as primary 1726 M. BECK M. BECK 1726 M. BECK M. BECK 1726 identifier for financialized banking comes at the expense of accounting for the pre- cise role of large European banks during financialization, and its uneven nature. Secondly, I detail the four features of the concept of EF. The conclusion empha- sizes the analytical and political implications for analyzing and responding to proc- esses of financialization. Market-based banking and the imperatives of financialization While there seems to be a widespread consensus that non-US banks have transi- tioned, at least somewhat, towards a market-based US form, scholars have struggled to conceptualize this transformation outside arguments of convergence or indefinite notions of hybrid financial systems (Deeg, 2012; L€utz, 2004; R€oper, 2018; Vitols, 2004). After all, all financial systems exhibit diverse forms of market-based finance and bank lending (Sissoko, 2017). As Hardie et al. argue (2013), scholars have been REVIEW OF INTERNATIONAL POLITICAL ECONOMY 1727 too reliant on this theoretical dichotomy to appreciate recent changes in banking. They show that since the 2000s, European banks have increasingly traded in whole- sale markets. In this way, MBB has realigned Zysman’s original typology with the fact that European banks operate in financial markets rather than in opposition to their logics. The significance of MBB is that any conception about contemporary banking should consider market pressures directly on the banks themselves. The previous long-term orientation of their banking models that was able to protect their cor- porate lenders from financial vulnerability has significantly shortened. The short- term nature of banks’ balance sheets means that banks struggle to manage the credit risk for themselves and corporations. As a result, banks have ‘undermined their financial power in lending’ (Hardie et al., 2013, p. 700). It is, however, questionable whether the concept of MBB allows us to fully understand the pressures of US-led financialization. The emphasis on generic market forces as core imperative has prevented an analysis of the concrete finan- cial practices that the processes of financialization are rooted in, and the con- straints they represent. To be sure, Hardie and colleagues demonstrate that financial actors are subject to different kinds of pressures based on maturity lengths or the depths of financial markets (Hardie et al., 2013; Hardie & Maxfield, 2013). However, they leave us with a paradoxical interpretation of the role and power of European banks: they have become central actors during finan- cialization but have lost the power to navigate financial markets ( Hardie et al., 2013; cf Hardie & Thompson, 2020). I contend that this puzzle could be solved by incorporating the uneven nature of financialization. If market dynamics are central to what banks do (Hardie & Howarth, 2013), and if markets are political economic institutions, we need a more precise understanding of the power relations within them. Market-based banking and the imperatives of financialization For if MBB diminishes the power of banks, why would European banks attempt to route themselves into US markets? This was a journey that many commentators would evaluate as less than successful (Crow, 2019; Goodhart & Schoenmaker, 2016; Noonan, 2019). By contrast, US banks have mostly surged in power, leverage and profits through the rise of financial markets (Sgambati, 2019). I propose the concept of extroverted financialization (EF) to foreground the power imbalances between US and European banks as a core driver of financiali- zation. It does so by focusing on the novel imperatives and constraints that the rise of US finance has imposed on European banks. EF situates the transform- ation of large European banks within a longer-term trajectory of their inter- national practices that incrementally institutionalized their funding networks into US money markets. This realigns the debate on the US Americanization of finance with the importance of European banks’ own role in producing USD funding structures and with the specific practices that have encouraged and com- pelled European banks to do so. My methodological approach rests on historicizing the precise competitive pres- sures emanating from US finance which are seen as a key aspect of European banks’ transformations (Bell & Hindmoor, 2015; Erturk & Solari, 2007; Konings, 2008). Changes in agents’ practices and strategies generally emerge from historically and contextually specific imperatives (Wood, 2017). Prioritizing the precise context will help to differentiate between some of the most dominant financial actors. This M. BECK 1728 M. BECK follows Knafo’s (2010, 2013b, 2017) methodology of radical historicism that aims to make visible how the role and changing practices of key agents produce large scale outcomes. These practices represent the attempts of actors to act upon and, indeed, (re)produce structural transformations, albeit not always successfully or as intended. The four factors of EF described below do not represent the only driving forces of the international transformations of European banks. The imperatives I am fore- grounding here did not fully determine but have rather constrained the extroverted strategies of large European banks. Within this framework, banks have reacted in various ways. There are differences, for example, in the specific institutional frame- works that have influenced how European banks respond to these pressures. French banks were more closely aligned with US networks than German banks in the 1960s and 1970s (Feiertag, 2005). Market-based banking and the imperatives of financialization The difference between British and continen- tal European banks is perhaps most obvious as London hosts a dominant financial center closely aligned with US finance. In this sense, I somewhat neglect the com- plex histories of individual banks and instead highlight the shared imperatives that guided their extroverted paths. Extroverted financialization: Four factors that shaped European banking This section makes the case to understand European financialization as a process of extroverted financialization (EF). Extroversion describes the process in which European banks left their own domestic context to anchor themselves in distant markets with a foreign currency. More specifically, they devised strategies of inter- nationalization to manage the novel pressures of LM from the 1960s onwards. Thus, as much as US banks forced their way into European finance (Konings, 2008), European banks actively contributed to globalizing US financial institutions in order to extract USDs. As I show below by historicizing four distinctive financial pressures, these efforts to operate in a different context posed differentiated con- straints to European banks in contrast to US banks, which demonstrates the uneven nature of USD markets. REVIEW OF INTERNATIONAL POLITICAL ECONOMY 1729 Historically, LM developed out of pragmatic responses of New York banks to a crisis of funding. Traditionally, in the US and in Europe, banks would fund their investments with customer deposits. Deposits were a convenient funding strategy for the banks because they accumulated steadily, if slowly, and were a cheap resource. They were considered as secure because customers tend to be loyal. This way of funding allowed the lenders to put their strategic emphasis on the accumulation of assets. In the 1960s, however, US corporations started to invest their surplus cash on the money markets, attracted by higher returns. An interest cap on deposits due to Regulation Q meant that banks could not increase interest rates to get their deposits back. This meant that banks could no longer fund themselves through cheap and secure customer deposits and they had to go to the money markets themselves to get funding (Cerpa Vielma et al., 2019; Konings, 2007; Stigum, 1990). The money market is a wholesale and retail market for very short-term, highly liquid IOUs. At the wholesale level, financial institutions trade large volumes of short-term debt securities, whilst at the retail level, individual customers invest into money market funds. Billions of USDs in Federal funds and Eurodollars are traded every day and the market comprises several different though interrelated segments and instruments, involving Treasury bills, federal agency securities, Certificates of Deposits (CDs), and repurchase agreements (repos)34. It involves short-term secur- ities, ranging from overnight to several months, through which banks fund them- selves and manage their liquidity. The rise of institutional investors and the corporate surplus cash meant that money markets grew into a deep pool of capital in the US and then evolved to be a central source of funding within global finance. These days, money market funding is central for capital market lending. Traditionally, however, banks would use customer deposits as their main source of funding. Money markets represented a back up to overcome short-term liquidity gaps. Since the 1960s, this dynamic has changed (Beck & Knafo, 2020). CDs and other money markets securities allowed bank credit to excel well beyond what would normally be possible with a traditional deposit base (Minsky, 1986). CDs were a crucial innovation that subsequently travelled to the Euromarkets where they started to affect the European banks (Schenk, 2002; Stigum, 1990). The rise of LM affected financial agents in several ways. The rise of liability management My starting point is the rise of liability management (LM). A hypothesis that Samuel Knafo and I developed is that LM represents a decisive innovation of finan- cialization (Beck & Knafo, 2020). It is a new funding strategy for banks connected to the rise of US money markets from the 1960s onwards that allowed US banks to leverage unprecedented amounts (Battilossi, 2010; Dutta, 2020; Knafo, 2013a; Konings, 2007). Financial actors could use LM to finance themselves much more dynamically by buying and selling short-term securities in money markets. The dif- ference between LM and MBB is that specifying LM as a core practice foregrounds the US funding techniques that would redefine models of commercial banking. This change on the liability side predates, and indeed largely influenced, the shift of banks towards ‘originate-to-distribute’ models in the 1980s. REVIEW OF INTERNATIONAL POLITICAL ECONOMY REVIEW OF INTERNATIONAL POLITICAL ECONOMY It allowed banks to transcend their previous funding constraints and fund much bigger projects. At the same time, this dynamic also created new pressures on banks. While money mar- kets allowed financial houses to raise funds very quickly, money market securities carried higher interests than deposits. These short-term securities can be sold easily by investors if they need cash and as a result, it is more difficult to manage fund- ing under these conditions. Funding became more expensive and insecure so that banks started to worry about it on an everyday level. They had to find new ways to manage that volatility and risk, and thus came to focus on the management of liabilities as a central strategy5. This new way of funding led banks to question a core function of their own business models, namely the validity of holding a loan on their own balance sheets. Using costly money market securities as funding meant that any asset became much more expensive to hold. Taking into consideration these increased costs exposed that the yields of corporate loans could not cover them anymore. In response, lenders started to sell loans so that they would use less capital on the bal- ance sheet. This transformation, according to Sanford (1996), chairman of Bankers M. BECK 1730 M. BECK Trust, ‘would affect the very foundations upon which commercial banking - at least wholesale commercial banking – relied’. These strategies spread to other banks as they needed to keep up with the rapid increase in financing power in the 1970s and 1980s (Cerpa Vielma et al., 2019). Lenders started to trade securities with each other to optimize their loan portfolios and keep the costs of capital to a minimum. Selling the loans would allow them to go on underwriting more. This innovation thus propelled a huge increase in the volume of financial transactions and the speed that US banks could issue loans (Battilossi, 2000, 2010). Securitization, the construction of tradeable securities out of loans and future income streams, has been identified as the ‘frontier of financial expansion’ (Bryan & Rafferty, 2014, p. 895). These financial innovations were cru- cial in allowing banks to break ‘the limits of financial production’ (Wigan, 2010, p. 111; cf. Nesvetailova, 2014). REVIEW OF INTERNATIONAL POLITICAL ECONOMY This brief history of the development of LM suggests that the rapid increase in securitization was an outcome of the pressures of the revolution in funding strategies in the 1960s. This reconceptualization of US finance is important to show the power it gave US banks and what European banks had to adjust to. Financial practices are devel- oped within and for a specific context, even if they are sometimes conceived of as universally adoptable. The short-term nature of LM evolved from the context of US markets and was difficult to develop elsewhere. While MBB emphasizes how European banks had to transform their business models in response to rising secur- ities markets in the 1990s, and particularly the 2000s, I zoom in on the precise technology that allowed US banks to increase their financial power to unprece- dented levels from the 1960s onwards. These distinct US imperatives and institu- tional foundations that shaped the Americanization of European finance gave US banks institutional advantages in contrast to European banks. As the next feature demonstrates, LM put new pressures onto European banks to change their extro- verted strategies towards raising higher volumes of USD, a key feature demonstrat- ing the uneven nature of financialization. The need for (Euro-) dollars The second feature is that banks need to get USD as their central funding currency. Access to USD as a foreign currency is a distinct pressure for European banks. ‘Once we recognize the importance of this issue of dollar access for these European banks, analytically pairing any other economies’ banks with those of the US is mis- leading.’ (Thompson, 2016, p. 218). Or in the words of global head of FX strategy for Societe Generale, Kit Juckes: ‘There’s no other currency anyone wants to buy’ (Szalay et al., 2019). The importance of USD for issues of global banking is recog- nized (Aldasoro & Ehlers, 2018; Borio et al., 2017; Fender & McGuire, 2010), but its impact on the extroverted strategies of European banks is rarely conceptualized. Access to USD represents a distinct pressure for European banks in contrast to US banks because their access to USD funding was more restricted. European deposits – the ‘easy’ and stable source of funding – were in GBP, DM, CHF, FF, and later EUR but not in USD. This prompted European banks to engineer new ways to fund themselves. Many foreign banks are legally precluded from USD deposits insured by the Federal Deposit Insurance Corporation (FDIC) because they operate via a branch in the US, rather than a separately capitalized subsidiary REVIEW OF INTERNATIONAL POLITICAL ECONOMY 1731 (Ivashina et al., 2015). They have to source USD in different ways (Eren et al., 2020). The issue of access to USD arose historically in the 1960s when US banks started to rival their European counterparts on European markets, an assault the Europeans perceived as particularly painful (Battilossi & Cassis, 2002; Feiertag, 2005). With the help of LM, US banks were able to take over European corporate assets via the Euromarkets. This differential in USD funding power was a decisive turning point that motivated European banks to embrace the Eurodollar markets (and later the US wholesale markets from the 1970s onwards) to gain more USD to catch up to US banks. When US banks started to expand into Europe in the 1960s, continental lenders could barely continue as primary underwriters of new loan projects (Bussiere, 2005). European banks were outraged that they were reduced to mere contributors while US banks became lead managers for large syndicated loans. The need for (Euro-) dollars In order to defend their dominant position as lenders to ‘their’ corporations, European banks planned to build a common capital market and a network of stock exchanges. Joint German, Belgian, Dutch and Swiss investment funds were to increase foreign investment in all European bourses. In this way, they wanted to ensure their financial power in their own home markets and indeed, international issues on the German market, for example, increased in the mid-1960s (Feiertag, 2005). However, foreign investment in the continental bourses remained marginal and this joint initiative never fully materialized because French and German inter- ests diverged. The French commercial banks had closer ties to US financial institu- tions and continued to cooperate with them, rather than engaging in a joint counter attack (Bussiere, 2005). Having failed to build alternative international structures, the German and other European banks had no option but to establish themselves on the Euromarkets to recoup their assets. This is a first step in how the rise of LM started to impact European strategies and set in motion their paths towards a US-form of banking. Once the European banks embraced the Euromarkets, they were initially rela- tively successful. They built international currency hubs and financial networks for sustained capital market transactions in Luxembourg (D€orry, 2014). As banking on the Euromarkets represented many unknown challenges, European banks jointly founded syndicate and club banks to tackle the Euromarkets together so as to limit the risks and share expensive resources (R. Roberts, 2001; Ross, 1998). Their strength was to pool their Eurodollars and other foreign currencies to boost the volumes of their loans and exploit exchange rate margins. Each parent bank would provide capital to the syndicates which leveraged their limited USD resources. Some big European banks such as Deutsche Bank were able to become heavy- weights in the market (Burk, 1992) and even rival some of the US institutions (Battilossi, 2002). According to The Economist (1976), the consortium banks were ‘the most important banking development for a generation’ (quoted in Roberts, 2001, p. 38). By the mid-1970s, however, the clubs and syndicates were already ‘dinosaurs’ (Ross, 1998, p. 354) because of the innovations coming from the US. European banks had not changed any of their traditional funding strategies to improve their access to USD in a systematic way. The need for (Euro-) dollars Even though they pooled their capital, the con- sortiums represented a strategy of relying on traditional methods of credit creation. 1732 M. BEC 1732 1732 M. BECK M. BECK These methods became a problem in the 1970s and 1980s for three reasons. Firstly, and most importantly, the US banks were advancing their capacity to do LM. Being positioned in the Eurodollar markets and the US money markets, they were in a unique position to arbitrage between these markets to raise large volumes of USD to finance increasingly large investments (Stigum, 1990). They could easily decide on their investment first, and then use the money markets to finance their loans (Dutta, 2020). Secondly, as a consequence of their pooling strategies, consortium banks struggled with a particularly vulnerable balance sheet that juggled various foreign currencies and complicated maturity transformations. While all banks borrow short and lend long, the currency transformations made the USD fluctuations in the 1970s particularly difficult to negotiate. Consortium banks had to fund themselves to 81% on expensive inter-banking currency markets in comparison to 46% for US banks (Ross, 1998, p. 183). The parent banks resented their continuous injections of Eurodollars because they were expensive to obtain. Thirdly, the banks’ capacities to exploit their strengths in providing syndicated loans was increasingly limited for several reasons. While multinational corporations often funded themselves through syndicated loans in the 1970s, by the 1980s, they started to issue their own secur- ities, whilst US financiers learned how to do foreign currency. Banks such as Goldman Sachs eventually offered to swap USD commercial papers (CPs) into the local currency for US corporations (Stigum, 1990). Money market funds started to invest in foreign CPs in the 1980s (Baba et al., 2009). That way, US banks increas- ingly dominated the credit business that European banks previously had their advantages in. g As a result, European banks had to start changing their strategies of how they did international banking and they had to find novel ways to raise more USD. Lacking this monetary resource was a major structural disadvantage and a core imperative that prompted global banks such as BNP Paribas (and its predecessors), Credit Suisse and Deutsche Bank to innovate in order to leverage their limited USD. This uneven nature of financial globalization thus triggered the extroverted paths of European lenders to build individual financial networks in the Eurodollar markets. The need for (Euro-) dollars In the 1970s, they started to establish branches in London because it was the main hub of financial innovations coming from the US (cf. Konings, 2008) and it was experiencing an unparalleled credit revolution, pushed predominantly by US banks with the help of LM (Dutta, 2020). It is during this decade that European banks, and their banking clubs, shifted their focus on the Eurodollar markets from Luxembourg, as an international currency hub, to London where the US banks were operating. European banks needed to connect to these practices to operate on their own, even as the joint initiatives were still continuing to exist (Roberts, 2001; Ross, 1998). Thus, while the challenge of banking on global markets could initially be met by organizing into European syndicates, the challenge of LM could only be met by changing their banking practices to acquire more USD. After embracing the Eurodollar markets in general, this turn to London and US financial practices is the second step in following the international path under the pressures of USD funding. The point of this brief history is to account for why European banks started to build international funding networks on Eurodollar markets, a key offshore funding REVIEW OF INTERNATIONAL POLITICAL ECONOMY 1733 architecture with contradictory outcomes for the Europeans, as I will describe below. The practices of LM exerted very specific pressures on European banks that they could not respond to by continuing their traditional strategies. Instead, they had to restructure their strategies towards finding better ways to get USDs. This represents an uneven competition, as the capacity to raise large quantities of short- term USDs became a central aspect of power in banking. The second feature, the need to capture USD liabilities, thus starts to account for the structural disadvan- tages European financial institutions have in contrast to US banks – their balance sheets, funding strategies and institutional frameworks work in different currencies. It was the need to change their portfolios of liabilities that set-in motion their adjustments to the practices of LM. Why this took such a profound turn towards a distinct US-form of finance, including its money market dependency, is accounted for in the next section. Accessing US money markets They rest on a large pool of capital from big financial institutions and corporations, but also from the wider population (Konings, 2011; Krippner, 2011). US financial mar- kets have integrated households particularly closely, linking a wide variety of social concerns to those of financial markets. Retail customers provide a large pool of pri- vate capital, held for example by large institutional investors such as pension funds (Harmes, 1998) or housing finance (Montgomerie, 2009; A. Roberts, 2013). As a result, US banks had access to an abundance of capital they could use to manage their liquidity requirements. Thirdly, against the common understanding of US finance as being closest to the ideal type of a ‘free market’ (cf. Zysman, 1983), US financial markets are insti- tutionally complex and highly restricted (Konings, 2011). European banks and financial authorities complained that their banks could not establish themselves within the US financial landscape (Feiertag, 2005). While by the 1980s, any major bank had a foreign subsidiary in the US (Stigum, 1990), European universal banks were restricted in their operations in the US by various financial regulations. For example, the legal set up of their institutions as universal banks was only slowly allowed to operate in US capital and money markets, and on restricted terms until the end of Glass-Steagall legislation in the 1990s. They had to adopt new ‘transparent’ practices such as credit ratings, new accounting standards and infor- mation about their equity holdings to attract new investors that wanted informa- tion about their financial performances (L€utz, 2005). While attempting to establish themselves in the US from the 1970s onwards, these institutional restrictions pre- vented the banks from expanding rapidly in the US to improve their access to USD funding. As Capone, chief operating officer of UBS North America, declares as late at the early 1990s, UBS was still building its US presence which was needed for any bank aiming to compete successfully on the global scale (Celarier, 1996). Finding banking in the US difficult, European banks expanded their Eurodollar market networks to institutionalize better access to USDs from outside the US. I highlight two key strategies by which they attempted to change their peripheral position. Building international USD funding networks in this way, the banks started a process that led to the adoption of the practices of LM. Accessing US money markets The third feature is the need for European banks to go to US money markets. Because those markets were especially short-term and deep, they uniquely accom- modated the liquidity requirements of LM. Stressing the institutional specificity of US money markets is important because financial practices require specific socio- economic contexts to function well. This was a crucial imperative for European banks to attempt to adjust to US regulations and build new networks as the practi- ces of LM could not initially be established in their home markets and this process, so I argue, meant that European banks turned towards a specifically US-style of banking. The US has the largest money markets, as cash rich corporations and other institutions in the US have channeled their funds through them, instead of deposit- ing them with banks directly (Sissoko, 2015; Stigum, 1990). US money market funds provide a major share of the USD funding of foreign banks (Aldasoro et al., 2018). As described above, the use of CDs allowed bank credit in the 1960s to expand to unprecedented levels (Minsky, 1986). By contrast, debt markets in Europe were smaller and more fragmented. European money markets are mainly comprised of funds between financial institutions, rather than corporate or house- hold investments. National regulations initially separated the markets, and money market funds invested in their home currencies so they could not provide the means for banks to access USD (Mai, 2015). US banks therefore had several unique institutional advantages over European banks. Firstly, while money market funds were initially in fierce competition over deposits with the banks in the 1960s, the expanding money markets worked to the advantage of the major US commercial banks and later investment banks (Cerpa Vielma et al., 2019). In the early phases, US banks in New York used the money markets to arbitrage between them and the Eurodollar markets (Stigum, 1990). This has evolved into a closely knit network as most money market funds lend and borrow through the banking network (Sissoko, 2015). It was only in the 1990s and particularly in the 2000s that European banks were able to attract huge volumes of funding from the US money market funds through repo funding (Gabor, 2016). 1734 M. BECK 1734 M. BECK Secondly, US financial markets are institutionally deep and highly liquid. Accessing US money markets p p p From the 1970s onwards, a primary strategy to capitalize on rising USD flows were offshore centers. In this way, the European lenders built elaborate USD fund- ing networks to fund their USD investments (Aldasoro & Ehlers, 2018; McGuire, 2004). One can think of various offshore financial centers that European banks set up throughout the 1970s and 1980s, particularly in their efforts to recycle petrodol- lars (Altamura, 2016). As discussed above, European banks embraced London as an offshore center to learn various swap and derivative techniques to turn their local currencies into USD (Battilossi & Cassis, 2002; Burk, 1992). What they found, however, was that European companies would prefer the ‘full scale’ service of the US banks that advised on various forms of business expansions, mergers and acquisitions and financing options (Kobrak, 2007; Stigum, 1990). Despite their growing Eurodollar networks, European lenders had to continuously innovate to catch up to US banks. Subsequently, the second set of strategies were foreign acquisitions to improve their expertise and operational capacities to offer corporate funding. European banks embarked on expensive shopping sprees for new US institutions and REVIEW OF INTERNATIONAL POLITICAL ECONOMY 1735 personnel, or for British merchant banks that had closer connections to US banks (Hardie & Howarth, 2009; Noonan, 2020). Credit Suisse made the first move into the US market by acquiring parts of First Boston in 1978, taking a controlling stake in 1988. As money market trading ‘had leapt in importance’ (Burk, 1998, p. 136) in the 1980s and early 1990s, money market connections in particular were valued by big European banks. For example, Deutsche bought Morgan Grenfell in 1989, a British merchant bank that it renamed Deutsche Morgan Grenfell (DMG). DMG was important for its strong position in London and its long-standing relationships to the Morgan bank family, connecting Deutsche to the US money markets indir- ectly (Kobrak, 2007). However, when it became apparent that DMG was struggling in US equity and money market funding, Deutsche bought US investment bank Bankers Trust, hoping it would enhance its wholesale banking (Wieandt & Moral y Santiago, 2006). Similarly, UBS bought S.G. Warburg (a British investment bank) in 1995, but in order to become part of the top rank in US investment banking, it needed a US presence (Celarier, 1996). It subsequently bought Dillon Read (1997), Kidder Peabody (2000), and PaineWebber (2000). Accessing US money markets These strategies were often followed by larger transformations of the business model of the parent bank. While some European banks, for example Credit Suisse and Deutsche Bank, attempted initially to keep their US investment banking separ- ate from the rest of the bank (Montagu-Pollock, 1995), the logics of US money market funding crept across those institutions. US traders and allied European bankers worked hard to adapt the systems and technologies of European banks so that the bankers could manage the risks and quick decisions needed on US money markets (‘New Tricks to Learn,’ The Economist, 1993; c.f. Kobrak, 2007, p. 323, for details on Deutsche Bank). Building US money market foundations had transformative effects for European banks. Since the late 1990s and 2000s, they were able to exploit their newly gained capacities to resemble their US competitors (cf. Erturk & Solari, 2007). Big European banks expanded their capacities to do derivatives, currency options and ‘dark pools’ (Mattli, 2019) in which they traded and brokered securities worth bil- lions of USD, bypassing official stock exchanges. In order to afford these expensive banking networks, European banks sold many of their previous equity investments (Lane, 2003) and significantly reduced their corporate loans from the 1990s onwards (Jackson & Deeg, 2012). Corporate lending no longer yielded sufficient profits given the increased costs of their extroverted strategies (cf. Janssen, 2009). g g Enhancing their capacities to do LM, big European banks were able to exploit US wholesale markets in the 2000s, predominantly through engaging in extensive repo trading that developed into a popular tool to access US money markets from the 2000s onwards (Baklanova et al., 2015; Bayoumi, 2017; Gabor, 2016). Repo funding tripled in size (Sissoko, 2019), with European banks as one of the main lenders (Bayoumi, 2017). European banks lent from US money markets in unprece- dented volumes, only to invest the USDs back into US capital markets, most not- ably in mortgage-backed securities (MBS) (Tooze, 2018a). US bankers selling MBS even reshaped their pricing method to meet the needs of European banks’ offshore financing (McCauley, 2018). In addition, European banks bought securities houses and started to originate vast amounts of MBS themselves, fueling the pre-crisis USD global financial bubble. 1736 M. BECK 1736 M. BECK The 2000s were a period of excessive leverage and high profits for the European banks (Hardie & Howarth, 2013; Hardie & Macartney, 2016). Accessing US money markets Having limited access to US money markets onshore, they built large offshore networks to enhance their money market funding of capital market lending (Mehrling et al., 2013). In this way, an original weakness (lack of access) turned into a temporary strength. The extroverted strategies of European banks have turned them into repo intermediaries and they occupy key roles within global USD supply channels. Engaging in reverse repo finance6, French and German banks developed broker-dealer capacities since the 2000s (see Figure 2 in Aldasoro et al., 2018). While German banks reduced their reverse repo positions since the GFC, French banks have assumed a central role in supplying other foreign banks such as Japanese banks with US money mar- ket funding. In the process of building links with US money markets, European banks first changed the way they raised their liabilities and, subsequently, how they con- structed and managed their asset side. The fact that European banks significantly changed towards securities trading from the 1990s onwards, and restructured the institutional connections with their home markets, is widely recognized by CPE scholars. I emphasize here that European banks took this turn towards a specific- ally US-form of finance because they needed to address the pressures of LM and US money markets. Progressively adopting the practices of LM created further imperatives to change asset-based strategies. In other words, the transformation towards a US-form of finance started with changes in funding portfolios that led to further changes in the banks’ asset-based strategies. g g This shift has made European banks central agents of global USD flows (Hardie & Thompson, 2020; Shin, 2016), and allowed them unprecedented volumes of leverage in the early 2000s (Bayoumi, 2017). Equally important, however, is that this development generated new problems that cast the ‘success story’ of their increased USD access in a new light. The next section therefore demonstrates a nuanced view of the power of LM that incorporates the contradictions of European banks’ shift to US-style finance. The contradictions of LM The fourth feature represents the structural contradictions that the European strat- egies of extroversion have created for their own business models. As I have argued throughout the article, gaining access to USD liabilities has been a core concern of the European banks’ extroverted strategies. While many large European banks have managed to integrate themselves firmly into USD funding structures, their commit- ment to LM has generated new problems which the banks need to manage. In this section, I show firstly the unstable short-term funding structures that resulted from the extroverted strategies. Secondly, I demonstrate the unevenness of financialized banking as European banks have entrenched their own precarious dependency on US money markets. Ultimately, the contradiction of LM is that while European banks have become central actors of US-led financialization, their own success in adopting LM has not reduced but entrenched their structural disadvantages vis- a-vis US banks. Global banking has become inherently complex and fragile (Aldasoro et al., 2018; Bell & Hindmoor, 2015). Banks hold low capital reserves and juggle their REVIEW OF INTERNATIONAL POLITICAL ECONOMY 1737 highly leveraged books with the help of short-term funds from US money markets such as FX swaps or repo finance. Studies from the Bank of International Settlements (BIS) have identified the dramatic growth of USD liabilities outside the US as being a crucial driver of global financial risk. Non-US banks collectively hold just under 13 trillion of USD denominated assets, a similar amount compared to US banks (Aldasoro & Ehlers, 2018; Borio et al., 2017; Fender & McGuire, 2010). Rather than representing bastions of stability by using their balance sheets to allevi- ate funding strains for corporations, banks have turned into super spreaders of risk (Gabor, 2015). The international connections of USD networks mean that a financial crisis in one place can easily spread and escalate through global balance sheets. Repo mar- kets, key nodes within global USD networks, often absorb, rather than provide, financial liquidity, particularly in times of crisis (Sissoko, 2019). Repo finance is procyclical because in times of stress the price volatility of collateral assets can trig- ger margin calls and fire sales causing further price declines. The 2008 financial cri- sis is a case in point, since the defaults of US mortgages led to write-downs and losses for European banks. The contradictions of LM Some of the banks went bankrupt because they could not access enough funds to roll over their short-term USD debt (Baba et al., 2009; Buch et al., 2011). While global banking practices have become more crisis prone (Bell & Hindmoor, 2015), causing problems for internationally active banks in gen- eral, the remainder of this feature examines how this problem has been more diffi- cult to manage for European banks in contrast to US banks. g p US money markets are an inherently unreliable source of funding for European banks. Although European banks’ USD liabilities have declined since the GFC, the length of money market funding for foreign banks has shortened (Aldasoro et al., 2017). Raising the majority of USD funds through short-term wholesale markets, rather than insured deposits, makes banks more vulnerable to shocks because the lack of insurance can trigger investors to withdraw their funds more quickly if there is stress in the system (Ivashina et al., 2015). This is more problematic for European banks in contrast to US banks, because European banks are less institu- tionally embedded in USD funding structures and are therefore less flexible in their management of USD liabilities. A study by the Bundesbank suggests that banks with better onshore USD bases are more resilient against USD funding squeezes (Abbassi & Br€auning, 2018). European banks have fewer alternatives to compensate the loss of USD when money markets dry up. Most notably, they do not have sta- ble USD deposits, and non-deposit liabilities are on average larger than their cus- tomer deposits since the end of the 1990s (Noeth & Sengupta, 2012). Presently, foreign banks mostly operate via branches that are legally precluded from raising insured deposits (Ivashina et al., 2015). US commercial banks, by contrast, can raise a wider variety of onshore funding sources, such as deposits, but also funds from Federal Home Loan Banks (FHLB) not available to European banks (V. V. Acharya et al., 2017). Since the 2008 financial crisis, US branches and agencies of European banks no longer supply their headquarters with USD funds. Instead, they have turned into net debtors, relying on their parent banks for USD, instead of supplying them with liquidity (Aldasoro et al., 2018; D€uwel & Frey, 2012). FX swaps represent a key financial tool for banks to manage their USD currency mismatch (Borio et al., 2017; Fender & McGuire, 2010). The contradictions of LM The FX markets are diverse, of course, and some European banks, for example Belgian, Dutch and 1738 738 M. BECK 1738 M. BECK French banks have become net-lenders of USD FX swaps, demonstrating their cap- acity to raise large amounts of USD liabilities. German, Spanish, Swiss and UK banks are net borrowers (see Borio et al., 2017, pp. 47–48 for disaggregated data). Nonetheless, FX swaps exemplify the structural power relations between US and European banks: European banks raise the majority of their deposits in euros, but a key part of their assets are in USD. Non-US banks’ net borrowing in the FX mar- ket is larger than net lending (Borio et al., 2017). European banks rely on USD but US banks do not need euros to a similar extent, causing a structural disparity in the needs of swapping currency (Baba et al., 2009). Moreover, the onshore conditions of US money markets are key to the supply of FX swaps, despite its offshore nature. As BIS studies have shown, on average, the better the options for banks to source USD and the smaller the USD funding gap, the less they have to pay for turning their EUR deposits into USD (Abbassi & Br€auning, 2018; Aldasoro et al., 2018). If MMF withdraw their funding, less fund- ing is available to finance FX swaps for foreign banks (Aldasoro et al., 2019; Baba et al., 2009). While standing swap lines from the Fed to the ECB alleviated large funding dry ups for the European banks during the 2008 and Covid-19 market freezes (cf. Hardie & Thompson, 2020), US money market funding strains affect FX markets and impact foreign banks more than US banks, notwithstanding the Fed’s global swap support (Avdjiev et al., 2019; Shin, 2016). Offshore USD markets do not seem to allow European banks to circumvent the institutional constraints of US money markets. y The risk of USD funding strains does not only create more difficulties in raising USD liabilities. It also affects European banks assets because they must manage them with a view to accommodating US money markets. For example, during the EU-Greek debt crisis in 2012, German and French banks needed to reassure US money market funds (MMF) that their troubled Greek assets would not pose a danger to the European banks’ solvency. This was important for fear that US MMF withdrew their USDs investment (Thompson, 2015, 2016). The contradictions of LM During the Eurozone crisis in 2011, the ‘euro basis’ (the cost of swapping into USD) rose exceptionally high because of a perceived credit quality erosion (Ivashina et al., 2015). European banks US money market dependence affects their asset management not only in the US but also in their home markets. This final feature highlights that an important part of the systemic risk and fra- gility of contemporary banking stems from the tensions that result from the process of extroversion, that is, from funding a major part of their activity with short-term wholesale USDs outside of their own home markets. This demonstrates the importance of incorporating institutional power structures into the analysis of banking. In their pursuit to catch up to US banks, European banks have helped to entrench a financial framework in which a crucial source for their own empower- ment (USD debt), and the markets required for it (US money markets), are designed in way that systematically disadvantages European vis-a-vis US banks. Conclusion This paper offers a novel concept to understand the transformation of large European banks: extroverted financialization (EF). I argue that a key aspect of the transformations of European banks towards a US-form of finance is its extroverted REVIEW OF INTERNATIONAL POLITICAL ECONOMY 1739 nature, that is to say the institutionalization of their own business models into USD funding markets. This process started in the Eurodollar markets as part of joint European initiatives in the 1960s. Subsequently, big European banks started their own international subsidiaries in London to partake more directly in US financial innovations. In the 1980s, most of the banks established a subsidiary in the US and throughout the 1990s, they acquired US institutions that enabled them to manage innovations such as derivatives, repo, swaps and asset-backed securities. By the 2000s, these practices enabled the mega banks to leverage huge volumes of USD debt, reaping unprecedented volumes of profits. Following the GFC, however, it became apparent that they had built highly risky global funding structures with crucial tensions and problems within their own business models. I propose the concept of EF to account for this process. The four features of the concept foreground key imperatives that invite us to think of financialization in terms of the specific constraints it imposed on European banks. More specifically, it frames the transformation of European banking as a response to the rise of LM from the 1960s onwards and the specific needs of USD funding imposed on inter- nationally active European banks. This longer-term perspective reveals the histor- ical needs that European banks might have had for US money market funding, and how their attempts to accommodate these USD needs prompted larger changes in their business models towards the practices of LM. Using this concept has clear analytical and political implications. Analytically, capturing the novel pressures of the rise of US finance helps to grasp the uneven nature of financialization. The pursuit of LM to catch up to US banks has resulted in an international financial framework largely centered on US money markets. This gives institutional advantages to US banks, even though European banks man- age large volumes of USD offshore. Incorporating these unequal power relations redirects the analytical gaze to the politics of banks and their attempts and failures to change market forces for their own benefit. Funding This work was supported by Foundation of German Business (SDW); Economic and Social Research Council (ES/R00787X/1). Acknowledgements I would like to thank Dylan Cassar, Emanuele De Girolamo, Sahil Dutta, Samuel Knafo, Fabian Pape, Stefano Sgambati, Matthew Watson, the participants of the MaxCPE seminar, the editors and three anonymous reviewers for helpful comments on earlier drafts, and Edward Dee for thorough copy-editing. Notes 1. I use the category of large European banks to depict a wide range of large European commercial banks that have been involved in off- and onshore USD wholesale banking (Acharya & Schnabl, 2010; Bayoumi, 2017; Hardie & Thompson, 2020 ). This is closely aligned with Bayoumi’s focus on the ‘mega-banks’ of the Euro area (see Bayoumi, 2017, p. 39, for a list), but includes large British banks as well. p g 2. As Maxfield et al. (2017) demonstrate, the hypothesis of ‘full convergence’ is controversially debated. There is, however, consent in the literature that a shift towards more market-based finance has taken place. p 3. See Stigum (1990) for a sophisticated explanation of the money markets. 4. A repo is a short-term, collateral – backed overnight loan. See Baklanova et al. (2015) for an overview of repo markets in the US. 5. Cf Sanford (1996), director of Bankers Trust, for a detailed description of this development at his own bank that pioneered some of these strategies. 6. A reverse repurchase agreement describes the transaction of buying a security with the agreement to sell back at a specific price. That way, European banks sourced securities on US capital markets as collateral to borrow from US money markets. Disclosure statement No potential conflict of interest was reported by the author(s). Notes on contributor Mareike Beck is a Leverhulme Early Career Fellow at the department of European and International Studies, King’s College London. Her research focuses on financialization, global banking, asset and debt-based inequalities and comparative political economy. Conclusion This recovers the role of European banks within USD wholesale markets while recognizing their peripheral position to USD funding. The concept of EF offers new avenues of research to understand globalized banking. It could help to clarify the complex hierarchies within USD funding struc- tures by applying the concept to other banking groups. For example, Japanese banks’ USD positions have superseded those of European banks in recent years. Japanese banks’ asset side relies on more ‘traditional’ loans. In contrast to many large European banks, Japanese banks have not yet developed a capacity as broker- dealers, that is to buy US securities from capital markets that can serve as collateral for US money market funding. Instead, Japanese banks rely on French and other European banks for their US money market funding (Aldasoro et al., 2018, p. 11, see Figure 2). Future research could explore how well EF captures the historical constraints that USD funding might have exerted on internationally active Japanese banks. What is at stake politically? The history presented here suggests that calls for better regulation to ensure a process of ‘de-financialization’, or the divestment from speculative assets, are perhaps too short-sighted. The expansion of speculative balance sheets is often explained as an outcome of inadequate financial rules that created incentives for regulatory arbitrage (V. V. Acharya et al., 2013; Aldasoro et al., 2018). From that perspective, European banks expanded their balance sheets 1740 M. BECK M. BECK 1740 in a way that took on more risk in return for higher profits wherever there were fewer restrictions against speculation (Bayoumi, 2017; Hardie & Thompson, 2020). 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The epithelial to mesenchymal transition (EMT) and cancer stem cells: implication for treatment resistance in pancreatic cancer
Molecular cancer
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Background such as accelerated metastasis to distant organs and tissues. Thus treatment resistance becomes the major challenge in clinical cancer therapies. The focus on the management of PC patients, especially those in advanced stages, is to understand the pathophysiological mecha- nisms of therapy resistance and overcome the resistance. Pancreatic cancer (PC) is one of the poorest prognosis malignancies with a 5-year survival rate of less than 5% and a median survival of no more than 6 months after diagnosis [1, 2]. Even among patients diagnosed with early-stage disease who undergo clean surgical margins re- section (R0 resection) followed by adjuvant chemotherapy, the median survival rate is approximately 2 years, with a 5-year survival of 15–20% [3–5]. This devastating situ- ation is due to several factors. First, due to the absence of effective tools for an early detection, most patients at the time of diagnose have locally advanced or metastatic dis- ease, and lose the opportunity of surgical resection. Sec- ond, even for those patients who undergo surgical resection, the prognosis is poor due to early relapse and distant metastasis. Metastasis is a characteristic of pancre- atic cancer and the leading cause of mortality among can- cer patients [6]. Finally, PC shows profound resistance to relative chemotherapy and radiation treatment. Cancer cells resistant to treatment usually show more aggressive, Cellular heterogeneity is a well-recognized property of both normal and malignant tissues. The difference is that heterogeneity in the normal tissues is an ordered developmental program. However, tumors are composed of a small set of distinct cells termed cancer stem cells (CSCs), which is capable of driving tumor initiation and development. The CSCs model, on the other hand, sug- gests that the biology process of the tumor is driven by a small population of cells with the stem cell properties of sustaining growth and an ability to differentiate into the entire heterogeneous tumor [7]. Dick and colleagues in 1997 identified the first cancer stem cell in hematopoietic malignancies, such as acute myelogenous leukemia and chronic myelogenous leukemia using cell surface marker expression [8, 9]. Hematopoietic stem cells (HSCs) can self-renew and differentiate into all the cells of the hematopoietic system, and are responsible for lifelong blood production [10]. REVIEW Open Access © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. The epithelial to mesenchymal transition (EMT) and cancer stem cells: implication for treatment resistance in pancreatic cancer Pingting Zhou1†, Bo Li2†, Furao Liu1, Meichao Zhang1, Qian Wang1, Yuanhua Liu3, Yuan Yao4 and Dong Li1* Abstract The mechanical properties of epithelial to mesenchymal transition (EMT) and a pancreatic cancer subpopulation with stem cell properties have been increasingly recognized as potent modulators of the effective of therapy. In particular, pancreatic cancer stem cells (PCSCs) are functionally important during tumor relapse and therapy resistance. In this review we have surveyed recent advances in the role of EMT and PCSCs in tumor progression, metastasis and treatment resistance, and the mechanisms of integrated with biochemical signals and the underlying pathways involved in treatment resistance of pancreatic cancer. These findings highlight the importance of confirming stem-cells markers and complex molecular signaling pathways controlling EMT and cancer stem cells in pancreatic cancer during tumor formation, progression, and response to therapy. Keywords: Pancreatic cancer, Cancer stem cell, Epithelial-to-mesenchymal transition, Resistance * Correspondence: lidong@shsmu.edu.cn †Equal contributors 1Department of Oncology, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Full list of author information is available at the end of the article Zhou et al. Molecular Cancer (2017) 16:52 DOI 10.1186/s12943-017-0624-9 Zhou et al. Molecular Cancer (2017) 16:52 DOI 10.1186/s12943-017-0624-9 Open Access Background After the discovery of CSCs in leukemias, the first CSCs in solid tumors were * Correspondence: lidong@shsmu.edu.cn †Equal contributors 1Department of Oncology, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Full list of author information is available at the end of the article Zhou et al. Molecular Cancer (2017) 16:52 Page 2 of 11 Page 2 of 11 Page 2 of 11 identified in breast tumors [11], leading to much re- search in a variety of tumors, including glioblastoma [12], pancreas [13, 14], melanoma [15], prostate [16] and colon [17]. PCSCs have been first discovered in 2007 and since then have conducted as a subpopulation of cancer cells with special functional features including self-renewal and exclusive in vivo tumorigenicity. Furthermore, the resistance of PC to standard chemo- therapy and radiation treatment may in part be due to the existence of CSCs, which can express multidrug- resistant membrane transporters, aberrantly activate proliferation signaling pathways and increase the cap- ability of repairing DNA. (MET), is believed to support metastatic progress once migratory cells have reached their destination [26, 27]. During EMT, cells lose their epithelial cell-cell adherens junction and apical-basal cell polarity and acquire mesen- chymal characteristics with spindle-like cell shape and with the ability to migrate [28]. A variety of markers have been used to demonstrate EMT. E-cadherin, intergrins, and cytokeratins are the most commonly used epithelial markers and N-cadherin, vimentin or fibronectin for the mesenchymal [26]. In recent years, the cadherin switch from E-cadherin to N- cadherin, have been increasingly used to monitor EMT during cancer development. However, cells do not gain mesenchymal traits in a partial EMT. Early EMT might only involve loss of E-cadherin and do not gain N- cadherin, this phenomenon may lead to different bio- logical results of the intermediate states and likely behave distinctly during migration and invasion to those that do gain N-cadherin [29, 30]. Hence, it is important to consider not only epithelial or mesenchymal traits during EMT but also other processes related to EMT, such as invasion, increased survival or decreased proliferation. Although there are a growing number of studies that support the CSCs model in cancer, diverging theories exist on the precise origin of cancer stem cells. It is not yet known whether they originate from the tissue’s normal stem cells by the accumulation mutations or the acquisition of the mutations in more-differentiated cells. Background Recent studies have implicated that the process termed epithelial-to-mesenchymal transition (EMT) is associ- ated with features of CSCs [18, 19]. This review focuses on recent research findings related the role of EMT and CSCs on chemotherapy and radiotherapy resistance in pancreatic cancer, helping understand the complex biol- ogy of treatment resistance for the more effective treat- ments for PC patients. p EMT change is triggered in a number of distinct molecular processes including the expression of specific cell-surface proteins and the activation of transcription factors (TFs). The list of potent EMT-inducing transcrip- tion factors (EMT-TFs) has been growing ever since [31], EMT-TFs belong to different families, including superfamily, SNAI1 (previously known as Snail) and SNAI2 (previously known as Slug), two ZEB factors, ZEB1 (also known as dEF1/TCF8) and ZEB2and Twist. Other TFs have been shown to be related with EMT, including Prrx1,Sox4 and Sox9 [32, 33], Klf4 [34] and members of the AP-1 (Jun/Fos) family [35]. It is reported that EMT-TFs have been involved not only in migration and invasion but also in the protection from senescence and apoptosis, regulation of cell progression and resistance to chemotherapy and radiotherapy [36–38]. These EMT regulators have been shown to repress the expression of E-cadherin [39–42] through binding to con- served E-box sequences (mainly of the CAGGTG type) in the promoter of E-cadherin [41, 43]. Loss of E-cadherin can drive certain epithelial cells toward a mesenchymal state [44]. EMT in cancer There is no doubt that one of the most concern is the miR-200 family, which has five members classified in to two groups; miR-200a, miR-200b and miR-429 on hu- man chromosome 1, miR-200c and miR-141 on human chromosome 12 [62]. Their low expression of individual family members results in EMT progression by enhan- cing levels of EMT-TFs Zeb1 and Zeb2, which blocks EMT or induces MET [63]. Conversely, Zeb1/2 directly binding to miR-200 promoters can repress the expres- sion of miRNAs. This interaction between Zeb1/2 and miR-200 family members not only determines cell morphology but also controls cell migration and inva- sion [64, 65]. Reversely, microRNA array analyses have suggested that members of miR-200 family were mark- edly downregulated in TGF-β–induced EMT and in can- cer cell lines displaying an EMT phenotype [63, 66]. In addition to the members of the miR200 family, miR-10b, miR-373, and miR-520c also play roles in the progres- sion of cancer [67, 68]. It`s reported that miR-21 is enhanced during the process of TGFβ-induced EMT [69]. The details of the interaction of all these factors with each other are stated in Fig. 1. and recurrence has led to the theory that tumorigenesis was facilitated by a distinct population of cancer cells with the properties of stem cells (Fig. 2) [70]. The presence of CSCs has been studied in a variety of hematopoietic and solid organ malignancies [8, 11]. The current consensus agreement describes that CSCs identi- fied based on specific cell-surface markers are able to self-renew and differentiate into the heterogeneous of cancer cells that comprise the tumor [71]. Opinions on the precise origin of cancer stem cells existed different. One theory is that CSCs originate from the accumula- tion mutations occurring in normal stem cells and these mutations ultimately trigger a malignant transformation. Alternatively, some studies show that mutations in more-differentiated cells may develop the capacity for unregulated self-renewal and with stem cell-like proper- ties [72, 73]. Hence, the term CSCs, does not indicate the origin of cancer cells, but to the cells that maintain the tumorigenesis [71, 74]. In 1997, the first CSC was isolated in myeloid leukemia using cell surface marker expression [8]. Isolat- ing CSCs in solid organs was based on studies in hematopoietic malignancies. However, cell surface markers used to identify CSCs in epithelial solid organ were distinct from those of leukemia CSCs. EMT in cancer These microRNAs can therefore promote EMT (blue) or repress EMT and enhance MET programs (orange) β-ligand binding, TGF-β receptors I and II (TGF-βRI/ TGF-βRII) which have a serine/threonine kinase activity lead to phosphorylation of Smad2 and Smad3, members of the Smad protein family [45]. After the phosphoryl- ation, a Smad signaling cascade is activated, resulting in nuclear translocation of Smad4, which drives a wide range of tumor-promoting factor genes transcription [46]. TGF-β additionally induces Smad-independent signaling. Among the non-Smad signaling responses, TGF-β can regulate EMT through the activation of Rho GTPases, MAP kinase (MAPK) pathways and the PI3 kinase-Akt-mTOR pathway [47, 48]. On the other hand, non-Smad signaling can impact the activation of Smad signaling in TGF-β induced EMT. For example, Akt acti- vation can sequester Smad3 through unphosphorylated Smad3, impacts Smad activation in response to TGF-β in EMT [49]. P38 MAPK cooperates with Smad3/4 in TGF-β associated EMT through the transcription factor ATF2 [50]. TGF-β can interact with other growth factors such as the epidermal growth factor (EGF) to influence the malignant transformation of CSCs as well as the activation of cancer-associated stromal fibrosis [51–58]. Fig. 1 The Core Regulatory Machinery of EMT. Tumorigenesis activate EMT-promoting transcription factors of the TWIST, SNAIL and ZEB families through pathways known to play critical roles in both embryogenesis and tumour development, including the WNT, NOTCH, TGF-β, RAS and NF-κB cascades. MicroRNAs suppress production of these transcription factors as well as multiple markers defining the epithelial or mesenchymal characteristics. These microRNAs can therefore promote EMT (blue) or repress EMT and enhance MET programs (orange) Fig. 1 The Core Regulatory Machinery of EMT. Tumorigenesis activate EMT-promoting transcription factors of the TWIST, SNAIL and ZEB families through pathways known to play critical roles in both embryogenesis and tumour development, including the WNT, NOTCH, TGF-β, RAS and NF-κB cascades. MicroRNAs suppress production of these transcription factors as well as multiple markers defining the epithelial or mesenchymal characteristics. These microRNAs can therefore promote EMT (blue) or repress EMT and enhance MET programs (orange) [ ] While transcriptional regulation of EMT has been ex- tensively studied, post-transcriptional, translational and post-translational regulators are recently appreciated in several studies [59, 60]. The number of miRNAs that has been reported to be associated with EMT and MET is becoming as extensive as the list of EMT-TFs [61]. EMT in cancer For example, Akt acti- vation can sequester Smad3 through unphosphorylated Smad3, impacts Smad activation in response to TGF-β in EMT [49]. P38 MAPK cooperates with Smad3/4 in TGF-β associated EMT through the transcription factor ATF2 [50]. TGF-β can interact with other growth factors such as the epidermal growth factor (EGF) to influence the malignant transformation of CSCs as well as the activation of cancer-associated stromal fibrosis [51–58]. While transcriptional regulation of EMT has been ex- tensively studied, post-transcriptional, translational and post-translational regulators are recently appreciated in several studies [59, 60]. The number of miRNAs that has been reported to be associated with EMT and MET is becoming as extensive as the list of EMT-TFs [61]. There is no doubt that one of the most concern is the miR-200 family, which has five members classified in to two groups; miR-200a, miR-200b and miR-429 on hu- man chromosome 1, miR-200c and miR-141 on human chromosome 12 [62]. Their low expression of individual family members results in EMT progression by enhan- cing levels of EMT-TFs Zeb1 and Zeb2, which blocks EMT or induces MET [63]. Conversely, Zeb1/2 directly binding to miR-200 promoters can repress the expres- sion of miRNAs. This interaction between Zeb1/2 and miR-200 family members not only determines cell morphology but also controls cell migration and inva- sion [64, 65]. Reversely, microRNA array analyses have suggested that members of miR-200 family were mark- edly downregulated in TGF-β–induced EMT and in can- cer cell lines displaying an EMT phenotype [63, 66]. In addition to the members of the miR200 family, miR-10b, miR-373, and miR-520c also play roles in the progres- sion of cancer [67, 68]. It`s reported that miR-21 is enhanced during the process of TGFβ-induced EMT [69]. The details of the interaction of all these factors with each other are stated in Fig. 1. Pancreatic cancer stem cells The hypothesis of PCSCs has been hotly controversial for many years A new understanding of PC progression Fig. 1 The Core Regulatory Machinery of EMT. Tumorigenesis activate EMT-promoting transcription factors of the TWIST, SNAIL and ZEB families through pathways known to play critical roles in both embryogenesis and tumour development, including the WNT, NOTCH, TGF-β, RAS and NF-κB cascades. MicroRNAs suppress production of these transcription factors as well as multiple markers defining the epithelial or mesenchymal characteristics. EMT in cancer In PC, CSCs have initially been identified by being characterized as CD44+CD24+ESA+cells and having the ability to form tumors at a much higher frequency than the bulk tumor EMT in cancer In addition to the field of EMT in normal embryonic de- velopment, there are numbers of new work on the role of EMT in tissue fibrosis and cancer metastasis [20–22]. In March 2008, at a Cold Spring Harbor Laboratory meeting about EMT, the scientists classified EMT into three general subtypes based simply on the different functional consequences [23]. Type 1 EMT can generate mesenchy- mal cells (primary mesenchyme) that have the potential to generate secondary epithelia by mesenchymal-epithelial transition (MET), which is associated with embryonic gastrulation and neuroepithelial giving rise to monile neural crest cells. Type 2 EMT is associated with wound healing, tissue regeneration, and organ fibrosis, which are in essence an unabated form of wound healing in response to persistent inflammation. Type 3 EMTs occur in epithe- lial neoplastic cells undergoing genetic and epigenetic changes, producing outcomes far from those observed in other two types EMT. Neoplastic cells undergoing type 3 EMT may migrate through the blood stream and generate secondary nodules, threatening manifestations of cancer progression. It is now widely accepted that in epithelial cancers, including pancreatic cancer, EMT is associated with the three major steps of cancer development: inva- sion, dissemination and metastasis [24, 25]. While the reverse process, a mesenchymal to epithelial transition EMT associated with tumors can be induced by vari- ous secreted factors from the stroma, such as transform- ing growth factor beta (TGF-β), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF). Among these, TGF-β has received substantial attention as a major inducer of EMT during embryogenesis devel- opment, cancer progression and fibrosis. Consequently, TGF-β-induced EMT has been better understood than EMT in response to other inducers. In response to TGF- Zhou et al. Molecular Cancer (2017) 16:52 Page 3 of 11 β-ligand binding, TGF-β receptors I and II (TGF-βRI/ TGF-βRII) which have a serine/threonine kinase activity lead to phosphorylation of Smad2 and Smad3, members of the Smad protein family [45]. After the phosphoryl- ation, a Smad signaling cascade is activated, resulting in nuclear translocation of Smad4, which drives a wide range of tumor-promoting factor genes transcription [46]. TGF-β additionally induces Smad-independent signaling. Among the non-Smad signaling responses, TGF-β can regulate EMT through the activation of Rho GTPases, MAP kinase (MAPK) pathways and the PI3 kinase-Akt-mTOR pathway [47, 48]. On the other hand, non-Smad signaling can impact the activation of Smad signaling in TGF-β induced EMT. Pancreatic cancer stem cells The hypothesis of PCSCs has been hotly controversial for many years. A new understanding of PC progression Page 4 of 11 Zhou et al. Molecular Cancer (2017) 16:52 Fig. 2 Contribution of EMT and related signaling to PCSCs. a PCSCs with tumor-initiating capability can be identified by the expression of a distinct set of marker proteins, such as CD44, CD24, CD133 or c-Met. These CSCs can self-renew and differentiate into a number of cell types to generate the heterogeneity of the originating tumor. Inducers of EMT such as TGF-β, HH or Notch cause cells to acquire a CD44+ CD24 + ESA+ phenotype, reminiscent of PCSCs. b The PCSC cell surface markers CD24, and CD44 likely promote cell–cell interactions, the c-Met respond to secreted ligands to active developmental pathways, such as β-catenin, Notch and Stat3 in PCSCs. These pathways stimulate the expression of genes that regulate stem-cell properties, such as self-renewal Fig. 2 Contribution of EMT and related signaling to PCSCs. a PCSCs with tumor-initiating capability can be identified by the expression of a distinct set of marker proteins, such as CD44, CD24, CD133 or c-Met. These CSCs can self-renew and differentiate into a number of cell types to generate the heterogeneity of the originating tumor. Inducers of EMT such as TGF-β, HH or Notch cause cells to acquire a CD44+ CD24 + ESA+ phenotype, reminiscent of PCSCs. b The PCSC cell surface markers CD24, and CD44 likely promote cell–cell interactions, the c-Met respond to secreted ligands to active developmental pathways, such as β-catenin, Notch and Stat3 in PCSCs. These pathways stimulate the expression of genes that regulate stem-cell properties, such as self-renewal [13]. The CD44+CD24+ESA+ cells as a reflection of stemness is generally confirmed with the fact that cells enriched for CD44+CD24+ESA+ were far more tumori- genical than other tumor cells. Half of the mice injected with 100 CD44+CD24+ESA+ cells formed tumors, com- pared with 10000 triple-negative cells, which only 1 in 12 mice developed a tumor. In addition, the tumors formed by the CD44+CD24+ESA+ cells had morpho- logical features similar to those of the original pancreatic ductal adenocarcinoma from patients. These supporting data pointed that a population of cells are responsible for tumor initiation and self-renewal. development in the early stage, but followed with liver metastases or no any trace of metastases 2 weeks later in CXCR4+ group and CXCR4−group respectively [14]. Pancreatic cancer stem cells Molecular Cancer (2017) 16:52 Page 5 of 11 and CSCs has recently been studied in many types of cancer including PC (Table 1) [52, 94–99]. Cells with an EMT phenotype effect molecular characteristics of CSCs; CSCs also express an EMT phenotype. This phenomenon indicated that EMT and CSCs are closely related [100–102]. In breast cancer, Mani and colleagues reported that the overexpression of Twist, Snail or FOXC2 not only made the breast cancer cells with more mesenchymal properties, but also with an increased ex- pression of CD44+/CD24-/low breast CSC markers and an increased mammosphere forming efficiency [18, 103]. The similar results also showed in prostate cancer. Pros- tate cancer cells with an EMT phenotype have increased expression of Sox2, Nanog, Pou5F1, lin28B and/or Notch1 and an enhanced sphere-forming ability [104]. tumor growth. Additional studies have shown that ALDH1 (aldehyde dehydrogenase 1) and Dclk1 (double- cortin and Ca2/ calmodulin-dependent kinase-like 1) high cells have enhanced sphere-forming ability, suggesting a propensity to be associated with PCSCs function [80]. p p y Several markers have been defined to discriminate PCSCs, but the signaling pathways that regulate these PCSC functions have not been fully studied. Notch signaling is an important regulator of self-renewal and differentiation in the normal and development pancreas, but the dysregulation of the Notch pathway will lead to uncontrolled self-renewal of CSCs [81–84]. In PC, inhib- ition of the Notch pathway by either knockdown of the Notch ligand Jagged-1 or blocking Notch pathway with inhibitor MRK-003 leads to reduction of tumor sphere formation [85, 86]. On the other hand, the activation of Notch pathway acts to maintain the pancreatic precursor state. These findings indicate that Notch signaling is needed for PCSC function [87]. Nodal and Activin belong to the TGF-β superfamily and they were shown to be important for human embryonic stem cell main- tenance [88, 89]. In pancreatic cancer, Nodal/Activin is strongly expressed in PCSCs. The Nodal/Activin path- way can affect the self-renewal capacity and stemness properties of pancreatic CSCs and promote invasion of PCSCs [90]. Several studies have identified other devel- opmental pathways such as mTOR, hedgehog for target- ing PCSCs [91, 92]. Compared with normal pancreatic epithelial cells, the expression of HH transcript was in- creased 46-fold in CD44+CD24+ESA+ cells, which sug- gests that hedgehog signaling ligand is significantly expressed in pancreatic CSCs [93]. ZEB1 (zinc finger E-box binding homeobox 1) is a cru- cial promoter of EMT. Pancreatic cancer stem cells The difference in cell populations was activation of CXCR4. These cells, CD133+CXCR4+, were noted to have in- creased in vitro migratory ability and were able to pro- duce metastasis, however CD133+CXCR4− did not produce distant metastases. Importantly, CXCR4 inhib- ition prevented tumor metastasis in mice. These findings may have clinical implications that CXCR4 might be a potential target for therapies to inhibit metastasis of PCSCs. After the identification of CD44+CD24+ESA+ cells and CD133+ cells as a population of PCSCs, Li et al. identified c-Met as a hunman pancreatic CSC marker [76]. c-Met, a member of the receptor tyrosine kinase for hepatocyte growth factor (HGF), has an important role in both normal and malignant development [77]. It was shown to be related with tumorigenesis and drug resistance through activating mutations of path- way components or HGF-dependent autocrine/para- crine in tumor development [78, 79]. They found that c-Met high cells had enhanced tumorigenic potential, whereas c-Met-negative cells did not. They compared the tumorigenicity of different sets of primary tumor cells and identified that c-Met high cancer cell popu- lation had the highest tumorigenic potential, com- pared with pancreatic cancer cells expressing CD44, CD24, ESA and CD133 [76]. Cabozantinib, the c-Met inhibitor, significantly inhibited tumor sphere forma- tion, reduced the population of PCSCs and slowed In another group, CD133 was used to identify CSCs from human primary PC samples and PC cell lines [14]. Like CD44+CD24+ESA+ cells, 500 CD133+ PC cells gen- erated visible tumors that histologically indistinguishable from the primary tumor. In contrast, 106 CD133−PC cells failed to induce tumor formation. By flow cytome- try analysis, they showed that CD133+ cells and CD44 +CD24+ESA+ cells are not identical but have a 14% over- lap. Further experiments will need to estimate the tumorigenic potential of this quadruple-positive subset. This study also investigated the role of pancreatic CSCs in metastasis and the results revealed that a subpopula- tion of CD133+ cells that co-express CXCR4 determined the metastatic ability of PC cells. CXCR4 is a chemokine receptor of the ligand stromal cell-derived factor 1 (SDF1), which is reported to be a mediator of tumor invasion and metastasis [75]. Hermann et al. showed that two groups of mice injected CD133+ CXCR4+ and CD133+ CXCR4− cells appeared similar tumor Page 5 of 11 Zhou et al. Pancreatic cancer stem cells ZEB1 can repress the expression of the miR-200 family and stemness-inhibiting miR-203, resulting in activation of EMT and tumor-initiating cap- acity in pancreatic cancer [96]. In addition, miR-200 family can suppress the expression of stem cell factors, such as Sox2 and Klf4. It is also suggested that ZEB1and PCSCs marker CD44 regulate each other. ZEB1 enforces CD44 isoforms (CD44s) splicing by repression of epithe- lial splicing regulator ESRP1 in pancreatic cancer. CD44s, in turn, increases the expression of ZEB1, result- ing in a self-sustaining ZEB1 and CD44s expression. The relationship of this novel CD44s-ZEB1 impacts on can- cer cell ability, including increased tumorsphere initi- ation capacity and tumor metastasis [105]. These results suggested that ZEB1 linked EMT and stemness- maintenance in PC. Nestin was first recognized as a functional stem cell marker in embryonic and adult central nervous system (CNS) stem cells [106]. In addition, recent studies have identified nestin as a CSC marker in brain tumors, ovar- ian, head and neck, prostate, and PC [107–111]. Com- pared with parental cells, PC cells with Nestin The relationship between EMT and PCSCs Of fundamental importance biologically, the activation of EMT process has been associated with the properties of stem cell traits for both normal and neoplastic cells [18, 19]. The biologic link between EMT phenotypes Table 1 Principal data regarding the relationship of EMT with PCSCs Experimental approach Molecular characteristics of PCSCs References Short hairpin RNA (shRNA)-mediated ZEB1-knockdown in the two cells with the highest levels of ZEB1(Panc–1 and MiaPaCa–2) Reduction of CD24+/CD44+ subpopulation, reduced sphere formation in the two cancer cell lines and sphere numbers in subsequent generations decreased expression of stem cell factors such as Sox2, Bmi1 and p63 [96] CD133 overexpression in Mia PaCa-2 cell Increased mRNA expression of several EMT-associated genes: SNAI1, ZEB1, Vimentin, CDH2 and MMP9. CD133hi-MIA cells show a more fibroblast-like morphology [97] Silenced Snail in Panc-1 cells A significant decrease in the ALDHhigh population, reduction initial formation of spheres and sphere numbers in subsequent generations. [98] Nestin shRNA in PANC-1 cell and nestin-overexpressing in MiaPaCa-2 cell Expression of mesenchymal markers, acquisition of invasive properties and high motility/opposite effects [99] Isolate the SP cell fraction (side population, a cancer stem cell enriched fraction from Panc-1,KP-1NL and Capan-2 cell lines), incubate SP cells in the presence of TGF-β Production of cells with mesenchymal-like morphology,alteration such as reduction of E- cadherin mRNA and induction of Snail mRNA and (MMP)-2 mRNA [52] Page 6 of 11 Zhou et al. Molecular Cancer (2017) 16:52 Zhou et al. Molecular Cancer (2017) 16:52 knockdown exhibited decreased sphere formation and regulated EMT by decreasing slug expression [112]. Overexpression of nestin induced TGF-ß1 and the ex- pression of its receptors through the Smad4-dependent pathway in PC and nestin overexpression induces the EMT of PDAC cells. Meanwhile the excessive TGF- β1 cytokine as a major EMT-inducing soluble factor results in increased nestin expression. Thus, nestin-positive cells apparently use an autocrine positive feedback loop to regulate EMT in PDAC through TGF-β/Smad pathway [99]. In addition, Hypoxia induction in- creased expression of the PCSCs markers Notch1, Notch4, c-Met, CD133 and the embryonic stem cell markers Nanog, SOX2, FOXA2, SOX17 and PDX-1 [113]. Among above markers, the upregulation of FOXA2 was accompanied by an EMT, with down- regulation of E-cadherin and upregulation of mesen- chyme markers Vimentin, Slug, Snail and Twist2. The relationship between EMT and PCSCs Previous experience in breast cancer showed that Twist2 overexpression not only promoted EMT sig- naling, but also enhanced colony-forming abilities of stem cells, which suggested that Twist2 may be a master inducer of both EMT and CSC features. In hematopoietic malignancies, Michor and col- leagues identified that a subpopulation of human leukemia stem cells showed resistant to the Abl tyro- sine kinase inhibitor imatinib, an effective drug against differentiated leukemic cells. The survived leukemic stem cells regenerated the tumor, providing further evidence supporting the important role of CSCs in tumor relapse [124]. Evidence of the breast CSCs resistance is supported by a study in which tumor biopsies taken from patients with breast cancer during the 12-week chemotherapy treatment con- ducted increased CSC markers (CD44+/CD24-/low and MFSE) [125]. The glioblastoma CSCs were also shown to be responsible to standard therapies resistance in brain tumor. It was identified that the CSC popula- tion expressing CD 133 was increased two to four folds in both primary tumors and xenografts after radiation, maybe due to a preferential activation of the DNA damage response [126]. Recently, some studies suggest that pancreatic CSCs may also be resistant to chemotherapy and radiation therapy. With regard to this, Hermann et al. have con- ducted that human CD133+ pancreatic CSCs isolated from pancreatic tumor are highly resistant to standard gemcitabine therapy, which is conventional chemothera- peutic agent against PC [14]. Gemcitabine-resistant cells, which have stronger sphere-forming ability and are more tumorigenic than gemcitabine-sensitive cells in vitro and vivo, were equipped with the similar properties as pan- creatic CSCs [127]. Furthermore, it is reported that al- though the cytotoxic agent gemcitabine can arrest proliferating of CD133 CSCs, the apoptosis of CSCs was not affected, leading to CSCs` returning to stem cell pool when gemcitabine withdrawn. On the contrary, the more differentiated cells (CD133−), the vast majority of the tumour cell, became apoptotic under the manage- ment of gemcitabine. It is obvious that only the more differentiated tumour cells can be targeted with standard therapy, leaving undifferentiated cancer stem cells resist- ant to therapy. These results suggest that therapeutic targeting of the activation of apoptosis might provide a tool to sensitize CSCs to therapy. PCSCs, EMT and treatment resistance In the clinic, the combination of radio- and chemother- apy with or without surgical intervention is the standard of care in many cancers. Although technical advances in radiation and chemotherapy have improved local control and patient survival. Cancer treatment resistance, including chemoresistance and radioresistance, is still a major challenge in cancer research and treatment [114, 115]. Treatment resistance has become a key obstacle in improving the effectiveness of tumor ther- apy, resulting in the high mortality in patients diag- nosed with PC. This disappointing situation strongly needs to improve on understanding the mechanisms of the treatment resistance, leading to find out novel thera- peutic strategies for overcoming the resistance and in- creasing the survival rate. Potential mechanisms of resistance to antiangiogenic therapy may result from the selection effect directly and indirectly on advantaged sub- populations of tumor and tumor- associated cells [116]. Tumor-infiltrating immune cells are feature of most solid tumors, can promote chemoresistance and metas- tasis in aggressive tumors, leading to awful clinical out- comes of cancer patients [6, 128]. It has been illustrated that tumor-infiltrating macrophages (TAMs) can directly induce PCSCs properties through the activation of STAT3 pathway. Conversely, STAT3+ CSCs enhance TAM-mediated immunosuppression. Furthermore, tar- geting TAMs by inhibiting either CSF1 receptor (CSF1R) or chemokine (C-C motif) receptor 2 (CCR2) decreases the numbers of pancreatic CSCs and improves chemo- therapeutic efficacy in vivo. The PCSCs response Accumulating researches clearly suggest that CSCs and EMT-type cells play important roles in chemoresis- tance and radioresistance (Table 2) [117–122]. The role of CSCs contributing to treatment resistance has been reported to be closely related with activation of the DNA damage checkpoint repair, thereby protecting cells from DNA damage and activating the cell survival signaling pathways [114, 115, 123]. Few of the current therapies can eliminate CSCs because they have critical roles in treatment resistance, which might interpret why cancer is difficultly eradicated completely. Zhou et al. PCSCs, EMT and treatment resistance Molecular Cancer (2017) 16:52 Page 7 of 11 Table 2 Principal data regarding the role of PCSCs in the induction of treatment resistance Experimental approach Molecular characteristics of PCSCs References Isolated SP(side population) cell fractions in L3.6pl cell‚ gemcitabine- and 5-FU-resistant L3.6pl cells were established Induce faster and more aggressive orthotopic tumor growth with higher rates of metastases‚ gemcitabine resulted in an increase of CD24 positive cells and the percentage of SP cells [117] Incubated in the presence of 5- fluorouracil (5-FU) for 24 h, and further incubated without 5-FU for 28 days to eliminate 5-FU-sensitive cells. Certain stemness-genes such as OCT4 and NANOG were enhanced and spheres arose [118] Treat Capan-1 and Panc-1 cells with serial concentrations of gemcitabine and counting surviving cells after 6 days, Stem markers CD44,CD24,CD133,EpCAM,Oct4 and PDX1 increased [119] Xenograft tumours were dissociated into single cells and identified SP cells using FACS analysis SP displayed higher sphere-forming capacity, epithelial-mesenchymal transition and gemcitabine reisistant [120] Konckdown of ALDH1 in MIA PaCa-2 cell The IC50 of gemcitabine decreased, induction of apoptosis and S-phase arrest by gemcitabine. [121] Enriched pancreatic cancer stem CD44+/CD24+ cells in PANC-1 cells under sphere forming conditions Increased resistance to gemcitabine, migration ability, exhibit Epithelial to Mesenchymal Transition (EMT) [122] Konckdown of ALDH1 in MIA PaCa-2 cell target CD44 might be developed to block post- radiotherapy recurrence in patients [132]. target CD44 might be developed to block post- radiotherapy recurrence in patients [132]. efficiency to chemotherapeutic is highly related with TAMs [129]. The self-renewal potential and resistance to traditional treatment suggest that maybe strategies targeting CSC will improve clinical outcomes. At first, surface proteins used to enrich for pancreatic CSCs maybe good targets for treatment of pancreatic CSCs. For example, inhibit- ing c-Met with XL18427 or Alk-4 and −7 with SB431542 can eliminate the CSCs in tumors and enhance the anti- tumor effect of gemcitabine, reducing tumor burden in mice [90]. In addition to reagents targeting surface proteins of PCSCs, several cellular signaling pathways regulating the self-renewal and proliferation ability of PCSCs may be a potential targets against CSCs. Feldmann and colleagues showed that inhibition of Hedgehog pathway reduces a process of PC metastases, which has been linked to the invasion of CSCs [130]. However, Mueller et al. have shown that neither hh inhibition alone nor rapamycin alone or as supple- ments to chemotherapy can effectively diminish the CSC pool [131]. PCSCs, EMT and treatment resistance Chemotherapy, only with the com- bined inhibition of hh and mTOR (mammalian target of rapamycin) pathway played a role in reducing the number of CSCs to virtually undetectable levels in vitro and in vivo and significantly prolonged survival of mice. However, CD24, CD44, ESA, CD133 and CXCR4 are also expressed on normal stem cells, which share many pathways with CSCs. Consequently, these reagents might lack important specificity in tar- geting CSCs. It is important to find out effective tar- geting strategies achieving the aim of tackling CSCs without harming normal stem cells. Besides, the en- richment of the CD44+CD24+ESA+ cells was observed after the ionizing radiation treatment in human pri- mary pancreatic cancer xenografts. CD44 is required for post-radiation recurrence of xenograft tumors in mice. Antibody against CD44 eliminated bulk tumor cells as well as TICs from the tumors. Strategies to Competing interests The authors declare that they have no competing interests. 18. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133: 704–15. Ethics approval and consent to participate Not applicable. 20. Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest. 2003;112:1776–84. Acknowledgments 13. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM. Identification of pancreatic cancer stem cells. Cancer Res. 2007;67:1030–7. This work was supported by National Natural Science Foundation of China (grants 81370600), The Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning(TP2015022)and Shanghai Pujiang Program (15PJ1404800). This work was supported by National Natural Science Foundation of China (grants 81370600), The Program for Professor of Special Appointment 14. Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, Bruns CJ, Heeschen C. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell. 2007;1:313–23. (Eastern Scholar) at Shanghai Institutions of Higher Learning(TP2015022)and Shanghai Pujiang Program (15PJ1404800). Authors’ contributions LD and ZPT provided direction and guidance throughout the preparation of this manuscript. ZPT and LB conducted the literature review and drafted the manuscript. LFR, ZMC, WQ, LYH, YY reviewed the manuscript and made significant revisions on the drafts. All authors read and approved the final manuscript. 16. Miki J, Furusato B, Li H, Gu Y, Takahashi H, Egawa S, Sesterhenn IA, McLeod DG, Srivastava S, Rhim JS. Identification of putative stem cell markers, CD133 and CXCR4, in hTERT-immortalized primary nonmalignant and malignant tumor-derived human prostate epithelial cell lines and in prostate cancer specimens. Cancer Res. 2007;67:3153–61. 17. O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007; 445:106–10. Availability of data and materials 15. Frank NY, Margaryan A, Huang Y, Schatton T, Waaga-Gasser AM, Gasser M, Sayegh MH, Sadee W, Frank MH. ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma. Cancer Res. 2005;65: 4320–33. All the authors confirm the availability of data and materials. Received: 16 December 2016 Accepted: 23 February 2017 26. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–90. Conclusion Since cancers are heterogeneous, future novel treatment targets aimed at increasing patient survival will undoubt- edly need to consider the heterogeneity of cancer cells. Heterogeneity among cancer cells within the same tumor arises from a consequence of environmental dif- ferences, genetic mutation, and reversible changes in cellular properties. Among them one origin of such het- erogeneity is EMT and the existence of dedifferentiated cells with CSC-like properties. A better understanding of the properties of EMT and CSCs in PC will play an im- portant role in developing emerging and effective ther- apies targeting not only the bulk tumor but also the residual cluster of cells that are responsible for the relapse, metastasis and treatment resistance of the tumor. CSC properties have been put forward to explain diverse unsolved clinical problems. However, difficulties confirming solid CSC markers in order to isolate PCSCs have hindered the research identifying their existence in some cancers and studying their biology in clinical applications. It is now widely accepted that the presence of EMT and a PC subpopulation, with stem cell properties, play important roles in escaping from current clinical specific therapies. In this review we have discussed the detailed process and complex molecular signaling pathways con- trolling EMT and CSCs in PC during tumor formation, progression, and response to therapy. The combined use of different gene products altered in EMT and PCSCs may represent potential strategies for improving the ef- fectiveness of the diagnostic/prognostic methods and treatments efficacy for cancer patients in the clinics. Tar- geting CSCs via modification of the Wnt, HH and Notch signaling pathways of these cells holds the promise of preventing treatment resistance. However, additional Page 8 of 11 Page 8 of 11 Zhou et al. Molecular Cancer (2017) 16:52 studies are required to further confirm stem-cells markers and abnormal signaling pathways in CSCs, which have significant correlation with the high treat- ment resistance of PC. 11. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003;100:3983–8. 12. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003;63:5821–8. 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W4249745382.txt
null
fr
Histoires extraordinaires
˜The œmodern language journal/˜The œModern language journal
1,946
public-domain
129,422
ŒUVRES COMPLETES DE CHARLES BAUDELAIRE V HISTOIRES EXTRAORDINAIRES PARIS. - J. CLAtE, iMlRlMÊDH, RVE SAiNt-BENOIT , 7. — [649] ?f HISTOIRES EXTRAORDINAIRES EDGAR POE TRADUCTION DE CHARLES BAUDELAIRE 'g| M . L \\k ee PARIS MICHEL LÉVY FRÈRES, ÉD[TEURS RUE VIVIENNE 2 BIS, ET BOULEVARD DES ITALIENS, là A LA LIBRAIRIE NOUVELLE 1869 Droits de reproduction et de traduction réservés ce. )8b9 CETTE TRADUCTION EST DÉDIÉE MARIA GLEMM A LA MERE ENTHOUSIASTE ET DEVOUEE CELLE POUR QUI LE POETE A A ÉCRIT CES VERS Parce que je sens que, là-haut dans les Cieux, Les Anges, quand ils se parlent doucement à l'oreille, Ne trouvent pas, parmi leurs termes brûlants d'amour, D'expression plus fervente que celle de 7nère, Je vous ai dès longtemps justement appelée de ce grand nom, Vous qui êtes plus qu'une mère pour moi Et remplissez le sanctuaire de moncœur où laMort vous a installée En affranchissant l'âme de ma Virginia. Ma mère, ma propre mère, qui mourut de bonne heure, N'était que ma mère, à moi ; mais vous, Vous êtes la mère de celle que j'aimais si tendrement, Et ainsi vous m'êtes plus chère que la mère que j'ai connue De tout un infini, — juste comme ma femme Était plus chère à mon âme que celle-ci à sa propre essence. MG'r5€^4 EDGAR POE SA VIE ET SES ŒUVRES Quelque maître malheureux à qui Fatalité' a donné une chasse l'inexorable acharnée, toujours plus acharnée, jusqu'à ce que ses chants n'aient plus qu'un unique refrain, jusqu'à ce que les chants funèbres de son Espérance aient adopté ce mélancolique refrain : « Jamais ! Jamais plus » ! Edgar Poe. — Le Corbeau. Sur son trône d'airain le Destin, qui s'en raille, Imbibe leur éponge avec du fiel amer. Et la nécessité les tord dans sa tenaille. Théophile Gautier. — Ténèbres. Dans- ces derniers temps, un malheureux fut amené devant nos tribunaux, dont le front était illustré d'un rare et singulier tatouage ses yeux : Pas de chance! Il portait ainsi au-dessus de l'étiquette de sa vie, comme un livre son titre, et l'interrogatoire prouva que ce bizarre écriteau était cruelle- ment véridique. Jl y a, dans l'histoire littéraire, des destinées analogues, de vraies damnations, le mot guignon écrit en — des hommes qui portent caractère*^ mystérieux dans les plis sinueux de leur front. L'Ange aveugle de l'expiation s'est emparé d'eux et les fouette à tour de bras pour l'édification EDGAR POE 4 des autres. cial, En vain leur vie montre-t-elle des talents, des de la grâce vertus, et ; la Société a pour eux un anathème spé- accuse en eux les infirmités que sa persécution leur a données. — Que ne fit pas Hoffmann pour désarmer la destinée, et que n'entreprit pas Balzac pour conjurer la for- tune? — Existe-t-il donc une Providence diabolique qui prépare le malheur dès le berceau, — qui jette avec prémé- ditation des natures spirituelles et angéliques dans des milieux hostiles, comme des martyrs dans les cirques? Y a-t-il donc des âmes sacrées, vouées à l'autel, condamnées à marcher à la mort et à la gloire à travers leurs propres ruines ? Le cauchemar des Ténèbres assiégera-t-il éternellement ces âmes de choix ? Vainement elles se elles elles se débattent, vainement forment au monde, à ses prévoyances, à ses ruses perfectionneront prudence, boucheront toutes la ; les issues, matelasseront les fenêtres contre les projectiles du ha- sard; mais le Diable entrera par une serrure; une perfection sera le défaut de leur cuirasse, et une qualité superlative le germe de leur damnation. L'aigle, pour le briser, du haut du firmament, Sur leur front découvert lâchera la tortue, Car ils doivent périr inévitablement. Leur destinée est écrite dans toute leur constitution, elle brille d'un éclat sinistre dans leurs regards et dans leurs gestes, elle circule dans leurs artères avec chacun de leurs globules sanguins. Un écrivain célèbre de notre temps a écrit un livre pour démontrer que le poëte ne pouvait trouver une bonne place ni dans une société démocratique ni dans une aristocratique, pas plus dans une république que dans une monarchie SA VIE ET SES OEUVRES. 5 absolue ou tempérée. Qui donc a su lui répondre péremptoi- rement? J'apporte aujourd'hui une nouvelle légende à l'ap. pui de sa thèse, j'ajoute j'ai un saint nouveau au martyrologe; à écrire l'histoire d'un de ces illustres malheureux, trop de poésie et de passion, qui riche est venu, après tant d'autres, faire en ce bas monde le rude apprentissage du gé- nie chez les âmes inférieures. Lamentable tragédie que la d'Edgar Poe vie Sa mort, ! dénùment horrible dont l'horreur est accrue par la lité! — De tous les documents que moi la j'ai lus est conviction que les États-Unis ne trivia- résultée pour furent pour Poe qu'une vaste prison qu'il parcourait avec l'agitation fiévreuse d'un èlre fait pour respirer dans un monde plus aromal, qu'une grande barbarie éclairée au gaz, intérieure, spirituelle de poëte qu'un effort ou — même d'ivrogne, perpétuel pour échapper a — que sa vie et n'était l'influence de cette atmosphère antipathique. Impitoyable dictature que celle de l'opinion dans les sociétés démocratiques ; n'implorez d'elle ni charité ni indulgence, ni élasticité plication de ses lois aux cas quelconque dans l'ap- multiples et complexes de la vie morale. On dirait que de l'amour impie de la liberté est née une tyrannie nouvelle, la tyrannie des bêtes, ou zoocratie, qui par son insensibilité féroce ressemble Jaggernaut. à l'idole de — Un biographe nous dira gravement — bien intentionné, le brave homme — que Poe, s'il il est avait voulu régulariser son génie et appliquer ses facultés créatrices d'une manière plus appropriée au sol américain,- aurait pu devenir — un autre, — un naïf cynique, celui-là, — que, quelque beau que soit un auteur à argent, a 7noney making aiilhor ; le génie de Poe, talent, le talent génie. il eût mieux valu pour lui n'avoir que du s'escomptant toujours plus facilement que le Un autre, qui a dirigé des journaux et des revues, un 1. EDGAR POE 6 ami du poëte, qu on était qu'il écrivait de l'employer et avotie qu'il était difficile obligé de le payer moins que d'autres, parce dans un style trop au-dessus de vulgaire.. Quelle odeur de magasin! comme disait Joseph de Maistre. Quelques-uns ont osé davantage, et, unissant l'inintelli- gence la plus lourde de son génie à la férocité de l'hypocriaprès sa soudaine sie bourgeoise, l'ont insulté à l'envi; et, disparition, ont rudement morigéné ce cadavre, ils — parti- culièrement M. Rufus Griswoid, qui, pour rappeler ici l'expression vengeresse de M. George Graham, a commis alors une immortelle infamie. Poe, éprouvant peut-être le sinistre pressentiment d'une fin subite, avait désigné MM. Griswoid et Willis pour mettre ses œuvres en ordre, écrire sa vie et restaurer sa mémoire. Ce pédagogue-vampire a diffamé lon- guement son ami dans un énorme article, plat et haineux, juste en tête de l'édition posthume de ses œuvres. — Il n'existe donc pas en Amérique d'ordonnance qui interdise aux chiens l'entrée des cimetières? — Quanta M. Willis, il a prouvé, au contraire, que la bienveillance et la décence marchaient toujours avec le véritable esprit, et que la charité envers nos confrères, qui est un devoir moral, était aussi un des com- mandements du goût. Causez de Poe avec un Américain, il avouera peut-être son génie, peut-être même s'en montrera-t-il fier un ton sardonique supérieur qui sent son ; mais, avec homme positif, il vous parlera de la vie débraillée du poète, de son haleine alcoolisée qui aurait pris feu à ses la flamme d'une chandelle, de habitudes vagabondes ; il vous dira que c'était un être erratique et hétéroclite, une planète désorbitée, qu'il roulait sans cesse de Baltimore à New- York, de New-York à Philadelphie, de Philadelphie à Boston, de Boston à Baltimore, de Baltimore à Kichmond. Et si, le cœur ému par ces préludes SA VIE ET SES OEUVRES. 7 d'une histoire navrante, vous donnez à entendre que l'indi- vidu n'est peut-être pas seul coupable et qu'il doit être difficile de penser et d'écrire commodément dans un pays où il y a des millions de souverains, un pays sans capitale à pro- prement parler, ses sans aristocratie, et — alors vous verrez yeux s'agrandir et jeter des éclairs, la bave du patrio- tisme souffrant lui monter aux lèvres, et l'Amérique, par sa bouche, lancer des injures à l'Europe, sa vieille mère, et à philosophie des anciens jours. la moi la persuasion s'est Je répète que pour faite qu'Edgar Poe et sa patrie n'étaient pas de niveau. Les États-Unis sont un pays gigantesque et enfant, naturellement jaloux du vieux continent. Fier de son développement matériel, anormal et presque monstrueux, ce nouveau venu dans foi naïve dans vaincu, finira la l'histoire a une toute-puissance de l'industrie; il est con- comme quelques malheureux parmi nous, qu'elle par manger le Diable. une valeur si grande Le temps et l'argent ont là-bas L'activité matérielle, ! exagérée jus- qu'aux proportions d'une manie nationale, laisse dans les esprits bien la terre. peu de place pour les choses qui ne sont pas de Poe, qui était de bonne souche, et qui d'ailleurs professait que le grand malheur de son pays était de n'avoir pas d'aristocratie de race, attendu, disait-il, que chez un peuple sans aristocratie le culte du Beau ne peut que se cor- rompre, s'amoindrir et disparaître, — qui accusait chez ses concitoyens, jusque dans leur luxe emphatique etcoùteux, tous lessymptômes du mauvais goûtcaractéristique des parvenus,— qui considérait le Progrès, la grande idée moderne, comme uneextasede gobe-mouches, et qui appelait les perfectionne- ments de l'habitacle humain des cicatrices et des abominations rectangulaires, — Poe lièrement solitaire. ne croyait qu'à l'immuable, à l'éternel Il était là-bas un cerveau singu- EDGAR POE 8 au self'-same, et il jouissait — cruel privilège dans une so— de ce grand bon sens à ciété amoureuse d'elle-même! la Machiavel qui marche devant lumineuse, à travers le le désert sage, de comme une colonne l'histoire. — Qu'eût-il pensé, qu'eùt-il écrit, l'infortuné, s'il avait entendu la théo- logienne du sentiment supprimer l'Enfer par amitié pour le genre humain, le philosophe du chiffre proposer un système d'assurances, une souscription à un sou par tête pour la sup- pression de la guerre, — et l'abolition de de l'orthographe, ces deux d'autres malades qui écrivent, la peine de mort et — et tant V oreille inclinée au vent, folies corrélatives ! des fantaisies giratoires aussi flatueuses que l'élément qui les leur dicte ? — Si vous ajoutez à cette vision impeccable du vrai, véritable infirmité dans de certaines circonstances, une délicatesse exquise de sens qu'une note fausse torturait, une finesse de goût que tout, excepté l'exacte proportion, révoltait, un amour insatiable du Beau, qui avait pris la puissance morbide, vous ne vous étonnerez pas que d'une passion pour un pareil homme la vie soit devenue un enfer, et qu'il ait mal fini; vous admirerez qu'il ait pu durer aussi long- temps. n La famille de Poe était une des plus respectables de Baltimore. Son grand-père maternel avait servi comme quartermaster- gêner al dans la guerre de l'indépendance, et la Fayette l'avait en haute estime et amitié. Celui-ci, lors de son dernier voyage aux États-Unis, voulut voir la veuve du général et lui témoigner sa gratitude pour les services que lui avait ren- SA VIE ET SES OEUVRES. dus son mari. Le bisaïeul avait épousé une fille de l'amiral anglais Mac Bride, qui était allié avec les plus nobles mai- sons d'Angleterre. David Poe, père d'Edgar et fils du général, s'éprit violemment d'une actrice anglaise^ Elisabeth Arnold, célèbre par sa beauté; s'enfuit avec elle et l'épousa. il Pour mêler plus intimement sa destinée à la sienne, il se fit co- médien et parut avec sa femme sur différents théâtres, dans les principales villes de l'Union. Les deux époux moururent à même temps, laissant dans l'abandon Richmond, presque en et le dénûment le plus complet trois enfants en bas- âge, dont Edgar. Edgar Poe était né à Baltimore, en 1813. — C'est d'après son propre dire que je donne cette date, car il a réclamé contre l'affirmation de Griswold, qui place sa naissance en 1811. — Si jamais l'esprit de roman, pour me servir d'une expression de notre poëte, a présidé à une naissance, prit sinistre et orageux ! — certes il — es- présida à la sienne. Poe fut véritablement l'enfant de la passion et de l'aventure. riche négociant de la ville, Un M. Allan, s'éprit de ce joli mal- heureux que la nature avait doté d'une manière charmante, et, comme il n'avait pas d'enfants, il l'adopta. Celui-ci s'ap- pela donc désormais Edgar Allan Poe. Il fut ainsi élevé dans une belle aisance et dans l'espérance légitime d'une de ces fortunes qui donnent au caractère une superbe certitude. Ses parents adoptifs l'emmenèrent dans un voyage qu'ils firent en Angleterre, en Ecosse et en Irlande, et, avant de retourner dans leur pays, ils le laissèrent chez le docteur Bransby, qui tenait une importante maison Stoke-Newington, près de Londres. William Wilson, décrit d'éducation à — Poe a lui-même, dans cette étrange maison bâtie dans le vieux style d'Elisabeth, et les impressions de sa vie d'écolier. EDGAR POE 10 Jl Richmond en 1822, et continua ses éludes en revint à Amérique, sous la direction des meilleurs maîtres de l'endroit. il A l'université de Gharlottesville, où se distingua non-seulement par il entra en 1825, une intelligence quasi mi- raculeuse, mais aussi par une abondance presque sinistre de passions, — une précocité vraiment américaine, — qui, lement, fut la cause de son expulsion. 11 fina- est bon de noter en passant que Poe avait déjà, à Gharlottesville, manifesté une aptitude des plus remarquables pour les sciences physiques et mathématiques. Plus tard, dans ses étranges contes, il en fera un usage fréquent et en tirera des moyens très -inat- tendus. Mais j'ai des raisons de croire que ce n'est pas à cet ordre de compositions qu'il attachait le plus d'importance, et que il — peut-être môme à cause de cette précoce aptitude — n'était pas loin de les ries, considérer comme de faciles jongle- comparativement aux ouvrages de pure imagination. — Quelques malheureuses dettes de jeu amenèrent une brouille momentanée entre lui et son père adoptif, et Edgar des plus curieux et — fait qui prouve, quoi qu'on ait dit, une dose de chevalerie assez forte dans son impressionnable cerveau, — conçut le projet de se mêler à la guerre des Hellènes et d'aller combattre les Turcs. Il partit donc pour la Grèce. Que devint-il en Orient? qu'y fit-il? étudia-t-il classiques de la Méditerranée? — pourquoi le retrouvons- nous à Saint-Pétersbourg, sans passe-port, compromis, dans quelle sorte d'affaire, — les rivages et obligé d'en appeler au ministre américain, Henry Middleton, pour échapper à la pénalité russe et retourner chez lui ? lui seul aurait — on l'ignore ; il y a là une lacune que pu combler. La vie d'Edgar Poe, sa jeunesse, ses aventures en Russie et sa correspondance ont été long- temps annoncées par les journaux américains et n'ont jamais paru. SA VIE ET SES OEUVRES. Revenu en Amérique en 1829, 11 manifesta le désir d'en- il trer à l'école militaire de West-Point; il y fut admis en effet, et, là comme ailleurs, il donna les signes d'une intelligence admirablement douée, mais indisciplinable, quelques mois, il fut rayé. sa famille adoptive un et, au bout de — En même temps se passait dans événement qui devait avoir les consé- quences les plus graves sur toute sa vie. Madame Allan, pour laquelle liale, il semble avoir éprouvé une affection réellement fi- mourait, et M. Allan épousait une femme toute jeune. Une querelle domestique prend ici place, — une histoire bi- zarre et ténébreuse que je ne peux pas raconter, parce qu'elle n'est clairement expliquée par aucun biographe. Il n'y a donc pas lieu de s'étonner qu'il se soit définitivement séparé de M. Allan, et que celui-ci, qui eut des enfants de son second mariage, l'ait complètement frustré de sa succession. Peu de temps après avoir un petit volume de poésies; quitté Richmond, Poe publia c'était en vérité une aurore éclatante. Pour qui sait sentir la poésie anglaise, il y a là déjà l'accent extraterrestre, le calme dans la mélancolie, la solennité délicieuse, l'expérience précoce, —j'allais, je crois, dire expérience innée, — qui caractérisent les grands poètes. La misère le fit quelque temps soldat, et il est présumable qu'il se servit des lourds loisirs de la vie de garnison pour préparer les matériaux de ses futures compositions, — com- positions étranges, qui semblent avoir été créées pour nous démontrer que l'étrangeté est une des parties intégrantes du beau. Rentré dans la vie littéraire, le seul élément où puis- sent respirer certains êtres déclassés, Poe se mourait dans une misère extrême, quand un hasard heureux le releva. Le propriétaire d'une revue venait de fonder deux prix, l'un pour le meilleur conte, l'autre pour le meilleur poëme. Une écri- ture singulièrement belle attira les yeux de M. Kennedy, qui EDGAR POE 42 présidait le comité, et lui donna l'envie d'examiner lui-même les manuscrits. Il trouva que Poe avait gagné se deux les prix; mais un seul lui fut donné. Le président de la commis- sion fut curieux de voir l'inconnu. L'éditeur du journal lui amena un jeune homme d'une beauté frappante, en guenilles, boutonné jusqu'au menton, et qui avait l'air d'un gentil- homme aussi fier qu'affamé. Kennedy se conduisit bien. Il fit faire à Poe la connaissance d'un M. Thomas White, qui fon- dait à Richmond le Southern Literary Messenger. M. White était un il lui homme d'audace, mais sans aucun talent littéraire; fallait un aide. Poe se trouva donc tout jeune, —à — directeur d'une revue dont la destinée re- posait tout entière sur lui. Cette prospérité, il la créa. vingt-deux ans, Le Southern JAterary Messenger a reconnu depuis lors que c'était à cet excentrique maudit, à cet ivrogne incorrigible, qu'il devait sa clientèle et sa fructueuse notoriété. C'est dans ce magasin que parut pour la première fois V Aventure sans pareille d'un certain Ilatis Pfaall, que nos lecteurs verront et plusieurs autres défller sous leurs contes yeux. Pendant près de deux ans, Edgar Poe, avec une ardeur merveilleuse, étonna son public par une série de compositions d'un genre nouveau et par des articles critiques dont la vivacité, la netteté, la sévérité raisonnées étaient bien faites pour attirer les yeux. Ces articles portaient sur des livres de tout genre, et la forte éducation que servit pas le médiocrement. jeune homme s'était faite ne le est bon qu'on sache que cette Il besogne considérable se faisait pour cinq cents dollars, c'està-dire deux mille sept cents francs par an. — Immédiate- — dit Griswold, ce qui veut dire se croyait donc ment, assez riche, l'imbécile! » — il : « Il épousa une jeune fille, belle, charmante, d'une nature aimable et héroïque, mais ne possé- dant pas un sou, — ajoute le même Griswold avec une SA VIE ET SES nuance de dédain. C'était ŒUVRES. 13 une demoiselle Virginia Clemm, sa cousine. Malgré les services rendus à son journal, M. White se brouilla avec Poe au bout de deux ans, à peu près. La raison de cette séparation se trouve évidemment dans les d'hypocondrie et les crises d'ivrognerie du poète, accès — ac- cidents caractéristiques qui assombrissaient son ciel spirituel, comme ces nuages lugubres qui donnent soudainement au plus romantique paysage un air de mélancolie en apparence irréparable. placer sa tenle, — Dès lors, nous verrons l'infortuné dé- comme un homme du désert, et transporter ses légers pénates dans les principales villes de l'Union. Partout, dirigera des revues ou y collaborera d'une manière il éclatante. ticles II répandra avec une éblouissante rapidité des ar- critiques, philosophiques, et des contes pleins de magie qui paraissent réunis sous le titre de Taies of the Grotesque and the Arabesque, — titre remarquable et inten- tionnel, car les ornements grotesques et arabesques repoussent la figure humaine, et l'on verra qu'à littérature de Poe est extra beaucoup d'égards la ou suprahumaine. Nous appren- drons par des notes blessantes et scandaleuses insérées dans les journaux, que M. Poe et sa femme se trouvent dangereu- sement malades à Fordham et dans une absolue misère. Peu de temps après la mort de madame Poe, le poëte subit les premières attaques du delirium tremens. Une note nouvelle paraît soudainement dans un journal, cruelle, — qui accuse son mépris lui fait — celle-là, plus que et son dégoût du monde, et un de ces procès de tendance, véritables réquisitoires de l'opinion, contre lesquels il eut toujours à se défendre, une des luttes les plus stérilement fatigantes que — je con- naisse. Sans doute il gagnait de l'argent, et ses travaux littéraires V U EDGAR POE pouvaient à peu près le faire vivre. Mais j'ai les preuves qu'il avait sans cesse de dégoûtantes difficultés à surmonter. Il rêva, comme tant d'autres écrivains, voulut être chez lui, et souffert le fait une Revue à lui, est qu'il avait pour désirer ardemment cet abri il suffisamment définitif pour sa pensée. Pour arriver à ce résultat, pour se procurer une somme d'argent suffisante, il eut recours aux lectures. On sait ce le que sont ces lectures, — une espèce de spéculation, Collège de France mis à la disposition de tous les littéra- teurs, l'auteur ne publiant sa lecture qu'après qu'il en a tiré toutes les recettes qu'elle peut rendre. Poe avait déjà donné à New- York une lecture A' Eurêka, son poëme cosmogonique, qui avait même soulevé de grosses discussions. Il imagina cette fois de donner des lectures dans son pays, dans la Virginie. Il comptait, comme il l'écrivit à Willis, faire une tour- née dans l'Ouest et le Sud, et il espérait le concours de ses amis littéraires et de ses anciennes connaissances de collège et de West-Point. Virginie, et jeune, si Il donc visita Richmond les revit celui principales villes de la qu'on y avait connu si pauvre, si délabré. Tous ceux qui n'avaient pas vu Poe depuis les jours de son obscurité accoururent en foule pour contempler leur illustre compatriote. Il apparut, beau, élégant, correct comme le génie. Je crois même que, depuis quelque temps, il avait poussé la condescendance jusqu'à se faire admettre dans une société de tempérance. Il choisit un thème aussi large qu'élevé le développa : le Principe de la Poésie, croyait, en vrai poëte qu'il était, Il est et il avec cette lucidité qui est un de ses privilèges. que le but de la poésie de même nature que son principe, et qu'elle ne doit pas avoir en vue autre chose qu'elle-même. Le bel accueil qu'on lui fit inonda son pauvre cœur d'orgueil et de joie; il se montrait tellement enchanté, qu'il par- SA VIE ET SES OEUVRES. ]ait 15 de s'établir définitivement à Richmond et de finir sa vie dans les lieux que son enfance lui avait rendus chers. Cependant, il avait affaire à New-York, et il partit le 4 octobre, se plaignant de frissons et de faiblesses. Se sentant toujours assez mal en arrivant à Baltimore, le 6, au soir, ses bagages à l'embarcadère d'oii il fit porter devait se diriger sur Phi- il dans une taverne pour y prendre un excitant quelconque. Là, malheureusement, il rencontra de ladelphie, et entra vieilles connaissances et s'attarda. les pâles ténèbres du petit jour, voie, Le lendemain matin, dans un cadavre fut trouvé sur la — est-ce ainsi qu'il faut dire? — non, un corps vivant encore, mais que la Mort avait déjà marqué de sa royale estampille. Sur ce corps, dont on ignorait le nom, on ne trouva ni papiers ni argent, et on le porta dans un hôpital. C'est là que Poe mourut, le soir même du dimanche, 7 octobre 1849, à l'âge de trente-sept ans, vaincu par le delirium tremens, ce terrible visiteur qui avait déjà hanté son cerveau une ou deux fois. Ainsi disparut de ce monde un des plus grands héros littéraires, l'homme -de génie qui avait écrit dans le Chat noir ces mots fatidiques : Quelle maladie est compa- rable à l'alcool ! Cette mort est presque un suicide, depuis longtemps. — un suicide préparé Du moins, elle en causa le scandale. La clameur fut grande, et la vertu donna carrière à son cant emphatique, librement et voluptueusement. Les oraisons funèbres les plus indulgentes ne purent pas ne pas donner place à l'inévitable morale bourgeoise qui n'eut garde de manquer une si admirable occasion. M. Griswold diffama; M. Willis, sincèrement affligé, fut mieux que convenable. — Hélas! celui qui avait franchi les hauteurs les plus ardues de l'esthétique et plongé dans les abîmes les moins explorés de l'intellect humain, celui qui, à travers une vie qui ressemble EDGAR POE 1G à une tempête sans accalmie, avait trouvé des moyens nou- veaux, des procédés inconnus pour étonner l'imagination, pour séduire les esprits assoifés de Beau, venait de mourir en qnelques heures dans un lit d'hôpital, tant de grandeur et tant de malheur, — quelle destinée! Et pour soulever un tour- billon de phraséologie bourgeoise pour devenir la pâture et le thème des journalistes vertueux ! Ut declamatio fias ! Ces spectacles ne sont pas nouveaux; il est rare qu'une sépulture fraîche et illustre ne soit pas un rendez-vous de scandales. D'ailleurs, la société n'aime pas ces enragés mal- heureux, et, soit qu'ils troublent ses fêtes, soit qu'elle les considère naïvement comme des remords, elle a incontesta- blement raison. Qui ne se rappelle les déclamations pari- siennes lors de la mort de Balzac, qui cependant mourut cor- rectement? — Et plus récemment encore, — y a aujourd'hui, — quand un écrivain d'une honnêteté il 26 janvier, juste un an, admirable, d'une haute intelligence, et qui fut toujours lucide, alla discrètement, sans déranger personne, — si dis- crètement que sa discrétion ressemblait à du mépris, délier son — âme dans la rue la plus noire qu'il put trouver, — quelles dégoûtantes homélies! — quel assassinat raffiné! Un journaliste célèbre, à qui Jésus n'enseignera jamais les manières généreuses, trouva l'aventure assez joviale pour la célébrer en un gros calembour. — Parmi l'énumération nom- breuse des droits de l'homme que la sagesse du xix« siècle recommence si souvent et si complaisamment, deux assez importants ont été oubliés, qui sont le droit de se contredire et le droit de s'e7i aller. Mais la société regarde celui qui s'en va comme un insolent; elle châtierait volontiers certaines SA VIE ET SES OEUVRES. 17 dépouilles funèbres, comme ce malheureux soldat, atteint de vampirisme, que fureur. — Et; la vue d'un cadavre exaspérait jusqu'à la cependant, on peut dire que, sous la pression de certaines circonstances, après un sérieux examen de certaines incompatibilités, avec de fermes croyances à de certains dogmes et métempsycoses, — on peut dire, sans emphase et sans jeu de mots, que le suicide est parfois l'action — Et raisonnable de la vie. ainsi se plus la forme une compagnie de fantômes déjà nombreuse, qui nous hante familièrement, et dont chaque membre vient nous vanter son repos actuel et nous verser ses persuasions. Avouons toutefois que la lugubre fin de l'auteur à^ Eurêka suscita quelques consolantes exceptions, sans quoi il faudrait désespérer, et la place ne serait plus tenable. M. Willis, comme je l'ai dit, parla honnêtement, et même avec émotion, des bons rapports qu'il avait toujours eus avec Poe. MM. John Neal et George Graham rappelèrent M. Griswold à la pudeur. M. Longfellow — et celui-ci est méritant que Poe l'avait cruellement maltraité d'autant plus — sut louer d'une manière digne d'un poëte sa haute puissance comme poëte et comme prosateur. Un inconnu écrivit que l'Amérique littéraire avait perdu sa plus forte tête. Mais le cœur brisé, le cœur déchiré, le cœur percé des sept glaives fut celui de madame Clemm. Edgar était à la fois son fils et sa fille. Rude destinée, dit Willis, à qui j'emprunte ces détails, presque qu'elle surveillait et mot pour mot, rude destinée que celle protégeait. Car Edgar homme embarrassant; outre qu'il Poe écrivait avec était une un fasti- dieuse difficulté et dans un slyle trop au-dessus du niveau intellectuel commun pour qu'on pût le payer cher, il était toujours plongé dans des embarras d'argent, et souvent lui et sa femme malade manquaient des chosi^^ les phis néces4- EDGAR POE 18 Un jour, Willis vit entrer dans son bureau saires à la vie. une femme, vieille, douce, grave. C'était madame Clemm. Elle cherchait de l'ouvrage pour son cher Edgar. graphe dit qu'il fut Le bio- sincèrement frappé, non pas seulement de l'éloge parfait, de l'appréciation exacte qu'elle faisait des talents de son fils, — mais aussi de tout son être extérieur, de sa voix douce et triste, de ses manières un peu surannées, mais belles et grandes. Et pendant plusieurs années, ajoutet-il, nous avons vu cet infatigable serviteur du génie, pau- vrement et insuffisamment vêtu, allant de journal en journal pour vendre tantôt un poëme, tantôt un article, disant quel- — unique explication, unique quefois qu'i7 était malade, raison, invariable excuse qu'elle donnait quand son fils se trouvait frappé momentanément d'une de ces stérilités que connaissent les écrivains nerveux, — et ne permettant jamais à ses lèvres de lâcher une syllabe qui pût être interprétée comme un doute, comme un amoindrissement de confiance dans le génie et la volonté de son bien-aimé. Quand sa fille mourut, elle s'attacha au survivant de la désastreuse avec une ardeur maternelle renforcée, prit soin de lui, le surveillant, contre lui-même. Certes, et impartiale raison, — si le elle bataille vécut avec défendant contre la lui, vie et — conclut Willis avec une haute le dévouement de la femme, né avec un premier amour et entretenu par la passion humaine, glorifie et consacre son objet, que ne dit pas en faveur de celui qui l'inspira un dévouement comme celui-ci, pur, dés- intéressé et saint comme une sentinelle divine? Les détrac- teurs de Poe auraient dû en effet remarquer qu'il est des séductions si puissantes qu'elles ne peuvent être que des vertus. On devine combien terrible fut la nouvelle pour heureuse femme. Elle écrivit à Willis une quelques lignes : lettre la mal- dont voici SA VIE ET SES OEUVRES. « J'ai appris ce 10 matin la mort de mon bien-aimé Eddie... Pouvez-voiis me transmettre quelques détails, quelques circonstances?... Oh! n'abandonnez pas votre pauvre amie dans cette amère affliction... Dites à M... de venir me voir; j'ai à m'acquitter envers lui d'une commission de la part de mon pauvre Eddie... Je n'ai pas besoin de vous prier d'annoncer sa mort, et de parler bien de lui. Je sais que vous le ferez. Mais dites bieji quel fils affectueux il était pour moi, sa pauvre mère désolée... Cette >> emme m'apparaît grande et plus qu'antique. Frappée d'un coup irréparable, elle ne pense qu'à la réputation de celui qui était tout pour elle, et il ne suffit pas, pour la con- tenter, qu'on dise qu'il était un génie, il faut qu'on sache qu'il était un homme de devoir et d'affection. Il est évident que cette mère haut ciel — flambeau et foyer allumé par un rayon du plus — a été donnée en exemple à nos races trop peu soigneuses du dévouement, de l'héroïsme, et de tout ce qui est plus que e devoir. N'était-ce pas justice d'inscrire au- dessus des ouvrages du poëte le nom de celle qui fut le soleil moral de sa vie? Il embaumera dans sa gloire le femme dont la tendresse savait panser ses nom de la plaies, et dont l'image voltigera incessamment au-dessus du martyrologe de la littérature. III La vie de Poe, ses mœurs, ses manières, son être physique, tout ce qui constitue l'ensemble de son personnage, nous apparaissent comme quelque chose de ténébreux et de brillant à la fois. Sa personne était singulière, séduisante et, EDGAR POE 20 comme ses ouvrages, marquée d'un indéfinissable cachet de mélancolie. Du reste, il toutes façons. Jeune, il était remarquablement bien avait montré doué de une rare aptitude pour tous les exercices physiques, et, bien qu'il fût petit, avec des pieds et des mains de femme, tout son être portant d'ailleurs ce caractère de délicatesse féminine, il était plus que robuste et capable de merveilleux traits de force.. 11 a, dans sa jeunesse, gagné un pari de nageur qui dépasse la mesure ordinaire du possible. On dirait que la Nature à ceux fait dont elle veut tirer de grandes choses un tempérament énergique, comme elle donne une puissante vitalité aux arbres qui sont chargés de symboliser le deuil et la douleur. Ces hommes-là, avec des apparences quelquefois chétives, sont taillés en athlètes, bons pour l'orgie et pour le travail, prompts aux excès et capables d'étonnantes sobriétés. est quelques points relatifs à Il Edgar Poe, sur lesquels il y a accord unanime, par exemple sa haute distinction naturelle, son éloquence et sa beauté, dont, à ce qu'on tirait un peu vanité. Ses manières, mélange singulier de dit, il hauteur avec une douceur exquise, étaient pleines de certitude. Physionomie, démarche, gestes, airs de tête, tout le désignait, surtout dans ses bons jours, comme une créature d'élection. Tout son être était réellement II respirait une solennité pénétrante. marqué par la nature, comme ces figures de passants qui tirent l'œil sa mémoire. Le pédant et aigre Grisvvold lui-même avoue de l'observateur et préoccupent que, lorsqu'il alla rendre visite à Poe, et qu'il il le trouva pâle malade encore de la -mort et de la maladie de sa femme, et fut frappé outre mesure, non-seulement de la perfection de ses manières, mais encore de la physionomie aristocratique, de l'atmosphère assez parfumée de son appartement, d'ailleurs modestement meublé. Griswold ignore que le poêle a SA VIE ET SES OEUVRES. 21 plus que tous les hommes ce merveilleux privilège attribué femme parisienne et à l'Espagnole, de savoir se parer à la avec un rien, et que Poe, amoureux du beau en toutes choses, aurait trouvé l'art de transformer une chaumière en un palais d'une espèce nouvelle. N'a-t-il pas écrit, avec l'esprit le plus original et le plus curieux, des projets de mobiliers, des plans de maisons de campagne, de jardins et de réformes de paysages? Il existe une lettre charmante de madame Frances Osgood, nous donne sur ses qui fut une des amies de Poe, et qui mœurs, sur sa personne et sur sa vie de ménage, curieux détails. Cette femme, qui était les plus elle-même un litté- rateur distingué, nie courageusement tous les vices et toutes les fautes reprochés au poëte. « Avec les hommes, dit-elle à Griswold, peut-être était-il que vous le dépeignez, tel et comme homme vous pouvez avoir raison. Mais je pose en fait qu'avec les tout autre, et que jamais femme n'a femmes il était pu connaître M. Poe sans éprouver pour lui un profond intérêt. Il ne m'a jamais apparu que comme un modèle d'élégance, de distmction et de générosité... » La première fois que nous nous vîmes, ce fut à Astor- Ilouse. Willis m'avait fait passer à table d'hôte le Corbeau, sur lequel l'auteur, me dit-il, désirait connaître mon opinion. La musique mystérieuse et surnaturelle de ce poëme étrange me pénétra si intimement, que, lorsque j'appris que Poe désirait m'etre présenté, j'éprouvai un sentiment singulier et qui ressemblait à de l'effroi. tête, ses yeux 11 parut avec sa belle et orgueilleuse sombres qui dardaient une lumière d'élection, une lumière de sentiment et de pensée, avec ses manières qui étaient un mélange intraduisible de hauteur et de suavité, — il me salua, calme, grave, presque froid; mais sous EDGAR POE 22 cette froideur vibrait une sympathie si marquée, que je ne pus m'empôcher d'en être profondément impressionnée. partir de ce moment jusqu'à sa mort, A nous fûmes amis..., et je sais que, dans ses dernières paroles, j'ai eu ma part de sou- m'a donné, avant que sa raison ne fût cul- venir, et qu'il butée de son trône de souveraine, une preuve suprême de sa fidélité » en amitié. C'était surtout dans son intérieur, à la fois simple et poétique, que le caractère d'Edgar Poe apparaissait pour moi dans sa plus belle lumière. Folâtre, tantôt docile et tantôt affectueux, spirituel, méchant comme avait toujours pour sa jeune, douce et un enfant adorée gâté, il femme, et pour tous ceux qui venaient, même au milieu de ses plus fatigantes besognes littéraires, un mot aimable, un sourire bienveillant, des attentions gracieuses et courtoises. sait d'interminables Il pas- heures à son pupitre, sous le portrait de sa Lenore, l'aimée et la morte, toujours assidu, toujours ré- signé et fixant avec son admirable écriture les brillantes étonnant cerveau incessam- fantaisies qui traversaient son ment en éveil. — Je me rappelle l'avoir vu un matin plus joyeux et plus allègre que de coutume. Virginia, sa douce femme, m'avait priée d'aller les voir et il m'était impossible de résister à ses sollicitations... Je le trouvai travaillant à la série d'articles qu'il a publiés sous le titre : Ihe Literali of New-York. ((Yoyez^ » me dit-il, en déployant avec 'un rire de triomphe plusieurs petits rouleaux de papier (il écrivait sur des bandes étroites, sans doute pour conformer sa copie à h juslifîcalio7i dos journaux), «je vais vous montrer par la » différence des longueurs les divers degrés d'estime que j'ai » pour chaque membre de votre gent littéraire. Dans chacun » de ces papiers, l'un de vous est peloté et proprement discuté. » — Venez ici, Virginia, et aidez-moi ! » Et ils les déroulèrent SA VIE ET SES OEUVRES. tous un à un. A la fin, y en avait il 23 un qui semblait inter- minable. Virginia, tout en riant, reculait jusqu'à un coin de la chambre le tenant par un bout, et son mari vers un. autre coin avec l'autre bout. « Et quel est l'heureux, dis-je, que » vous avez jugé digne de cette incommensurable douceur? » L'entendez-vous, s'écria-t-il, » cœur ne lui avait pas déjà dit que c'est elle-même » — comme si son vaniteux petit ! » Quand je fus obligée de voyager pour ma santé, j'entre- tins une correspondance régulière avec Poe, obéissant en cela aux vives sollicitations de sa pouvais obtenir sur taires... lui femme, qui croyait que je une influence et un ascendant salu- Quant à l'amour et à la existaient moi un spectacle avec trop délicieux, je n'en saurais parler qui confiance entre sa femme et lui, et qui étaient pour de conviction, avec trop de chaleur. Je néglige quelques petits épisodes poétiques dans lesquels le jeta son tempérament romanesque. Je pense qu'elle était la seule femme qu'il ait toujours vérita- blement aimée... » Dans les Nouvelles de Poe, n'y a jamais d'amour. il Du moins Ligeia, Eleonora, ne sont pas, à proprement parler, dos histoires d'amour, l'idée principale sur laquelle pivote que l'œuvre étant tout autre. Peut-être croyait-il n'est pas une langue à la hauteur de ce bizarre et presque la prose intraduisible sentiment; car ses poésies, en revanche, en sont fortement saturées. La divine passion y apparaît magnifique, étoilée, et toujours voilée d'une irrémédiable mélancolie. Dans ses articles, il parle quelquefois de l'amour, et même comme d'une chose dont le nom fait frémir la plume. Dans the Domain of Arnhaim, il affirmera que les quatre conditions élémentaires du bonheur sont : la vie en plein air, Va?nour d'une femme, le détachement de toute ambition et la création d'un Beau nouveau. — Ce qui corrobore l'idée de M"'"'Frances EDGAR POE 24 Osgood relativement au respect chevaleresque de Poe pour les femmes, c'est que, malgré son prodigieux talent pour le grotesque et l'horrible, il n'y a pas dans tout son œuvre un môme aux jouis- seul passage qui ait trait à la lubricité ou sances sensuelles. Ses portraits de femmes sont, pour ainsi dire, auréolés; ils brillent au sein d'une et sont peints à la vapeur surnaturelle manière emphatique d'un adorateur. Quant aux pelils épisodes romanesques ^ y — de a-t-il lieu s'étonner qu'un être aussi nerveux, dont la soif du Beau était peut-être le trait principal, ait parfois, avec une ardeur pas- sionnée, cultivé la galanterie, cette fleur quée pour qui le volcanique et mus- cerveau bouillonnant des poètes est un terrain de prédilection? De sa beauté personnelle singulière dont parlent plusieurs biographes, l'esprit peut, je crois, se faire une idée approxi- mative en appelant à son secours toutes les notions vagues, mais cependant caractéristiques, contenues dans le mot ro- mantique, mot qui sert généralement à rendre les genres de beauté consistant surtout dans l'expression. Poe avait un front vaste, dominateur, où certaines protubérances trahis- saient les facultés débordantes présenter, qu'elles sont chargées de ré- — construction, comparaison, causalité, — trônait dans un orgueil calme le sens de l'idéalité, le et où sens esthétique par excellence. Cependant, malgré ces dons, ou même à cause de ces privilèges exorbitants, cette tête vue de Comme dans profil n'offrait peut-être pas un aspect agréable. toutes les choses excessives résulter de l'abondance, par un sens, un déficit pouvait une pauvreté de l'usurpation. Il avait de grands yeux à la fois sombres et pleins de lumière, d'une couleur indécise nez noble et solide, meni souriante, le et ténébreuse, la bouche teint poussée au fine et triste, brun clair, la violet, le quoique légère- face généralement SA VIE ET SES OEUVRES. physionomie un peu distraite pâle, la 25 et imperceptiblenient grimée par une mélancolie habituelle. Sa conversation était des plus remarquables et essentielle- ment nourrissante. Il n'était pas ce qu'on appelle un beau parleur, sa — une chose horrible, — et d'ailleurs sa parole plume avait horreur du convenu comme mais un vaste savoir, ; une linguistique puissante, de fortes études, des impressions ramassées dans plusieurs pays feisaient de cette parole un enseignement. Son éloquence, pleine de méthode, et se méthode connue, essentiellement mouvant toutefois un arsenal d'images peu fréquenté par la foule des esprits, hors poétique, de tirées d'un un art toute monde prodigieux à déduire d'une proposition évidente et absolument acceptable des aperçus secrets et nouveaux, à ouvrir d'étonnantes perspectives, et, en un mot, l'art de ravir, de faire penser, de faire rêver, d'arracher les âmes des bourbes de la routine, telles étaient les éblouissantes facultés dont souvenir. Mais gardé le moins, — que destructeur, le poëte, rappelait il beaucoup de gens ont arrivait parfois se —on le dit, du complaisant dans un caprice brusquement ses amis à par la terre un cynisme affligeant et démolissait brutalement son œuvre de spiritualité. C'est d'ailleurs une chose à noter, qu'il fort peu difficile que le dans le était choix de ses auditeurs, et je crois lecteur trouvera sans peine dans l'histoire d'autres intelligences grandes et originales, pour qui toute compa- gnie était bonne. Certains esprits, solitaires au milieu de la foule, et qui faire de la se repaissent délicatesse dans le en matière monologue, n'ont que de public. C'est, en somme, une espèce de fraternité basée sur le mépris. De cette ivrognerie, — célébrée et reprochée avec une in- sistance qui pourrait donner à croire que tous les écrivains des États-Unis, excepté Poe, sont des anges de sobriété, — , EDGAR POE 26 il cependant faut sibles, et en parler. Plusieurs versions sont plau- aucune n'exclut les autres. Avant tout, suis je obligé de remarquer que Willis et madame Osgood affirment qu'une quantité fort minime de vin ou de liqueur suffisait pour perturber complètement son organisation. Il est d'ailleurs facile de supposer qu'un homme aussi réellement solitaire, aussi profondément malheureux, et qui a pu souvent envisager tout le système social comme un paradoxe et une imposture, un homme qui, harcelé par une destinée sans pitié, répétait souvent que la société n'est Griswold qui rapporte (c'est qu'une cohue cela, de misérables qu'un aussi scandalisé homme qui peut penser la même chose, mais qui ne la dira jamais), — il e>t naturel, dis-je, jeté tout enfant dans les hasards de supposer que ce poëte de la vie libre, le cerveau cerclé par un travail âpre et continu, ait cherché parfois une volupté d'oubli dans les bouteilles. Rancunes littéraires, vertiges de l'infini, douleurs de ménage, insultes de sère, Poe fuyait tout dans le la noir de l'ivresse comme midans une tombe préparatoire. Mais, quelque bonne que paraisse cette explication, je ne la trouve pas suffisamment large, et je m'en défie à cause de sa déplorable sim])licité. J'apprends qu'il ne buvait pas en gourmand, mais en barbare, avec une activité et une économie de temps tout à fait comme accomplissant une fonction homicide, comme ayant en lui quelque chose à tuer, a worm Ihat ivould noldie. On raconte d'ailleurs qu'un jour, au moment de se remarier (les bans étaient publiés, et, comme on le américaines, félicitait sur une union qui mettait dans ses mains les plus hautes conditions de bonheur et de bien-être, il avait dit « Il est possible ceci : je ne : que vous ayez vu des bans, mais notez bien me marierai pas! » ), il alla, épouvantablement ivre, scandaliser le voisinage de celle qui devait être sa femme, SA VIE ET SES OEUVRES. 27 ayant ainsi recours à son vice pour se débarrasser d'un parjure envers pauvre morte dont l'image vivait toujours en la lui et qu'il avait admirablement chantée dans son Annahel Lee. Je considère donc, dans un grand nombre de cas, le fait infi- niment précieux de préméditation comme acquis et constaté. Je lis d'autre part, dans un long article du Southern Li- terary Messenger, — cette même revue dont mencé la — que jamais style, fortune, jamais travail, la netteté il la pureté, le avait comfini de son de sa pensée, jamais son ardeur au ne furent altérés par cette terrible habitude; que la confection de la plupart de ses excellents morceaux a précédé ou suivi une de ses crises; qu'après la publication à' Eurêka^ penchant, et qu'à New-York, il sacrifia déplorablement à son le matin même où paraissait le CorbeaUj pendant que le nom du poëte était dans toutes les bouches, il traversait Broadway en trébuchant outrageusement. Remarquez que précédé ou suivi, les mots : impliquent que l'ivresse pouvait servir d'excitant aussi bien que de repos. Or, il est incontestable que — semblable à ces impressions fugitives et frappantes, d'autant plus frappantes dans leurs retours qu'elles sont plus fugitives, qui suivent quelquefois un symptôme extérieur, une espèce d'avertissement comme un son de cloche, une note musicale ou un parfum oublié, et qui sont elles-mêmes suivies d'un événement semblable à un événement déjà connu et qui occupait la même place dans une chaîne antérieurement révélée, — semblables à ces sin— guliers rêves périodiques qui fréquentent nos sommeils, il existe dans l'ivresse non-seulement des enchaînements de rêves, mais des séries de raisonnements, qui ont besoin, pour se reproduire, du milieu qui leur a donné naissance. Si lecteur m'a suivi sans répugnance, il a déjà le deviné ma con- clusion : je crois que, dans beaucoup de cas, non pas certai- EDGAR POE 28 nement dans tous, l'ivrognerie de Poe était un moyen mnéméthode énergique monique, une méthode de mortelle, mais appropriée à sa nature passionnée. Le poète avait appris à boire, travail, et comme un littérateur soigneux s'exerce à faire des cahiers de notes. Il ne pouvait résister au désir de retrouver les visions merveilleuses ou effrayantes, les conceptions subtiles qu'il avait rencontrées dans une tempête pré- cédente; c'étaient de vieilles connaissances qui l'attiraient impérativement, et, pour renouer avec elles, il prenait le chemin le plus dangereux, mais le plus direct. Une partie de ce qui fait aujourd'hui notre jouissance est ce qui l'a tué. IV Des ouvrages de ce singulier génie, j'ai peu de chose à dire le public fera voir ce qu'il en pense. Il ; me serait difficile, peut-être, mais non pas impossible de débrouiller sa méthode, d'expliquer son procédé, surtout dans la partie de ses œuvres dont le principal effet gît dans une analyse bien ménagée. Je pourrais introduire le lecteur dans les mystères de sa fabrication, m'étendre longuement sur cette portion de génie américain qui le fait se réjouir d'une difficulté vaincue, d'une énigme expliquée, d'un tour de force réussi, — qui le pousse à se jouer avec une volupté enfantine et presque perverse dans le monde des probabilités et des conjectures, et à créer des canards auxquels son art subtil a donné une vie vraisemblable. Personne ne niera que Poe ne soit un jongleur merveilleux, et je sais qu'il donnait surtout son estime à une autre partie de ses œuvres. J'ai quelques remarques plus importantes à faire, d'ailleurs très-brèves. SA VIE ET SES OEUVRES. 29 Ce n'est pas par ses miracles matériels, qui pourtant ont fait sa renommée, qu'il lui sera donné de conquérir l'admira- amour du Beau, par tion des gens qui pensent, c'est par son des conditions harmoniques de sa connaissance la beauté, profonde et plaintive, ouvragée néanmoins, par sa poésie transparente et correcte comme un bijou de cristal, — par — serré comme minutieux, — et mailles d'une armure, — complaisant son admirable style, pur et bizarre, les et dont la plus légère intention sert à pousser doucement le lecteur vers un but voulu, par spécial, — et enfin surtout par ce génie tout qui lui a permis de ce tempérament unique peindre et d'expliquer, d'une manière impeccable, saisissante, terrible, V exception dans l'ordre moral. prendre un exemple entre cent, est l'écrivain — des nerfs, et est un — Diderot, pour auteur sanguin ; Poe même de quelque chose de plus, et le meilleur que je connaisse. Chez lui, toute entrée en matière estattirante sans violence, comme un tourbillon. Sa solennité surprend et tient l'esprit en éveil. On sent tout d'abord qu'il s'agit de quelque chose de grave. Et lentement, peu à peu, se déroule une histoire dont tout l'intérêt repose sur une imperceptible déviation de l'intellect, sur une hypothèse audacieuse, sur un dosage imprudent de la Nature dans l'amalgame des facultés. Le lecteur, lié par le vertige, est contraint de suivre l'auteur dans ses entraînantes déductions. Aucun homme, je le répète, n'a raconté avec plus de magie les exceptions de la vie humaine et de la nature; deurs de curiosité de la convalescence; chargées de splendeurs énervantes, — les — les ar- les fins de saisons temps chauds, hu- mides et brumeux, où le vent du sud amollit et détend les nerfs comme les cordes d'un instrument, où les yeux se remplissent de larnaes qui ne viennent pas du cœur; ****** . — l'hallu2 EDGAR POE 30 cination laissant d'abord place au doute, bientôt convaincue — l'absurde s'installant dans et raisonneuse comme un livre; l'intelligence et la gouvernant avec une épouvantable logique; — l'hystérie usurpant la place do la volonté, la contradiction établie entre les- nerfs et l'esprit, et l'homme désaccordé au point d'exprimer la douleur par le rire. analyse ce qu'il Il y a de plus fugitif, il soupèse l'impondérable et décrit, avec cette manière minutieuse et scientifique dont les effets sont tout cet imaginaire qui flotte autour de l'homme terribles, nerveux et le conduit à mal. L'ardeur même avec laquelle il se jette dans le grotesque pour l'amour du grotesque de l'horrible, et dans l'horrible pour l'amour me sert à vérifier la sincérité de son œuvre et l'accord de l'homme avec le poëte. — J'ai déjà remarqué que, chez plusieurs hommes, cette ardeur était souvent le résultat d'une vaste énergie vitale inoccupée, quelquefois d'une opiniâtre chasteté, et aussi d'une profonde sensibilité refoulée. La volupté surnaturelle que l'homme peut éprouver à voir couler son propre sangj les inutiles^ grands les cris mouvements soudains, violents^ jetés en l'air^ sans que l'esprit ait commandé au gosier^ soiit des phénomènes à ranger dans le même ordre» Au sein de Cette littérature oii l'air est raréfié^ l'esprit peut éprouver cette vague angoisse, cette peur prompte aux larmes et ce malaise du cœur qui habitent les lieux immenses et singuliers. Mais l'admiration l'art est si grand ! est la plus forte, et d'ailleurs Les fonds et les accessoires y sont appro- priés aux sentiments des personhagës. Solitude de la riature du agitation deâ villes, tout y est décrit nerveusement et fantastiquement. Comme notre Eugène Delacroix, qui a élevé son art à la hauteur de la grande poésie, Edgar Poe aime à agiter ses figures sur des fonds violàtres et verdâtres où SA VIE ET SES OEUVRES. 31 se révèlent la phosphorescence de la pourriture et la senteur de l'orage. La nature dite inanimée participe de la nature des êtres vivants, et, comme eux, frissonne d'un frisson par l'o- donne un sens magique à toutes les surnaturel et galvanique. L'espace est approfondi pium ; l'opium y teintes, et fait vibrer cative sonorité. tous les bruits avec une plus signifi- Quelquefois, des échappées magnifiques, gorgées de lumière et de couleur, s'ouvrent soudainement dans ses paysages, et l'on voit apparaître au fond de leurs horizons des villes orientales et des architectures, vaporisées par la distance, oià le soleil jette des pluies d'or. Les personnages de Poe, ou plutôt le personnage de Poe, l'homme aux facultés suraiguës, l'homme aux nerfs relâchés, l'homme dont la volonté ardente et patiente jette un défi aux celui difficultés, dont le regard est tendu avec la roideur d'une épée sur des objets qui grandissent à mesure qu'il regarde, -- c'est Poe lui-même. les — Et ses femmes, toutes lumineuses et malades, mourant de maux bizarres et parlant avec une voix qui ressemble à une musique, c'est encore lui; ou du moins, par leurs aspirations étranges, par leur savoir, par leur mélancolie inguérissable, elles participent fortement do la nature de leur créateur. Quant à sa femme idéale, à sa Titanide, elle se révèle sous différents portraits éparpilles dans ses poésies trop peu nombreuses, portraits, ou plutôt manières de sentir la beauté, que le tempérament de l'auteur rapproche et confond dans une unité vague mais sensible, et où vit plus insatiable délicatement peut-être qu'ailleurs cet du Beau, qui est son grand titre, amour c'est-à dire le ésumé de ses titres à l'affection et au respect des poètes. Nous rassemblons sous le titre Histoires extraordmaires, : divers contes choisis dans l'œuvre général de Poe. Cet œuvre se compose d'un nombre considérable de Nouvelles, d'une EDGAR POE. 32 quan*tité non moins divers, d'un poëme forte d'articles critiques et d'articles philosophique [Eurêka), de poésies et d'un roman purement humain [la Relation d'Arthur Gordon Pym). Si je trouve encore, comme je l'espère, l'occasion de parler de ce poëte, je donnerai l'analyse de ses opinions philosophiques et littéraires, ainsi œuvres dont la traduction complète que généralement des aurait peu de chances de succès auprès d'un public qui préfère de beaucoup l'amu- sement et l'émotion à la plus im})ortante vérité philosophique. C. B. HISTOIRES EXTRAORDINAIRES DOUBLE ASSASSINAT DANS LA RUE MORGUE Quelle chanson chantaient les sirènes ? quel nom Achille avait-il pris, quand il se cachait parmi les femmes? embarrassantes, est il — Questions yrai, mais qui ne sont pas situées au delà de toute conjecture. Sir Thomas Browne. Les facultés de l'esprit qu'on définit par le terme analytiques sont en elles-mêmes fort peu susceptibles d'analyse. tats. Nous ne les apprécions que par leurs résul- Ce que nous en savons, entre autres choses, c'est qu'elles sont pour celui qui les possède à un degré extraordinaire une source de jouissances des plus vives. De même que Tliomme fort se réjouit dans son apti- tude physique, se complaît dans les exercices qui pro- voquent les muscles à l'action, de même l'analyse HISTOIRES EXTRAORDINAIRES. 34 prend sa gloire dans cette activité fonction est de débrouiller. Il tire spirituelle dont la du plaisir même des plus triviales occasions qui mettent ses talents en jeu. Il raffole des énigmes, des rébus, des hiéroglyphes; il déploie dans chacune des solutions une puissance de perspicacité qui, dans l'opinion vulgaire, prend un caractère surnaturel. Les résultats, habilement déduits par l'âme même et l'essence de sa méthode, ont réel- lement tout l'air d'une intuition. Cette faculté de résolution tire peut-être une grande force de l'étude des mathématiques, et particulièrement de la très-haute branche de cette science, qui, improprement et fort simplement en raison de ses opé- rations rétrogrades, a été nommée l'analyse, comme si en somme, tout Un joueur d'échecs elle était l'analyse par excellence. Car, calcul n'est pas en soi une analyse. par exemple, fait fort bien l'un sans l'autre. là que le jeu d'échecs, dans ses effets spirituelle, est fort mal apprécié. Je ici sur Il suit de la nature ne veux pas écrire un traité de l'analyse, mais simplement mettre en tête d'un récit passablement singulier, quelques obser- vations jetées tout à fait à l'abandon et qui lui serviront de préface. Je prends donc cette occasion de proclamer que la haute puissance de la réflexion est bien plus activement et plus profitablement exploitée par le modeste jeu de dames que par toute la laborieuse futilité des échecs. Dans ce dernier jeu, où les pièces sont douées de mouvements divers et bizarres, et représentent des valeurs DOUBLE ASSASSINAT DANS LA RUE MORGUE. diverses et variées, la complexité est prise 3a — erreur fort commune — pour de la profondeur. L'attention y est puissamment mise en jeu. Si elle se relâche d'un instant, on commet une erreur, d'où il résulte une perte ou une défaite. Comme les mouvements possibles sont, non-seulement variés, mais inégaux an puissance, les chances dépareilles erreurs sont très-multipliées; et dans neuf cas sur dix, c'est le joueur le plus attentif qui gagne et non pas le plus habile. Dans les dames, au contraire, où le mouvement espèce et ne subit que peu bilités est simple dans son de variations, les proba- d'inadvertance sont beaucoup moindres, et l'at- tention n'étant pas absolument et entièrement accaparée, tous les avantages remportés par chacun des joueurs ne peuvent être remportés que par une perspicacité supérieure. Pour laisser là ces abstractions, supposons un jeu de dames où dames, la et où totalité des pièces soit réduite à quatre naturellement il n'y ak^pas lieu de s'at- tendre à des étourderies. 11 est évident qu'ici la victoire — deux parties étant abso— que par une tactique habile, résultat ne peut être décidée, lument égales, les de quelque puissant effort de l'intellect. Privé des ressources ordinaires, l'analyste entre dans l'esprit de son adversaire, s'identifie avec lui, et souvent découvre d'un seul coup d'œil l'unique moyen ~ un moyen quelquefois absurdement simple l'attirer — de dans une faute ou de le précipiter dans un faux calcul. On a longtemps cité le whist pour son action sur Id HISTOIRES EXTRAORDI AIRES. 36 i\ faculté du calcul ; et on a connu des hommes d'une haute intelligence qui semblaient y prendre un plaisir incompréhensible et dédaigner les échecs jeu frivole. En effet, il n'y a aucun jeu comme un analogue qui fasse plus travailler la faculté de l'analyse. Le meilleur joueur d'échecs delà chrétienté ne peut guère être autre chose que le meilleur joueur d'échecs; mais la force au whist implique la puissance de réussir dans toutes les spéculations bien autrement importantes où l'esprit lutte avec l'esprit. Quand je dis la force, j'entends cette perfection dans le jeu qui comprend l'intelligence de tous les cas dont on peut légitimement faire son profit. Ils sont non-seulement divers, mais complexes, et se dérobent souvent dans des profondeurs de la pensée ah solument inaccessibles à une intelligence ordinaire. Observer attentivement, c'est se rappeler distinc- point de d'échecs tement;, et, à ce vue, le joueur capable d'une attention très-intense jouera fort bien au whist, puisque les règles de Hoyle, basées elles-mêmes sur le simple mécanisme du jeu, sont facilement et généralement intelligibles. Aussi, avoir une mémoire fidèle et procéder d'après le livre le sont des points qui constituent pour le vulgaire summum du bien jouer. Mais c'est dans les cas situés au delà de la règle que le talent de l'analyste se manifeste; il fait en silence une foule d'observations et de déductions. Ses partenaires en font peut-être autant ; et la différence d'étendue dans les renseignements ainsi DOUBLE ASSASSINAT DANS La RUE MORGUE. 37 acquis ne gît pas tant dans la validité de la déduction que dans la qualité de l'observation. L'important, principal est de savoir ce qu'il faut observer. le Notre joueur ne se confine pas dans son jeu, et, bien que ce jeu soit l'objet actuel de son attention, il ne rejette pas pour cela les déductions qui naissent d'objets étrangers au jeu. 11 examine la physionomie de son partenaire, avec il la compare soigneusement ses adversaires. Il celle de chacun de considère la manière dont chaque partenaire distribue ses cartes; il compte souvent, grâce aux regards que laissent échapper les joueurs satisfaits, les atouts et un à un. Il note les ho7ineurs, chaque mouvement de la physionomie, à mesure que le jeu marche, et recueille un capital de pensées dans les expressions variées de certitude, de surprise, de triomphe ou de mauvaise humeur. A ramasser une levée, il devine peut faire une autre dans si la la suite. manière de la même personne en reconnaît ce qui II est joué par feinte à l'air dont c'est jeté sur la table. Une parole accidentelle, une carte qui involontaire, tombe, ou qu'on retourne par hasard, qu'on ramasse avec anxiété ou avec insouciance et l'ordre ; le compte des levées dans lequel elles sont rangées l'hésitation, la vivacité, la trépidation, lui ; l'embarras, — tout est pour symptôme, diagnostic, tout rend compte à cette perception, état des — intuitive en apparence, — du véritable choses. Quand les deux ou tours ont été faits, il possède à fond trois premiers jeu qui est le dans chaque main, et peut dès lors jouer ses cartes en -38 HISTOIRES EXTRAORDINAIRES. parfaite connaissance comme si de cause, tous les autres joueurs avaient retourné les leurs. La faculté d'analyse ne doit pas être confondue avec la simple ingéniosité; car, pendant que l'analyste ingénieux, nécessairement souvent arrive il est que l'homme ingénieux est absolument incapable d'analyse. La faculté de combinaison, ou par constructivité, laquelle se manifeste généralement cette ingéniosité, phrénologues — ont selon — assignent un organe à part, — en supposant et à laquelle moi, les qu'elle soit une faculté êtres dont l'intelligence assez souvent pour tort, ils primordiale, a paru dans des était limitrophe attirer l'attention de l'idiotie, générale des écrivains psychologistes. Entre l'ingéniosité et l'aptitude analytique, il y a une différence beaucoup plus grande qu'entre l'Imaginative et l'imagination, mais d'un caractère rigoureusement analogue. En somme, on verra que l'homme ingénieux est toujours plein d'imaginative, et que l'homme vraiment imaginatif n'est jamais autre chose qu'un analyste. Le récit qui suit sera pour le lecteur un commen- taire lumineux des propositions que je viens d'avancer. — pendant printemps une — connaissance d'un Je demeurais à Paris, partie de l'été de 18.., le et et j'y fis la certain G. Auguste Dupin. Ce jeune gentleman appartenait à une excellente famille, une famille illustre même; mais, par une série d'événements malencontreux, se trouva il réduit à une telle pauvreté, que l'énergie de son caractère y succomba, et qu'il cessa de se DOUBLE ASSASSINAT DANS LA RUE MOIIGLE. pousser dans le monde sement de sa fortune. Hl) de s'occuper du rétablis- et Grâce à la courtoisie de ses créanciers, il resta en possession d'un petit reliquat de son patrimoine; et, sur la rente qu'il en tirait, il trouva moyen, par une économie rigoureuse, de subvenir aux nécessités de la vie, sans s'inquiéter autrement des superfluités. Les livres étaient véritablement son seul luxe, et à Paris on se les procure facilement. Notre première connaissance se fit dans un obscur cabinet de lecture de la rue Montmartre, par ce fortuit que nous fait étions tous deux à la recherche d'un même livre, fort remarquable et fort rare; cette coïncidence nous rapprocha. Nous nous vîmes toujours de plus en plus. Je fus profondément intéressé par sa petite histoire de famille, qu'il me raconta minutieu- sement avec cette candeur et cet abandon, façon du moi, — qui est le propre — ce sans- de tout Français quand il parle de ses propres affai-res. Je fus aussi fort étonné de la prodigieuse étendue de ses lectures, et par-dessus tout je me sentis l'âme prise par l'étrange chaleur et la vitale fraîcheur de son imagination. Cherchant dans Paris certains objets qui mon unique étude, je vis que la société d'un pareil homme serait pour moi un trésor inappréciable, et dès lors je me livrai franchement à lui. Nous décifaisaient dâmes enfin que nous vivrions ensemble tout le temps démon séjour dans cette ville; et, comme mes affaires étaient un peu moins embarrassées que les siennes, je me chargeai de louer et de meubler, dans un style HISTOIRES EXTRAORDINAIRES. 40 approprié à la mélancolie fantasque de nos deux caractères, une maisonnette antique et que des bizarre superstitions dont nous ne daignâmes pas nous enquérir avaient fait déserter, — tombant presque en ruine, et du faubourg située dans une partie reculée et solitaire Saint-Germain. Si la routine de notre vie dans ce lieu avait été connue du monde, nous eussions passé pour deux fous, — peut-être pour des fous d'un genre réclusion était complète; inoffensif. Notre nous ne recevions aucune — — soigneusement gardé pour mes anciens camarades visite. Le lieu de notre retraite était resté un secret ; et il y avait plusieurs années que Dupin avait cessé de voir du monde et de se répandre dans Paris. Nous 'ne vivions qu'entre nous. — Mon ami avait une bizarrerie d'humeur, car comc'était d'aimer la nuit pour l'ament définir cela? mour de la nuit; la nuit était sa passion et je tombai moi-même tranquillement dans cette bizarrerie, — ; comme dans toutes les autres qui lui étaient propres, me laissant aller au courant de toutes ses étranges originalités avec un parfait abandon. La noire divinité ne pouvait pas toujours nous en faisions du jour, nous la demeurer avec nous contrefaçon. fermions tous les ; mais Au premier point lourds volets de notre masure, nous allumions une couple de bougies fortement parfumées, qui ne jetaient que des rayons très-faibles et très-pâles. Au sein de cette débile clarté, nous livrions chacun notre âme à ses rêves, nous li- DOUBLE ASSASSINAT DANS LA RUE MORGUE. U sions, nous écrivions, ou nous causions, jusqu'à ce que la pendule nous avertît curité. Alors, du retour de la véritable obs- nous nous échappions à travers les rues, bras dessus bras dessous, continuant la conversation du jour, rôdant au hasard jusqu'à une heure très- avancée, et cherchant à travers les lumières désordon- nées et les ténèbres de la populeuse cité ces innom- brables excitations spirituelles que l'étude paisible ne peut pas donner. Dans ces circonstances, de remarquer lité je et d'admirer, ne pouvais m'empêcher — quoique la riche idéa- dont il était doué eût dû m'y préparer, aptitude analytique particulière chez Dupin. — une 11 sem- prendre un délice acre à l'exercer, — peut-être — avouait sans façon tout même à blait et l'étaler, le plai- me disait à moi, avec un petit rire tout épanoui, que bien des hommes avaient pour lui sir qu'il en tirait. 11 une fenêtre ouverte à l'endroit de leur cœur, et d'habitude il accompagnait une pareille assertion de preuves immédiates et des plus surprenantes, tirées d'une connaissance profonde de ma propre personne. Dans ces moments-là, ses manières étaient glaciales et distraites; ses yeux regardaient dans le vide, et sa voix, — une riche voix de ténor, habituellement, — montait jusqu'à la voix de tête; c'eût été de lance, sans l'absolue délibération de la pétu- son parler et la parfaite certitude de son accentuation. Je l'observais dans ses allures, et je rêvais souvent à la vieille philosophie de rd?7ierfou^/e, — je m'amusais à l'idée d'un Dupin HISTOIRES EXTRAORDINAIRES. 12 doiibje, — un Dupin créateur et un Dupin analyste. Qu'on ne s'imagine pas, d'après ce que je viens de dire, que je vais dévoiler un grand mystère ou écrire un roman. Ce que j'ai remarqué dans ce singulier Français était simplement le résultat d'une intelligence — malade peut-être. Mais un exemple surexcitée, donnera une meilleure idée de la nature de ses observations à l'époque dont il s'agit. Une nuit, nous flânions dans une longue rue sale, avoisinant le Palais-Royal. Nous étions plongés chacun dans nos propres pensées, en apparence du moins, et, depuis près d'un quart d'heure, nous n'avions pas soufflé une syllabe. Tout à coup Dupin lâcha ces paroles — C'est un bien petit garçon, : en vérité; et il serait mieux à sa place au théâtre des Variétés. — Cela ne pas l'ombre d'un doute, répliquai-je fait sans y penser et sans remarquer d'abord, tant j'étais absorbé, la singulière façon dont l'interrupteur adaptait sa parole à ma propre rêverie. Une minute après, je revins à moi, et mon étonne- ment fut profond. — Dupin, dis-je très-gravement, voilà qui passe mon intelligence. Je vous avoue, sans ambages, que j'en suis stupéfié et que j'en peux à peine croire mes sens. Comment a-t-il pu se faire que vous ayez de- viné que je pensais à...? Mais je m'arrêtai pour m' assurer indubitablement qu'il avait réellement deviné à qui je pensais. — A Chantilly ? dit-il ; pourquoi vous interrompre ? DOUBLE ASSASSINAT DANS LA RUE MORGUE. 43 Vous faisiez en vous-même la remarque que sa petite taille le rendait' impropre à la tragédie. C'était précisément ce qui faisait le sujet de mes ré- un ex-savetier de la rue Saint- flexions. Chantilly était Denis qui avait la rage du théâtre, et avait abordé le rôle de Xercès dans la tragédie de Crébillon ; ses préten- tions étaient dérisoires : on en faisait des gorges chaudes. si l'aide — Dites-moi, pour l'amour de Dieu! méthode — de laquelle vous avez pu méthode y a — à la il pénétrer mon âme, dans le cas actuel ! En réalité, j'étais encore plus étonné que je n'aurais voulu le confesser. — C'est le fruitier, répliqua mon ami, qui vous a amené à cette conclusion que le raccommodeur de semelles n'était pas de taille à jouer Xercès et tous les rôles de ce genre. — Le fruitier! vous m'étonnez! je ne connais de d'aucune espèce. fruitier — L'homme qui s'est jeté contre vous, quand nous sommes entrés dans la rue, il y a peut-être un quart d'heure. Je me rappelai alors qu'en effet un fruitier, portant sur sa tête un grand panier de pommes, m'avait pres- que jeté par terre par maladresse, comme nous passions de la rue C... dans l'artère principale où nous étions alors. Mais quel rapport cela avait-il avec Chantilly? 11 Il m'était impossible de m'en rendre compte. n'y avait pas mon ami Dupin. un atome de charlatanerie dans HISTOIHKS EXTRAO IIDIA AIRES. •44 — Je vais vous expliquer cela, dit-il, et, pour que vous puissiez comprendre tout très-clairement, nous allons d'abord reprendre la série de vos réflexions, de- puis le moment dont je vous parle jusqu'à la ren- contre du fruitier en question. Les anneaux principaux de la chaîne se suivent ainsi CliantUly, Orion, : le doc- teur I^lchols, Epicure, la stéréotomie, les pavés, le fruitier. Il est peu de personnes qui ne se soient amusées, à un moment quelconque de leur vie, à remonter le cours de leurs idées et à rechercher par quels chemins leur esprit était arrivé à de certaines conclusions. Souvent cette occupation est pleine d'intérêt, et celui qui l'es- saye pour la première fois est étonné de l'incohérence et de la distance, immense en apparence, entre le point de départ et le point d'arrivée. Qu'on juge donc de mon étonnement quand j'en- tendis mon Français parler comme il avait fait, et que je fus contraint de reconnaître qu'il avait dit la pure vérité. Il continua : — Nous causions de chevaux — ma mémoire ne me trompe pas — juste avant de quitter rue si G... la Ce fut notre dernier thème de conversation. Comme nous passions dans cette rue-ci, un fruitier, avec un gros panier sur la tête, passa précipitamment devant nous, vous jeta sur un tas de pavés amoncelés dans un endroit où la voie est en réparation. Vous avez mis le pied sur une des pierres branlantes vous vous êtes légèrement foulé ; vous avez glissé, la cheville ; vous avez DOUBLE ASSASSINAT DANS LA RUE MORGUE. 45 paru vexé, grognon; vous avez marmotté quelques pavous vous roles; êtes retourné pour regarder le tas, puis vous avez continué votre chemin en silence. Je n'étais pas absolument attentif à tout ce que vous fai- siez; mais, pour moi, l'observation est devenue, vieille date, » Vos yeux sont restés attachés sur le sol, lant avec de une espèce de nécessité. une espèce d'irritation les — surveil- trous et les or- nières du pavé ( de façon que je voyais bien que vous pensiez toujours aux jusqu'à ce pierres), que nous eûmes atteint le petit passage qu'on nomme le passage Lamartine \ où Ton vient de faire Tessai du pavé de bois, un système de blocs unis et solidement assemblés. Ici votre physionomie s'est éclaircie, j'ai vu vos lèvres remuer, et j'ai deviné, à n'en pas douter, que vous vous murmuriez le mot stéréotomie, un terme appliqué fort prétentieusement à ce genre de pavage. Je savais que vous ne pouviez pas dire stéréotomie sans être induit à penser d'Épicure; et, eûmes, fait il aux atomes, et comme dans la de là aux théories discussion que nous n'y a pas longtemps, à ce sujet, je vous avais remarquer que les vagues conjectures de l'illustre Grec avaient été confirmées singulièrement, sans que personne y prît garde, par les dernières théories sur les nébuleuses et les récentes découvertes cosmo- goniques, je sentis que vous ne pourriez pas empêcher 1. Ai-je besoin d'avertir à propos de la rue Morgue, du passage Lamartine, etc., qu'Edgar Poe n'est jamais venu à Paris? 3. — C. B. HISTOIRES EXTRAORDINAIRES. 4(j VOS yeux de se tourner vers la grande nébuleuse d'Orion je m'y attendais certainement. ; Vous n'y avez pas man- qué, et je fus alors certain d'avoir strictement emboîté le pas de votre rêverie. Or, dans cette amère boutade sur Chantilly, qui a paru hier dans le Musée, l'écrivain satirique, en faisant des allusions désobligeantes au changement de nom du savetier quand il a chaussé le cothurne, citait un vers latin dont nous avons souvent causé. Je veux parler du vers : Perdidit antiquum littera prima sonum. Je VOUS avais dit qu'il avait trait à Orion, qui s'écrivait primitivement Urion; et, à cause d'une certaine acri- monie mêlée à cette discussion, j'étais sûr que vous ne l'aviez pas oubliée. Il était clair, dès lors, que vous ne pouviez pas manquer d'associer les deux idées d'Orion et de Chantilly. Cette association d'idées, je la vis au siyle du sourire qui traversa vos lèvres. Vous pensiez à l'immolation du pauvre savetier. Jusque-là, vous aviez marché courbé en deux, mais alors je vous vis vous redresser de toute votre hauteur. J'étais bien sûr que vous pensiez à la pauvre petite taille de Chantilly. C'est dans ce moment que j'interrompis vos réflexions pour vous faire remarquer que c'était un pauvre petit avorton que ce Chantilly, et qu'il serait bien mieux à sa place au théâtre des Variétés. Peu de temps après cet entretien, nous parcourions l'édition du soir de la Gazette des tribunaux, quand les paragraphes suivants attirèrent notre attention : DOUBLE ASSASSINAT DANS LA RUE MORGUE. u Double assassinat des plus singuliers. 47 — Ce matin, vers trois heures, les habitants du quartier Saint- Koch furent réveillés par une suite de cris effrayants, qui semblaient venir du quatrième étage d'une maison de la rue Morgue, que Ton savait occupée en totalité par une dame TEspanaye et sa fille, mademoiselle Camille TEspanaye. Après quelques retards causés par des efforts infructueux pour se faire ouvrir à l'amiable, la grande porte fut forcée avec une pince, et huit ou dix voisins entrèrent, accompagnés de deux gendarmes. Cependant, les cris avaient cessé; mais, au moment )) où tout ce monde arrivait pêle-mêle au premier étage, on distingua deux fortes voix, peut-être plus, qui semdisputer violemment et venir de la partie blaient se supérieure de la maison. Quand on arriva au second palier, ces bruits avaient également cessé, et tout était parfaitement tranquille. Les voisins se répandirent de chambre en chambre. Arrivés à une vaste pièce située sur le derrière, au quatrième étage, et dont on force la porte qui était fermée, avec la clef en dedans, ils se trouvèrent en face d'un spectacle qui les assistants d'une terreur non frappa tous moins grande que leur étonnement. » les La chambre était dans le plus étrange désordre ; meubles brisés et éparpillés dans tous les sens. 11 n'y avait qu'un lit, les matelas en avaient été arrachés et jetés au trouva un milieu du parquet. Sur une chaise, on rasoir mouillé de sang; dans l'âtre, trois longues et fortes boucles de cheveux gris, qui semblaient HISTOIRES EXTRAORDINAIRES. 48 avoir été violemment arrachées avec leurs racines. Sur parquet le gisaient ornée d'oreille quatre d'une une boucle napoléons, topaze, trois grandes cuillers d'argent, trois plus petites en métal d'Alger, et deux sacs contenant environ quatre mille francs en or. Dans un coin, les tiroirs d'une commode étaient ouverts et avaient sans doute été mis au pillage, bien qu'on y ait trouvé plusieurs articles intacts. Un petit coffret de fer fut trouvé sous la literie (non pas sous le bois de lit) il était' ouvert, contenait que avec la clef dans quelques vieilles la serrure. lettres et Il ; ne d'autres papiers sans importance. » On ne trouva aucune trace de madame l'Espanaye; mais on remarqua une quantité extraordinaire de suie dans le foyer; on fit une recherche dans la cheminée, et — chose horrible à dire — on en ! tira le corps de la demoiselle, la tête en bas, qui avait été introduit de force et poussé par l'étroite ouverture jusqu'à une distance assez considérable. Le corps était tout chaud. En l'examinant, on découvrit de nombreuses excoriaoccasionnées sans doute par tions, la violence avec laquelle il y avait été fourré et qu'il avait fallu employer pour le dégager. La figure portait égratignures, et la gorge était quelques fortes stigmatisée par des meurtrissures noires et de profondes traces d'ongles, comme si la mort avait eu lieu par strangulation. )) Après un examen minutieux de cl^feque partie de la maison, qui n'amena voisins aucune découverte nouvelle, les s'introduisirent dans une petite cour pavée, DOUBLE ASSASSINAT DANS LA RUE MORGUE. 4!) située sur le derrière du bâtiment. Là gisait le cadavre (le la vieille dame, avec la gorge si parfaitement cou- quand on essaya de pée, que, le relever, la tête se détacha du tronc. Le corps, aussi bien que la tête, terriblement mutilé, et celui-ci à ce point qu'il était gardait à peine une apparence humaine. )) Toute cette affaire resta un horrible mystère, et jusqu'à présent on n'a pas encore découvert, que nous sachions, le moindre fil conducteur. » Le numéro suivant portait ces détails additionnels «Le drame de la rue Morgue. — Bon nombre : d'in- dividus ont été interrogés relativement à ce terrible et extraordinaire événement, mais rien n'a transpiré qui puisse jeter quelque jour sur l'affaire. Nous donnons ci-dessous les dépositions obtenues )) : Pauline Dubourg, blanchisseuse, dépose qu'elle a connu les deux victimes pendant trois ans, et qu'elle a blanchi pour elles pendant tout ce temps. La vieille dame et sa fille semblaient en bonne très-affectueuses l'une envers intelligence, — C'étaient de l'autre. bonnes pmjes. Elle ne peut rien dire relativement leur genre de vie et à leurs croit que moyens d'existence. à Elle madame l'Espanaye disait la bonne aventure pour vivre. Cette dame passait pour avoir de l'argent de côté. Elle n'a jamais rencontré personne maison, quand elle linge. Elle est sûre venait dans la rapporter ou prendre le que ces dames n'avaient aucun do- mestique à leur service. 11 lui a semblé qu'il n'y avait HISTOIBES EXTRAORDINAIRES. 50 de meubles dans aucune partie de la maison, excepté au quatrième étage. » Pierre Moreau, marchand de tabac, dépose qu'il madame l'Espanaye, et lui fournissait habituellement vendait de petites quantités de tabac, quelquefois en poudre. est 11 né dans le quartier et y a toujours de- meuré. La défunte et sa fille occupaient depuis plus de six ans la maison où l'on a trouvé leurs cadavres. Pri- mitivement était elle sous-louait les habitée par un bijoutier, qui appartements supérieurs à différentes personnes. La maison appartenait à madame l'Espanaye. Elle s'était montrée très-mécontente de son locataire, qui endommageait les lieux ; elle était venue habiter sa propre maison, refusant d'en louer une seule partie. La bonne dame fille était en enfance. Le témoin a vu la cinq ou six fois dans l'intervalle de ces six années. Elles menaient toutes deux une vie excessivement retirée; elles passaient pour avoir de quoi. 11 a entendu dire chez les voisins que madame l'Espanaye disait la bonne aventure; il ne le croit pas. Il n'a jamais vu personne franchir la porte, excepté la vieille dame et sa fille, un commissionnaire une ou deux fois, et un médecin huit ou dix. » Plusieurs autres personnes du voisinage déposent dans le même sens. On ne cite personne comme ayant fréquenté fille la maison. On ne sait pas si la dame et sa avaient des parents vivants. Les volets des fenêtres de face s'ouvraient rarement. Ceux de derrière étaient toujours fermés, excepté aux fenêtres de la grande DOUBLE ASSASSINAT DAiSS LA RUE MORGUE. arrière-pièce du quatrième étage. La maison était 51 une assez bonne maison, pas trop vieille. » Isidore Muset, gendarme, dépose qu'il a été mis en réquisition, vers trois heures du matin, et qu'il a trouvé à la grande porte vingt ou trente personnes qui s'effor- çaient de pénétrer dans la maison. Il l'a forcée avec une baïonnette et non pas avec une pince. Il n'a pas eu grand'peine à l'ouvrir, parce qu'elle était à deux battants et n'était verrouillée ni par en haut, ni par en bas. Les cris ont continué jusqu'à ce que la porte fût On eût dit enfoncée, puis ils ont soudainement cessé. les cris d'une ou de plusieurs personnes en proie aux plus vives douleurs; des cris très-hauts, très-prolongés, — non pas des grimpé cris brefs, l'escalier. ni précipités. Le témoin a En arrivant au premier palier, il a entendu deux voi-x qui se disputaient très-haut et très- — aigrement; plus aiguë, l'une, une voix rude, l'autre une voix très-singulière. II beaucoup a distingué quelques mots de la première, c'était celle d'un Français. Il 11 est certain que ce n'est pas une voix de femme. a pu distinguer les mots sacré et diable. La voix aiguë était celle c'était d'un étranger. Il ne sait pas précisément si une voix d'homme ou de femme. Il n'a pu deviner ce qu'elle disait, mais il présume qu'elle parlait espagnol. Ce témoin rend compte de l'état de la cham- bre et des cadavres dans les mêmes termes que nous l'avons fait hier. » Henri Du val, un voisin, et orfèvre de son état, dépose qu'il faisait partie du groupe de ceux qui sont HISTOIRES EXTRAORDINAIRES. 52 entres les premiers dans 'la maison. Confirme généra- lement le témoignage de Muset. Aussitôt qu'ils se sont introduits dans la maison, ils ont refermé la porte pour barrer le passage à la foule qui s'amassait considé- rablement, malgré l'heure plus que matinale. La voix aiguë, à en croire le témoin, était une voix d'Italien. A coup sûr, ce n'était pas une voix française. Il ne sait pas au juste si c'était une voix de femme; cependant, cela pourrait bien être. Le témoin n'est pas familiarisé avec la langue italienne; il n'a pu distinguer les pa- mais il roles, d'après l'intonation que est convaincu l'individu qui parlait était un Italien. Le témoin a connu madame l'Espanaye et sa fille. Il a fréquemment causé avec elles. Il est certain que la voix aiguë n'était celle d'aucune des victimes. )) Odenheimer, restaurateur. Ce témoin s'est offert de lui-même. 11 ne parle pas français, et on l'a interrogé parle canal d'un interprète. Il est né à Amsterdam. 11 passait devant la maison au moment des cris. ont duré quelques minutes, dix minutes Ils peut-être. C'étaient des cris prolongés, très-hauts, très-effrayants, — des cris navrants. Odenheimer est un de ceux qui ont pénétré dans la maison. Il confirme le témoignage précédent, à l'exception d'un seul point. Il est sûr que la voix aiguë était celle d'un 11 et n'a homme, — d'un Français. pu distinguer les mots articulés. On parlait haut vite, — d'un ton inégal, — crainte aussi bien et qui exprimait la que la colère. La voix était âpre, plutôt âpre qu'aiguë. 11 ne peut appeler cela précisément DOUBLE ASSASSINAT DANS LA RUE MORGUE. 53 une voix aiguë. La grosse voix dit à plusieurs reprises Sacré, — diable, — et une fois : Mon Dieu : ! D.Jules Mignaud, banquier, de la maison Mignaud et lils, Madame rae Deloraine. 11 est l'aîné des Mignaud. i'Espanaye avait quelque fortune. 11 lui avait ouvert un compte dans sa maison, huit ans auparavant, au printemps. Elle a souvent déposé chez lui de petites sommes d'argent. Il ne kii délivré jusqu'au rien a troisième jour avant sa mort, où elle est venue lui demander en personne une somme de quatre mille francs. Cette somme lui a été payée en or, et un commis a été chargé de la lui porter chez elle. )) Adolphe commis chez Mignaud Lebon, et fils, dépose que, le jour en question," vers midi, il a accom- pagné madame I'Espanaye à son logis, avec les quatre mille francs, en deux sacs. Quand la porte s'ouvrit, mademoiselle I'Espanaye parut, et lui prit des mains l'un des deux sacs, pendant que la vieille dame le dé- chargeait de l'autre. Il les salua et partit. Il n'a vu per- sonne dans la rue en ce moment. C'est une rue borgne, très-solitaire. )) William Bird, tailleur, dépose qu'il est un de ceux qui se sont introduits dans la maison. 11 est Anglais. Il a vécu deux ans à Paris. ont monté l'escalier. Il II est un des premiers qui a entendu les voix qui se dis- putaient. La voix rude était celle d'un Français. distinguer quelques pas. Il mots, mais il Il a pu ne se les rappelle a entendu distinctement sacré et mon Dieu. C'é- tait en ce moment un bruit comme de plusieurs per- HISTOIRES EXTRAORDINAIRES. 54 — sonnes qui se battent, le tapage d'une lutte et d'objets qu'on brise. La voix aiguë était très-forte, plus forte que la voix rude. voix d'Anglais. Il Elle lui est sûr que ce n'était pas une sembla une voix d'Allemand; peut-être bien une voix de femme. Le témoin ne sait pas l'allemand. )) Quatre des témoins ci-dessus mentionnés ont été assignés de nouveau, et ont déposé que la porte de la chambre où fut trouvé le corps de mademoiselle l'Espanaye était fermée en dedans quand ils y arrivèrent. Tout était parfaitement silencieux; ni gémissements, ni bruits d'aucune espèce. Après avoir forcé la porte, ils ne virent personne. )) Les fenêtres, dans la chambre de derrière et dans celle de face, étaient fermées et solidement assujetties en dedans. Une porte de communication était fermée, mais pas à clef. La porte qui conduit de la chambre du devant au corridor était fermée à clef, et la clef en dedans; une petite pièce sur le devant de la maison, au quatrième étage, à l'entrée du corridor, ouverte, et la porte entre-bâillée; cette pièce, encombrée de vieux bois de lit, de malles, etc. On a soigneusement dérangé et visité tous ces objets. Il n'y a pas un pouce d'une partie quelconque de la maison qui n'ait été soigneu- sement visité. On a fait pénétrer des ramoneurs dans les cheminées. La maison est à quatre étages avec des mansardes. Une trappe qui donne sur le toit était con- damnée et solidement fermée avec des clous; elle ne semblait pas avoir été ouverte depuis des années. Les DOUBLE ASSASSINAT DANS LA RUE MORGUE. 55 témoins varient sur la durée du temps écoulé entre le moment où l'on a entendu les voix qui se disputaient et celui où l'on a forcé la porte de la chambre. Quel- ques-uns l'évaluent trop court, deux ou trois minutes, — d'autres, cinq minutes. La porte ne fut ouverte qu'à grand'peine. )) Alfonso Garcio, entrepreneur des pompes funèbres, dépose qu'il demeure rue Morgue. Il est né en Espagne. 11 un de ceux qui ont pénétré dans est n'a pas monté l'escalier. 11 maison. la Il a les nerfs très-délicats, et redoute les conséquences d'une violente agitation nerveuse. 11 a entendu les voix qui se disputaient. La grosse voix était celle d'un Français. guer ce qu'elle disait. II n'a pu distin- La voix aiguë était celle d'un Anglais, il en est bien sûr. Le témoin ne sait pas l'an- mais il juge d'après l'intonation. glais, ') Alberto Montani, confiseur, dépose qu'il fut des premiers qui montèrent l'escalier.. 11 a entendu les voix en cjuestion. La voix rauque était celle d'un Français. Il a distingué quelques mots. L'individu qui parlait semblait faire des remontrances. Il n'a pas pu deviner ce que disait la voix aiguë. Elle parlait vite et par sac- cades. 11 l'a prise pour la voix d'un Russe. II confirme en général les témoignages précédents. Il est Italien ; il avoue qu'il n'a jamais causé avec un Russe. » Quelques témoins, rappelés, certifient que les che- minées dans toutes les chambres, au quatrième étage, sont trop étroites pour livrer passage à un être humain. Quand ils ont parlé de ramonage, ils voidaient parler HISTOIRES EXTRAORDINAIRES. 56 de ces brosses en forme de cylindres dont on se sert pour nettoyer les chemine'es. On brosses da haut au bas dans tous maison. Il a fait passer les ces tuyaux de la n'y a sur le derrière aucun passage qui ait pu favoriser la fuite d'un assassin, pendant que les té- moins montaient l'escalier. Le corps de mademoiselle l'Espanaye était si solidement engagé dans la cheminée, qu'il a fallu, pour le retirer, que quatre ou cinq des té- moins réunissent leurs forces. )) Paul Dumas, médecin, dépose qu'il a été appelé au point du jour pour examiner les cadavres. Ils gisaient tous les deux sur le fond de sangle du lit dans la chambre où avait été trouvée mademoiselle TEspanaye. Le corps de la jeune dame était fortement meurtri et excorié. Ces particularités s'expliquent suffisamment par le fait de son introduction dans la cheminée. La gorge était singulièrement écorchée. Il y avait, juste au-dessous du menton, plusieurs égratigures profondes, ave(? une rangée de taches livides, résultant évidem- ment de la pression des doigts. La face était affreusement décolorée, et les globes des yeux sortaient de la tête. La langue était coupée à moitié. Une large meur- trissure se manifestait au creux de l'estomac, produite, selon toute apparence, par la pression d'un genou. Dans l'opinion de M. Dumas, mademoiselle l'Espanaye avait été étranglée par un ou par plusieurs individus inconnus. )) Le corps de la mère était horriblement mutilé. Tous les os de la jambe et du bras gauche plus ou moins DOUBLE ASSASSINAT DAx\S LA RUE MORGUE. 57 gauche brisé en que fracassés les ; côtes le tibia du esquilles, ainsi même côté. Tout le corps affreusement meurtri et décoloré. 11 était impossible de dire comment de pareils coups avaient été portés. Une lourde massue de bois ou une large pince de fer, une arme grosse, pesante et contondante aurait pu produire de pareils résultats, et encore, maniée par les mains d'un homme excessivement robuste. Avec n'importe quelle arme, aucune femme n'aurait pu frapper de tête de la défunte, quand le rement séparée du tronc, tels coups. La témoin la vit, était entiè- et, comme le reste, singu- lièrement broyée. La gorge évidemment avait été tran- chée avec un instrument très-affilé, très-probablement un rasoir. » Alexandre Etienne, chirurgien, a été appelé en même temps que M. Dumas pour visiter les cadavres; il confirme le témoignage et l'opinion de M. Dumas. )) Quoique plusieurs autres personnes aient été inter- rogées, on n'a pu obtenir aucun autre renseignement d'une valeur quelconque. Jamais assassinat embrouillé, n'a été commis à Paris, rieux, si fois y a eu assassinat. » il La police est absolument déroutée, si mysté- si toute- — cas fort usité dans les affaires de cette nature. Il est vraiment impossible de retrouver le fil de cette affaire. » L'édition du soir constatait qu'il régnait une agitation permanente dans le quartier Saint-Roch ; que les lieux avaient été l'objet d'un second examen, que les témoins avaient été interrogés de nouveau, mais tout cela sans HISTOIRES EXTRAORDINAIRES. 58 résultat. Cependant, unpost-scriptum annonçaitqu'Adol- phe Lebon, le commis de la maison de banque, avait été arrêté et incarcéré, bien que rien dans les faits déjà connus ne parût saflisant pour l'incriminer. Dupin semblait marche de cette s'intéresser affaire, singulièrement autant, du moins, à que la j'en pouvais juger par ses manières, car il ne faisait aucun commentaire. Ce fut seulement après que le journal eut annoncé l'emprisonnement de Lebon qu'il me de- manda quelle opinion j'avais relativement à ce double meurtre. Je ne pus que lui confesser que j'étais comme tout Paris, et que je le considérais comme luble. Je ne voyais un mystère inso- aucun moyen d'attraper la trace du meurtrier. — Nous ne devons pas juger des moyens possibles, dit Dupin, par une instruction embryonnaire. La police parisienne, si vantée pour sa pénétration, est très- rusée, rien de plus. Elle procède sans méthode, elle n'a pas d'autre méthode que celle du moment. ici On fait un grand étalage de mesures, mais il arrive souvent qu'elles sont si intempestives et si mal appropriées au but, qu'elles font penser à M. Jourdain, qui demandait sa robe de chambre — pour mieux entendre Les résultats obtenus sont la musique. quelquefois surprenants, mais ils sont, pour la plus grande partie, simplement dus à la diligence et à l'activité. Dans le cas où ces facultés sont insuffisantes, les plans ratent. Vidocq, par exemple, était bon pour deviner; c'était un homme de DOUBLE ASSASSINAT DANS LA RUE MORGUE. 59 mais sa pensée n'étant pas suffisamment éda- patience ; quée, il faisait continuellement fausse route, par Far- deur même de ses investigations. 11 diminuait la force de sa vision en regardant Tobjet de trop près. Il pouvait peut-être voir un ou deux points avec une netteté singulière, mais, par le fait même de son procédé, perdait l'aspect de l'affaire prise Cela peut s'appeler le moyen vérité n'est pas toujours il dans son ensemble. La d'être trop profond. dans un puits. En somme, quant à ce qui regarde les notions qui nous intéressent de plus près, je crois qu'elle est invariablement à la surface. vallée : Nous la cherchons dans c'est du sommet la profondeur de la des montagnes que nous la découvrirons. On trouve dans la contemplation des corps célestes )) des exemples et des échantillons excellents de ce genre d'erreur. Jetez sur une étoile un rapide coup d'œil, re- gardez-la obliquement, en tournant vers elle la partie latérale de la rétine (beaucoup plus sensible à une lu- mière faible que l'étoile la partie centrale), et vous verrez distinctement; vous aurez Tappréciation la plus juste de son éclat, éclat qui s'obscurcit à proportion que vous dirigez votre point de vue en plein sur elle. )) Dans le dernier cas, il tombe sur l'œil un plus grand nombre de rayons; mais, dans le premier, il y a une réceptibilité plus complète, une susceptibilité beaucoup plus vive. Une profondeur outrée affaiblit la pensée et la rend perplexe; et il est possible de faire disparaître HISTOIRES EXTRAORDINAIRKS. CO Vénus elle-même du firmament par une attention trop soutenue, trop concentrée, trop directe. nous-mêmes un exa- » Quant à cet assassinat, faisons men avant de nous former une opinion. Une enquête nous procurera de l'amusement (je trouvai cette expression bizarre, appliquée au cas en question, mais je ne dis mot) vice ; en outre, Lebon m'a rendu un ser- et, pour lequel je ne veux pas me montrer ingrat. Nous irons sur les lieux, nous les examinerons de nos propres yeux. Je connais G..., le préfet de police, et nous obtiendrons sans peine l'autorisation nécessaire. L'autorisation fut accordée, et nous allâmes tout droit à la rue Morgue. C'est un de ces misérables pas- sages qui relient la rue Richelieu à la rue Saint-Roch. C'était dans l'après-midi, et il était déjà tard quand nous y arrivâmes, car ce quartier est situé à une grande distance de celui que nous habitions. Nous trouvâmes bien vite la maison, car il y avait une multitude do gens qui contemplaient de l'autre côté de volets fermés, avec la rue les une curiosité badaude. C'était une maison comme toutes les maisons de Paris, avec une porte cochère, et sur l'un des côtés une niche vitrée avec un carreau mobile, représentant la loge du concierge. Avant d'entrer, nous remontâmes la rue, nous tournâmes dans une allée, les derrières et nous passâmes ainsi sur de la maison. Dupin, pendant ce temps, examinait tous les alentours, aussi bien que la maison, avec une attention minutieuse dont je ne pouvais pas deviner l'objet. DOUBLE ASSASSINAT DANS LA RUE MORGUE. 01 Nous revînmes sur nos pas vers la façade de la maison; nous sonnâmes, nous montrâmes notre }3ouvoir, et les agents nous permirent d'entrer. Nous montâmes jusqu'à la chambre où on avait trouvé le corps de ma- demoiselle l'Espanaye, et où gisaient encore les deux cadavres. Le désordre de la chambre avait été respecté, comme cela se pratique en pareil cas. Je ne vis rien de plus que ce qu'avait constaté la Gazette des trilninaux. Dupin analysait minutieusement toutes choses, sans en excepter les corps des victimes. Nous passâmes ensuite dans les autres chambres, et nous descendîmes dans les cours, toujours accompagnés par un gendarme. Cet examen dura fort longtemps, et il était nuit quand nous quittâmes la maison. En retournant chez nous, mon camarade s'arrêta quelques minutes dans les bureaux d'un journal quotidien. J'ai dit que mon ami avait toute sorte de bizarre- ries, et que je les ménageais (car ce mot n'a pas d'équi- valent en anglais). taisie de se Il entrait maintenant dans sa fan- refuser à toute conversation relativement à l'assassinat, jusqu'au lendemain à midi. Ce fut alors qu'il me demanda brusquement si j'avais remarqué quelque chose de particulier sur le théâtre du crime. 11 y eut dans sa manière de prononcer le mot parti- culier un accent qui me donna le frisson sans que je susse pourquoi. —Non, rien de particulier, dis-je, rien autre, du moins, que ce que nous avons lu tous deux dans — La Gazette, le journal. reprit-il, n'a pas, je le crains, pénétré HISTOIRES EXTRAORDINAIRES. (')•> l'horreur insolite de l'affaire. Mais laissons là les opi- nions niaises de ce papier. Il me semble que le mystère est considéré comme insoluble, par la raison même qui devrait le faire regarder comme facile à résoudre, je veux parler du caractère excessif sous lequel il — ap- paraît. Les gens de police sont confondus par l'absence apparente, de motifs légitimant, non le meurtre en lui- même, mais l'atrocité du meurtre. Ils se sont embarrassés aussi par l'impossibilité apparente de concilier les voix qui se disputaient avec ce fait qu'on n'a trouvé en haut de l'escalier d'autre personne que mademoiselle l'Espanaye, assassinée, et qu'il n'y avait aucun moyen de sortir sans être vu des gens qui montaient l'escalier. — corps fourré, — cheminée, mutidame, — ces considérations, L'étrange désordre de la chambre, la tête en bas, dans la le l'effrayante lation du corps de la vieille jointes à celles que j'ai mentionnées et à d'autres dont je n'ai pas besoin de parler, ont suffi pour paralyser l'action des agents du ministère et pour dérouter com- plètement leur perspicacité si vantée. Ils ont commis la très-grosse et très-commune faute de confondre l'ex- traordinaire avec l'abstrus. Mais c'est justement en suivant ces déviations du cours ordinaire de la n'ature que la raison trouvera son chemin, si la chose est possible, et marchera vers la vérité. Dans des investiga- tions du genre de celle qui nous occupe, il ne faut pas tant se demander comment les choses se sont passées, qu'étudier en quoi elles se distinguent de tout ce qui est arrivé jusqu'à présent. Bref, la facilité avec laquelle DOUBLE ASSASSINAT DANS LA UUE MORGUE. j'arriverai, — ou du mystère, est je suis déjà arrivé, —à 03 la solution en raison directe de son insolubilité apparente aux yeux de la police. Je fixai mon homme avec un étonnement muet. — J'attends maintenant, continua-t-il en jetant un regard sur la porte de notre chambre, j'attends un individu qui, bien qu'il ne soit peut-être pas l'auteur de cette boucherie, doit se trouver en partie impliqué dans sa perpétration. Il est probable qu'il est innocent de la partie atroce du crime. J'espère ne pas me tromper dans cette hypothèse ; car c'est sur cette hypothèse que je fonde l'espérance de déchiffrer l'énigme entière. J'attends l'homme ici, — dans cette chambre, — d'une minute à l'autre. Il est vrai qu'il peut fort bien ne pas venir, mais il y a quelques probabilités pour qu'il vienne. S'il vient, il sera nécessaire de le garder. Voici des pistolets, et nous savons tous deux à quoi ils servent quand l'occasion l'exige. Je pris les pistolets, sans trop savoir ce que je fai- sais, pouvant à peine en croire mes oreilles, — pendant que Dupin continuait, à peu près comme dans un monologue. J'ai déjà parlé de ses manières distraites dans ces moments-là. Son discours s'adressait à moi sa voix, quoique montée à un diapason avait cette intonation que l'on ; mais fort ordinaire, prend d'habitude en parlant à quelqu'un placé à une grande distance. Ses yeux, d'une expression vague, ne regardaient que le mur. — Les voix qui se disputaient, disait-il," les voix en- HISTOIRES EXTRAORDINAIRES. (54 tendues par les gens qui montaient l'escalier n'étaient pas celles de ces malheureuses femmes, — cela est plus que prouvé par l'évidence. Cela nous débarrasse pleinement de la question de savoir si la vieille dame aurait assassiné sa fille et se serait ensuite suicidée. )) Je ne parle de ce cas que par amour de la méthode; car la force de madame l'Espanaye eût été absolument insuffisante pour introduire le corps de sa fille dans la cheminée, delà façon où on l'a découvert; et la nature des blessures trouvées sur sa propre personne exclut entièrement l'idée de suicide. Le meurtre a donc été commis par des tiers, et les voix de ces tiers sont celles qu'on a entendues se quereller. )) Permettez-moi maintenant d'appeler votre atten- — non pas sur dépositions relatives à ces — mais sur ce y a de particulier dans ces déposivoix. les tion, qu'il tions. Y avez-vous remarqué quelque chose de particu- lier? — Je remarquai que, pendant que tous les témoins s'accordaient à considérer la grosse voix comme étant celle d'un Français, il y avait un grand désaccord re- lativement à la voix aiguë, ou, comme l'avait définie un seul individu, à la voix âpre. — Cela constitue l'évidence, la particularité de l'évidence. de distinctif; — cependant il dit Dupin, mais non Vous n'avez rien observé y avait quelque chose à observer. Les témoins, remarquez-le bien, cord sur la grosse voix; là-dessus, il sont d'ac- y a unanimité. Mais relativement à la voix aiguë, il y a une particula- DOUBLE ASSASSINAT DANS LA RUE MORGUE. rite, — elle ne consiste pas dans leur désaccord, mais en ceci que, quand un 65 — un Anglais, un Italien, Espagnol, un Hollandais, essayent de la décrire, chacun en parle comme d'une voix d'étranger, chacun est sûr que ce n'était pas la voix d'un de ses compatriotes. )) Chacun la compare, non pas à la voix d'un individu dont la langue lui serait familière, mais justement au contraire. Le Français présume que d'Espagnol, et il aurait s'il était affirme familiarisé que c'était pu avec la voix établi que le témoin, c'était une voix distinguer quelques mots Vesjjagnol. Le Hollandais d'un Français; mais il est ne sachant pas le français, a été interrogé par le canal d'un interprète. L'Anglais pense que c'était la voix d'un Allemand, et il n'entend pas V allemand. L'Espagnol est positivement sur la voix d'un Anglais, mais il que c'était en juge uniquement par l'intonation, car il n'a aucune connaissance de F anglais. L'Italien croit à une voix de Russe, mais il n'a jamais causé avec une personne native de Russie. Français, cependant, diffère du premier, et Un il autre est cer- tain que c'était une voix d'Italien; mais, n'ayant pas la connaissance de cette langue, il fait comme l'Espagnol, il lire sa certitude de Vintonation. Or, cette voix était donc bien insolite et bien étrange, qu'on ne pût obtenir à son égard que de pareils témoignages? Une voix dans les intonations de laquelle des citoyens des cinq grandes parties de l'Europe n'ont rien pu recon- naître qui leur fût familier! Vous me direz que c'était peut-être la voix d'un Asiatique ou d'un Africain. Les m HISTOIRES EXTRAORDINAIRES. Africains et les Asiatiques n'abondent pas à Paris ; mais, sans nier la possibilité du cas, j'appellerai simplement votre attention sur trois points. )) Un témoin dépeint la voix ainsi : plutôt âjrreqiC ai- guë. Deux autres en parlent comme d'une voix brève et saccadée. Ces témoins n'ont distingué aucune parole, — aucun son ressemblant )) Je à des paroles. ne sais pas, continua Dupin, quelle impression pu faire sur votre entendement; mais je n'hésite j'ai pas à affirmer qu'on peut tirer des déductions légitimes de cette partie même des dépositions, aux deux voix, — lative — la partie re- grosse voix et la voix aiguë la — très-suffisantes en elles-mêmes pour créer un soupçon qui indiquerait la route dans toute investigation ultérieure du mystère. » J'ai dit : déductions légitimes, mais cette expression ne rend pas complètement ma pensée. Je voulais faire entendre que ces déductions sont les seules convenables, et que ce soupçon en surgit inévitablement comme le seul résultat possible. Cependant, de quelle nature est ce soupçon, je ne vous le dirai pas immédiatement. Je désire simplement vous était plus que suffisant démontrer que ce soupçon pour donner un caractère dé- cidé, une tendance positive à l'enquête que je voulais faire dans la chambre. )) Maintenant, transportons-nous en imagination dans cette chambre. Quel sera le premier objet de notre re- cherche? Les moyens d'évasion employés par les meurtriers. Nous pouvons affirmer, — n'est-ce pas, — que DOUBLE ASSASSINAT DANS LA RUE MORGUE. 07 nous ne croyons ni l'un ni l'autre aux événements surnaturels? Mesdames l'Espanaye n'ont pas été assas- sinées par les esprits. Les auteurs du meurtre étaient des êtres matériels, et ils ont fui matériellement. » Or, comment? Heureusement, il n'y a qu'une ma- nière de raisonner sur ce point, et cette manière nous conduira à une conclusion positive. un à un les moyens possibles que les assassins étaient dans Examinons donc d'évasion. la Il est chambre où clair l'on a trouvé mademoiselle l'Espanaye, ou au moins dans la chambre adjacente quand lier. la foule a monté l'esca- Ce n'est donc que dans ces deux chambres que nous avons à chercher des issues. La police a levé les parquets, ouvert les plafonds, sondé la maçonnerie des murs. Aucune issue secrète n'a pu échapper à sa perspicacité. Mais je ne j'ai examiné avec d'issue secrète. les me suis pas fié à ses yeux, et miens; il n'y a réellement pas Les deux portes qui conduisent des chambres dans le corridor étaient solidement fermées et les clefs en dedans. Voyons les cheminées. Celles-ci, qui sont d'une largeur ordinaire jusqu'à une distance de huit ou dix pieds au-dessus du foyer, ne livreraient pas au delà un passage suffisant à un gros chat. » L'impossibilité de la fuite, du moins par les voies ci-dessus indiquées, étant donc absolument établie, nous en sommes réduits aux fenêtres. Personne n'a pu fuir par celles de la chambre du devant sans être vu par la foule du dehors. 11 a donc fallu que les meurtriers s'échappassent par celles de la chambre de derrière. HISTOIRES EXTRAORDINAIRES. 68 Maintenant, amenés, » comme nous le sommes, à cette conclusion par des déductions aussi irréfragables, nous n'avons pas le droit, en tant que raisonneurs, de la rejeter en raison de son apparente impossibilité. 11 ne nous reste donc qu'à démontrer que cette impossibilité apparente n'existe pas en réalité. » Il y a deux fenêtres dans la chambre.' L'une des deux n'est pas obstruée par l'ameublement, restée l'autre est cachée par le chevet du première la On solidement était qui lit, massif et qui est poussé tout contre. que et entièrement visible. La partie inférieure a est est de fort constaté assujettie en dedans. Elle a résisté aux efforts les plus violents de ceux qui ont essayé de la lever. On avait percé dans son châssis, à gauche, un grand trou avec une vrille, et on y trouva un gros clou enfoncé presque jusqu'à la En examinant l'autre fenêtre, on y a trouvé fiché tête. un clou semblable; et un vigoureux le châssis côté. effort n'a pas eu plus de succès La police était dès lors pour lever que de l'autre pleinement convaincue qu'aucune fuite n'avait pu s'effectuer parce chemin. 11 fut donc considéré comme superflu de retirer les clous et d'ouvrir les fenêtres. » Mon examen fut un peu plus minutieux, et cela par la raison que je vous ai donnée tout à l'heure. C'était le cas, je le savais, où il fallait démontrer que l'impos- sibilité n'était qu'apparente. )) Je continuai à raisonner ainsi, —à posteriori. — Les meurtriers s'étaient évadés par l'une de ces fenê- 'DOUBLE ASSASSINAT DANS LA RUE MORGUE. très. G'J Gela étant, ils ne pouvaient pas avoir réassujetti en dedans, comme on les a trouvés; consi- les châssis dération qui, par son évidence, a borné les recherches de la police dans ce sens-là. Cependant, ces châssis étaient bien fermés. Il faut donc qu'ils puissent se fer- n'y avait pas moyen mer d'eux-mêmes. Il d'échapper à cette conclusion. J'allai droit à la fenêtre non bouchée, avec quelque difficulté, et j'essayai retirai le clou je de lever châssis. le Il a résisté à tous mes efforts, comme je m'y attendais. Il y avait donc, j'en étais sûr maintenant, un ressort caché ; et ce fait, corroborant mon idée, me convainquit au moins de la justesse de mes prémisses, quelque mystérieuses que m' apparussent toujours les circonstances relatives aux clous. Un examen minutieux me fit bientôt découvrir le ressort secret. Je le poussai, et, satisfait de ma décou- verte, je m'abstins de lever le châssis. )) Je remis alors le clou en place et l'examinai atten- tivement. Une 'personne passant par la fenêtre pouvait l'avoir refermée, et le ressort aurait fait son office; mais le clou n'aurait pas été replacé. Cette conclusion était nette et vestigations. fuis par fallait que les assassins se fussent en- l'autre fenêtre. ressorts des il encore le champ de mes in- rétrécissait Il En supposant donc que les deux croisées fussent semblables, comme était probable, il fallait cependant trouver une diffé- rence dans les clous, ou au moins dans la manière dont ils avaient été montai sur le fond de fixés. Je sangle du lit, et je regardai minutieusement l'autre fe- HISTOIRES EXTRAORDINAIRES. ÎO nôtre par-dessus le chevet ma main du lit. Je passai derrière, je découvris aisément le ressort, et je le fis jouer; — il était, comme je l'avais deviné, identique au premier. Alors, j'examinai. le clou. Il était aussi gros que l'autre, et fixé de la même manière, enfoncé pres- que jusqu'à la tête. » Vous direz que j'étais avez une pareille pensée, embarrassé; mais, si vous c'est que vous vous êtes mépris sur la nature de mes inductions. Pour me servir d'un terme de jeu, je n'avais pas commis une seule perdu la piste un seul instant; il faute; je n'avais pas n'y avait une lacune d'un anneau à la chaîne. pas J'avais suivi le secret jusque et cette phase, c'était le clou. dans sa dernière phase, Il ressemblait, dis-je, sous tous les rapports, à son voisin de l'autre fenêtre; mais ce fait, quelque concluant qu'il fût en apparence, devenait absolument nul, en face de cette considération dominante, à savoir conducteur. Il faut, que là, à ce clou, finissait le fil me dis-je, qu^il y ait dans ce clou quelque chose de défectueux. Je le touchai, et la tête, avec un petit morceau de la tige, un quart de pouce environ, me resta dans les doigts. Le reste de la tige était dans le trou, oii elle s'était cassée. Cette fracture était fort ancienne, car les bords étaient incrustés de rouille, et elle avait été opérée par un coup de marteau, qui avait enfoncé en partie la tête du clou dans le fond du châssis. morceau qui Je rajustai soigneusement la tête avec le la continuait, et le tout intact; la fissure était inappréciable. figura Je un clou pressai le J)01:BLE assassinat DA?>^S la rue morgue. ressort, je pouces ; levai la tête doucement nouveau de quelques croisée du clou vint avec elle, sans bouger de son trou. Je refermai le la 71 la croisée, et le clou offrit de semblant d'un clou complet. Jusqu'ici l'énigme était débrouillée. L'assassin avait « par fui la fenêtre qui touchait au lit. Qu'elle fût re- tombée d'elle-même après la fuite ou qu'elle eût été fermée par une main himiaine, elle était retenue par le ressort, et la police avait attribué cette résistance au clou; aussi toute enquête ultérieure avait été jugée superflue. La question, maintenant, était » celle du mode de descente. Sur ce point, j'avais satisfait mon esprit dans notre promenade autour du bâtiment. A cinq pieds et demi environ do la fenêtre en question une court chaîne de paratonnerre. De cette chaîne, il eût été impossible à n'importe qui d'atteindre la fenêtre, à plus forte raison, d'entrer. » Toutefois, j'ai remarqué que les volets du quatrième étage étaient du genre particulier que les menuisiers parisiens appellent fer racles, genre de volets fort usité peu aujourd'hui, mais qu'on rencontre fréquemment dans de vieilles maisons de Lyon et de Bordeaux. sont faits Ils comme une porte ordinaire (porte simple, non pas à double battant), que la partie inférieure est façonnée à jour et treillissée, ce et à Fexcepiion qui donne aux mains une excellente prise. » Dans le cas en question, ces volets sont larges de trois bons pieds et demi. Quand nous les avons exa- HISTOIRES EXTRAORDINAIRES. 72 minés du derrière de la maison, ils étaient tous les deux ouverts à moitié, c'est-à-dire qu'ils faisaient angle droit avec le mur. Il est présumable que la police a examiné comme moi les derrières du bâtiment; mais, en regardant ces ferrades dans le sens de leur largeur (comme elle les a vues inévitablement), elle n'a sans doute pas pris garde à cette largeur même, ou du a pas attaché agents, moins elle n'y l'importance nécessaire. En somme, les quand il a été démontré pour eux que la fuite n'avait pu s'effectuer de ce côté, ne leur ont appliqué qu'un examen succinct. » Toutefois , il était évident pour moi que le volet appartenant à la fenêtre située au chevet du lit, si on le supposait rabattu contre le mur, se trouverait à deux pieds de la chaîne du paratonnerre. Il était clair aussi que, par l'effort d'une énergie et d'un courage insolites, on pouvait, à l'aide de la chaîne, avoir opéré une invasion par la fenêtre. Arrivé à cette distance de deux pied et demi (je suppose maintenant le volet complè- tement ouvert), un voleur aurait pu trouver dans treillage une prise solide. Il aurait pu dès lors, le en lâchant la chaîne, en assurant bien ses pieds contre le mur et en s' élançant vivement, tomber dans la chambre, et attirer violemment le volet avec lui de manière à le fermer, — en supposant, toutefois, la fenêtre ouverte en ce moment-là. Remarquez bien, je vous prie, que j'ai parlé d'une énergie très-peu commune, nécessaire pour réussir » dans une entreprise aussi difficile, aussi hasardeuse. DOUBLE ASSASSINAT DANS LA RUE MORGUE. 73 but est de vous prouver d'abord que la chose a pu iMoii — en second se faire, votre attention lieu et principalement, sur le caractère d'attirer très- extraordinaire, presque surnaturel, de l'agilité nécessaire pour l'ac- complir. » Vous direz sans doute, en vous servant de la langue judiciaire, que, pour donner ma preuve à fortiori, je devrais plutôt sous-évaluer l'énergie nécessaire dans ce cas que réclamer son exacte estimation. C'est peut-être pratique la des tribunaux, mais cela ne rentre pas dans les us de la raison. Mon objet final, c'est la vérité. Mon but actuel, cette énergie culière, c'est de vous induire à rapprocher tout à fait insolite de cette voix si parti- de cette voix aiguë (ou âpre), de cette voix saccadée, dont la nationalité n'a pu être constatée par l'accord de deux témoins, et dans laquelle personne n'a saisi de mots articulés, de syllabisation. A ces mots, une conception vague et embryonnaire de la pensée de Dupin passa dans mon esprit. Il me semblait être sur la limite de la compréhension sans pouvoir comprendre; comme les gens qui sont quel- quefois sur le bord du souvenir, et qui cependant ne parviennent pas à se rappeler. argumentation Mon ami continua son : — Vous voyez, dit-il, que j'ai transporté la question du mode de sortie au mode d'entrée. Il était dans mon plan de démontrer qu'elles se sont effectuées de la même manière et sur le même point. Retournons maintenant dans l'intérieur de la chambre. Examinons HISTOIRES EXTRAORDINAIRES. 74 toutes les particularités. Les tiroirs dit-on, ont été mis au pillage, et de la commode, cependant on y a trouvé plusieurs articles de toilette intacts. Cette conclusion est absurde; c'est une simple conjecture, une conjecture passablement — niaise, et rien de plus. Gomment pouvons-nous savoir que les articles trouvés dans les tiroirs ne représentent pas tout ce que les tiroirs contenaient? Madame l'Espanaye et sa fille me- naient une vie excessivement retirée, ne voyaient pas le monde, sortaient rarement, avaient donc peu d'oc- casions de changer de toilette. étaient au moins d'aussi ceux que possédaient Et, si Ceux qu'on a trouvés bonne qualité qu'aucun de vraisemblablement ces dames. un voleur en avait pris quelques-uns, pourquoi n'aurait-il aurait-il pas pris les meilleurs, — pourquoi ne les pas tous pris? Bref, pourquoi aurait-il aban- donné les quatre mille francs en or pour s'empêtrer d'un paquet de linge? L'or a été abandonné. La presque (otalité de la somme désignée par le banquier Mignaud a été trouvée sur le parquet, dans les sacs. Je tiens donc à écarter de votre pensée l'idée saugrenue d'un intérêt, idée engendrée dans dépositions qui le parlent cerveau de d'argent la police par les délivré à la porte même de la maison. Des coïncidences dix fois plus remarquables que celle-ci (la livraison de l'argent meurtre commis trois jours après sur se présentent dans chaque heure de notre vie sans attirer notre attention, les et le le propriétaire) même nih minute. En général, coïncidences sont de grosses pierres d'achoppement DOUBLE ASSASSINAT DANS LA RUE MORGUE. dans la 75 route de ces pauvres penseurs mal éduqués qui ne savent pas le premier mot de théorie des la probabilités, théorie à laquelle le savoir humain ses plus glorieuses conquêtes et ses plus belles doit décou" Dans le cas présent, si l'or avait disparu, le fait vertes. qu'il avait été délivré trois jours auparavant créerait quelque chose de plus qu'une coïncidence. Cela corroborerait l'idée d'intérêt. Mais, dans les circonstances réelles où nous l'or a été le sommes placés, si nous supposons que mobile de l'attaque, il nous faut supposer ce criminel assez indécis et assez idiot pour oublier h son or et le mobile qui l'a fait agir. la fois » Mettez donc bien dans votre esprit les points sur lesquels j'ai attiré votre attention, culière, cette agilité — cette voix sans pareille, et parti- cette absence frappante d'intérêt dans un meurtre aussi singuliè- rement atroce que celui-ci. la — Maintenant, examinons boucherie en elle-même. Voilà une femme étranglée par la force des mains, et introduite dans une che- minée, la tête en bas. Des assassins ordinaires n'emploient pas de pareils procédés pour tuer. Encore moins cachent-ils ainsi les cadavres de leurs victimes. Dans cette façon de fourrer le corps dans la cheminée, vous admettrez qu'il y a quelque chose d'excessif et de bizarre, avec — quelque chose d'absolument inconciliable tout ce que nous connaissons actions bumaines, en général des même en supposant que les auteurs fussent les plus pervertis des hommes. Songez aussi quelle force prodigieuse il a fallu pour pousser ce corps HISTOIRES EXTRAORDINAIRES. 70 dans une pareille ouverture, et l'y pousser puis- si samment, que les efforts réunis de plusieurs personnes furent à peine suffisants pour Ten retirer. )) Portons maintenant notre attention sur d'autres in- dices de cette vigueur merveilleuse. Dans le foyer, on a trouvé des mèches de cheveux, — des mèches très- épaisses de cheveux gris. Ils ont été arrachés avec leurs racines. Vous savez quelle puissante force il faut pour arracher seulement de la tête vingt ou trente cheveux à la fois. Vous avez vu les mèches en question aussi bien que moi. A leurs racines grumelées — affreux — adhéraient des fragments de cuir chevelu, — preuve certaine de prodigieuse puissance a spectacle! qu'il la fallu déployer pour déraciner peut-être cinq cent mille cheveux d'un seul coup. » Non-seulement le cou delà vieille dame était coupé, mais la tête absolument séparée du corps; l'instrument était un simple rasoir. Je vous prie de remarquer cette férocité bestiale. Je ne parle pas des meurtrissures du corps de madame l'Espanaye; M. Dumas et son honorable confrère, M. Etienne, ont affirmé qu'elles avaient été produites par un instrument contondant; et en cela ces messieurs furent tout à fait dans le vrai. L'instru- ment contondant a été évidemment le pavé de la cour sur laquelle la victime est tombée de la fenêtre qui donne sur le lit. Cette idée, quelque simple qu'elle apparaisse maintenant, a échappé à la police par la même raison qui Ta empêchée de remarquer la largeur des volets; parce que, grâce à la circonstance des DOUBLE ASSASSINAT DANS LA RUE MORGUE. clous, sa perception était l'idée hermétiquement 'bouchée à que les fenêtres eussent jamais pu être ouvertes. Si )) 77 maintenant, convenablement — subsidiairement, — vous avez réfléchi au désordre bizarre de la chambre, nous sommes allés assez avant pour combiner idées d'une agilité bestiale, d'une boucherie sans les merveilleuse, motif, d'une férocité d'une grotes- querie dans l'horrible absolument étrangère à l'humanité, et d'une voix dont l'accent est inconnu à l'oreille d'hommes de plusieurs nations, d'une voix dénuée de toute syllabisation distincte et intelligible. Or, pour vous, qu'en ressort-il? Quelle impression ai-je faite sur votre imagination? Je sentis un frisson courir dans ma chair quand Dupin me fit cette question. — Un fou, dis-je, aura commis ce meurtre, — quelque maniaque furieux échappé à une maison de santé du voisinage. — Pas trop mal, répliqua-t-il, votre idée est presque applicable. Mais les voix des fous, môme dans leurs plus sauvages paroxysmes, ne se sont jamais accordées avec ce qu'on dit de cette singulière voix entendue dans l'escalier. Les fous font partie d'une nation quelconque, et leur langage, pour incohérent qu'il soit* dans les pa- roles, est toujours syllabifié. En outre, le cheveu d'un fou ne ressemble pas à celui que je tiens maintenant dans ma main. J'ai dégagé cette petite touffe des doigts rigides et crispés de madame l'Espanaye. Dites-moi ce que vous en pensez. HISTOIRES EXTRAORDINAIRES. 78 — Diipin! complètement dis-je^ cheveux sont bien extraordinaires, bouleversé, ces — ce ne sont pas là des cheveux humains ! — Je n'ai pas affirmé qu'ils fussent tels, dit-il ; mais, avant de nous décider sur ce point, je désire que vous jetiez un coup d'œil sur le petit dessin que j'ai {racé sur ce bout de papier. C'est un fac-similé qui représente ce que certaines dépositions définissent les meurtrissures noirâtres (jles et les profondes marques cVon- trouvées sur le cou de mademoiselle l'Espanaye, et que MM. Dumas et Etienne appellent une série de taches livides, évidemment causées par l'impression des doigts. — Vous voyez, continua mon ami en déployant papier sur la table, que ce dessin donne le d'une l'idée poigne solide et ferme. Il n'y a pas d'apparence que les doigts aient glissé. Chaque doigt a gardé, peut-être jusqu'à la mort de la victime, la terrible prise s'était faite, et dans laquelle il s'est qu'il moulé. Essayez maintenant de placer tous vos doigts, en même temps, chacun dans la marque analogue que vous voyez. J'essayai, — 11 mais inutilement. est possible, dit Dupin, que nous ne fassions pas cette expérience d'une manière décisive. Le papier est déployé sur une surface plane, et la gorge humaine est cylindrique. Voici un rouleau de bois dont la cir- conférence est à peu près celle d'un cou. Étalez le dessin tout autour, et recommencez l'expérience. J'obéis; mais la difficulté fut que la première fois. encore plus évidente DOUBLE ASSASSINAT DANS LA RUE MORGUE. — Ceci, dis-je, n'est pas la trace d'une 70 main hu- maine. — Maintenant dit Dupin , , lisez ce passage de Cu- vier. C'était l'histoire minutieuse, tive, anatomique et descrip- du grand orang-outang fauve des îles de l'Inde orientale. Tout le monde connaît suffisamment la gi- gantesque stature, la force et l'agilité prodigieuses, la férocité sauvage et les facultés d'imitation de ce mam- mifère. Je compris d'un seul coup tout l'horrible du meurtre. — La description des doigts, lecture, la dis-je, quand j'eus fini s'accorde parfaitement avec le dessin. Je — excepté un orang-outang, de l'espèce en question, — n'aurait pu des marvois qu'aucun animal, et faire ques telles que celles que vous avez dessinées. Cette touffe de poils fauves est aussi d'un caractère identique à celui de l'animal de Cuvier. Mais je ne me rends pas facilement compte des détails de cet effroyable mys- on a entendu deux voix se disputer, et tère. D'ailleurs, l'une d'elles était incontestablement la voix d'un Français. — C'est vrai ; et vous sion attribuée presque vous rappellerez une expres- unanimement à cette voix, — l'expression Mon Dieu! Ces mots, dans les circonstances présentes, ont été caractérisés par l'un des témoins (Montani, le confiseur) comme exprimant un reproche et une remontrance. C'est donc sur ces j'ai fondé l'espérance de deux mots que débrouiller complètement HISTOIRES EXTRAORDINAIRES. 80 l'énigme. Il Un Français a eu connaissance du meurtre. est possible, est innocent — il est même plus que probable qu'il de toute participation à cette sanglante L'orang-outang a pu affaire. lui échapper. Il est pos- chambre, mais sible qu'il ait suivi sa trace jusqu'à la que, dans les circonstances terribles qui ont suivi, n'ait Je pu s'emparer de lui. L'animal est encore il libre. ne poursuivrai pas ces conjectures, je n'ai pas le droit d'appeler ces idées d'un autre nom, puisque les ombres de réflexions qui leur servent de base sont d'une profondeur à peine suffisante pour être appréciées pas ma propre raison, et que je ne prétendrais pas qu'elles fussent appréciables pour une autre intel- Nous les nommerons donc des conjectures, et ligence. nous ne les prendrons que pour en question est, cette atrocité, soir, telles. Si le Français comme je le suppose, innocent de cette annonce que j'ai laissée hier au pendant que nous retournions au logis, dans les bureaux du journal le Monde (feuille consacrée aux intérêts maritimes, et très-recherchée par les marins), l'amènera chez nous. Il me tendit un papier, et je lus AVIS. — On a trouvé dans lo : bois de Boulogne, le malin du... courant (c'était le matin de l'assassinat), de fort bonne heure, un énorme orang-outang fauve de l'espèce de Bornéo. Le propriétaire (qu'on sait l'équipage d'un maltais) après en l:)0ursé navire avoir donné quelques frais à être un marin appartenant un signalement la à peut retrouver l'animal, satisfaisant et rcm- personne qui s'en est emparée et DOUBLE ASSASSINAT DANS LA RUE MORGUE. qui l'a gardé. S'adresser rue faubourg Saint-Ger- n".., , 81 main, au troisième. — Comment avez-vous pu, demandai-je à Dupio, savoir que l'homme était un marin, et qu'il apparte- suis pas sûr. nait à un navire maltais ? — Je ne n'en le sais pas, dit-il, un petit morceau de ruban qui, Voici toutefois je j'en juge par sa forme et son aspect graisseux, a évidem- ment servi à nouer les cheveux en une de ces longues queues qui rendent les marins si fiers et si farauds. En outre, ce nœud est un de ceux que peu de personnes savent faire, excepté les marins, et il est particulier aux Maltais. J'ai ramassé le ruban au bas de la chaîne du paratonnerre. à l'une 11 est impossible qu'il ait appartenu des deux victimes. Après tout, si je me suis trompé en induisant de ce ruban que le Français est marin appartenant à un navire maltais, fait de mal à personne avec dans l'erreur, je mon annonce. Si je suis supposera simplement que il n'aurai j'ai été fourvoyé par quelque circonstance dont il ne prendra pas la peine de s'enquérir. Mais, si je suis dans le vrai, il y a un grand point de gagné. Le Français, qui a connaissance du meurtre, bien qu'il en hésitera soit innocent, —à naturellement à répondre à l'annonce, réclamer son orang-outang. Il raisonnera ainsi : <( Je suis innocent; je suis pauvre; mon orang-outang est d'un grand prix — une situation ; c'est presque une fortune dans comme la mienne ; — pourquoi le per- m HISTOIRES EXTRAORDINAIRES. drais-je par quelques niaises appréliensions de danger? Le voilà, il est du meurtre. ma main. On Fa trouvé dans le sous bois de Boulogne, — à une grande distance du théâtre Soupçonnera-t-on brute ait pu faire jamais qu'une bête coup? La police est dépistée, le elle n'a pu retrouver le plus petit fil conducteur. — Quand même on serait sur la piste de Fanimal, il serait impossible de me prouver que j'aie eu connaissance de ce meurtre, ou de m'incriminer en raison de cette connaissance. Enfin, e(, avant tout, je suis connu. Le rédacteur de l'annonce me désigne comme le proprié- taire de la bête. Mais je ne sais pas jusqu'à quel point s'étend sa certitude. j'évite Si de réclamer une pro- priété d'une aussi grosse valeur, qui est m' appartenir, attirer sur je puis connue pour l'animal un dange- reux soupçon. Ce serait de ma part une mauvaise politique d'appeler l'attention sur moi ou sur la bête. Je répondrai décidément à l'avis du journal, je reprendrai mon orang-outang, et je l'enfermerai solidement jus- qu'à ce que cette affaire soit oubliée. » En ce moment, nous entendîmes un pas qui montait l'escalier. — Apprêtez-vous, dit Dupin, prenez vos pistolets, mais ne vous en servez pas, — ne les montrez pas avant un signal de moi. On avait laissé ouverte la porte cochère, et le visiteur était entré sans sonner et avait gravi plusieurs marches de l'escalier. Mais on cCit dit maintenant qu'il hésitait. Nous l'entendions redescendre. Dupin se DOUBLE ASSASSINAT DANS LA RUE MORGUE. quand nous dirigea vivement vers la porte, 83 l'enten- dîmes qui remontait. Cette fois, il ne battit pas en retraite , mais s'avança délibérément et frappa à la porte de notice chambre. — Entrez, dit Dupin d'une voix gaie et cordiale. Un homme se présenta. C'était évidemment un marin, — un grand, robuste et musculeux individu, avec une expression d'audace de tous les diables qui n'était pas du tout déplaisante. Sa figure, fortement hàlée, était plus d'à moitié cachée par les favoris et les mous- taches. Il portait un gros bâton de chêne, mais ne semblait pas autrement armé, 11 nous salua gauche- ment, et nous souhaita le bonsoir avec un accent français qui, bien que légèrement bâtarde de suisse, rap- pelait suffisamment une origine parisienne. — Asseyez-vous, mon ami, dit Dupin; je suppose que vous venez pour votre orang-outang. Sur ma parole, vous l'envie presque je ; il est remarquablement beau et c'est sans doute une bête d'un grand prix. Quel âge lui donnez-vous bien? Le matelot aspira longuement, de l'air d'un homme qui se trouve soulagé d'un poids intolérable, et répliqua d'une voix assurée : — Je ne saurais trop vous dire cependant, ; peut guère avoir plus de quatre ou cinq ans. il ne Est-ce que vous l'avez ici? — Oh! non; nous n'avions pas de lieu commode pour l'enfermer. Il est dans une écurie de manège près d'ici, rue Dubourg. Vous pourrez l'avoir demain matin. HISTOIRES EXTRAORDINAIRES. 84 vous êtes en mesure de prouver votre droit de Ainsi propriété? — Oui, monsieur, certainement. — vraiment peiné de m'en séparer, — Je serais Dupin. dit — Je n'entends pas, pris tant de peine dit pour rien l'homme, que vous ayez ; je n'y ai pas compté. Je payerai volontiers une récompense à la personne qui a retrouvé Tanimal, une récompense raisonnable s'en- tend. — Fort bien, répliqua mon ami, tout cela en Voyons, — que donneriez- vous bien? est fort jusle, Ah ! vérité. je vais pense : vous le dire. Voici quelle sera ma récom- vous me raconterez tout ce que vous savez re- lativement aux assassinats de la rue Morgue. Dupin prononça ces derniers mots d'une voix trèsbasse et fort tranquillement. Il se dirigea vers la porte avec la même placidité, la ferma, et mit sa poche. 11 alors un pistolet tira la clef dans de son sein, et le posa sans le moindre' émoi sur la table. La figure du marin devint pourpre, comme était s'il en aux agonies d'une suffocation. Il se dressa sur ses pieds et saisit son bâton; mais, une seconde après, il se laissa retomber sur son siège, tremblant violem- ment et la mort sur le visage. Il nepouvait articulerune parole. Je le plaignais du plus profond de — Mon ami, dit mon cœur. Dupin d'une voix pleine de bonté, vous vous alarmez sans motif, —je vous assure. Nous ne voulons vous faire aucun mal. Sur mon honneur DOUBLE ASSASSINAT DANS LA RUE MORGUE. 85 homme et de Français, nous n'avons aucun de galant mauvais dessein contre vous. Je sais parfaitement que vous êtes innocent des horreurs de la rue Morgue. Cependant, cela ne veut pas dire que vous n'y soyez pas quelque peu impliqué. Le peu que je vous ai dit doit vous prouver que j'ai eu sur cette affaire des moyens d'information dont vous ne vous seriez jamais douté. Maintenant, la chose est claire pour nous. Vous n'avez rien fait que vous ayez pu éviter, — rien, à coup sûr, qui vous rende coupable. Vous auriez pu voler impu- nément; vous n'avez même pas été coupable de vol. Vous n'avez rien à cacher ; vous n'avez aucune raison de cacher quoi que ce soit. D'un autre côté, vous êtes contraint par tous les principes de fesser tout ce que vous savez. l'honneur à con- Un homme innocent est actuellement en prison, accusé du crime dont vous pouvez indiquer l'auteur. Pendant que Dupin prononçait ces mots, le matelot avait recouvré, en grande partie, sa présence d'esprit ; mais toute sa première hardiesse avait disparu. — Que, Dieu — tite pause, me soit en aide ! dit-il après une pe- je vous dirai tout ce que je sais sur cette affaire; mais je n'espère pas que vous en croyiez la moitié, — je serais vraiment un sot, Cependant, je suis innocent, j'ai sur le cœur, quand si je l'espérais! ce que et je dirai tout même il m'en coûterait la vie. Voici en fait substance ce qu'il nous raconta : Il avait dernièrement un voyage dans l'archipel indien. — HISTOIRES EXTRAORDINAIRES. 80 Une bande de matelots, dont il qua à Bornéo faisait partie, débar- dans l'intérieur pour y faire et pénétra une excursion d'amateurs. Lui et un de ses camarades avaient pris l'orang-outang. Ce camarade mourut, et devint donc sa l'animal propriété exclusive, à lui. Après bien des embarras causés par l'indomptable fé- du rocité captif pendant la traversée, il réussit à la longue à le loger sûrement dans sa propre demeure à pour ne pas attirer sur lui-même l'insuppor- Paris, et, table curiosité des voisins, il avait soigneusement en- fermé l'animal, jusqu'à ce qu'il l'eût guéri d'une blessure au pied qu'il s'était faite abord avec une esquille. Son projet, finalement, était de le vendre. Comme il revenait, une nuit, ou .plutôt un matin, — d'une petite orgie de mate- le matin du meurtre, lots, trouva la bête installée dans sa chambre il à coucher; elle s'était échappée du cabinet voisin, où il la croyait solidement enfermée. Un rasoir à la main et toute barbouillée de savon, elle était assise devant un miroir, et essayait de se raser, comme sans doute elle l'avait vu faire à son maître la serrure. Terrifié en l'épiant par le trou de en voyant une arme si dangereuse dans les mains d'un animal aussi féroce, parfaitement capable il l'homme, pendant quelques de s'en servir, instants, n'avait su quel avait dompté l'animal, parti prendre. D'habitude, même dans ses accès les plus furieux, par les coups de fouet, et il voulut y recourir cette fois encore. Mais, en voyant le fouet, l'orang-ou- tang bondit à travers la porte de la chambre, dégringola DOUBLE ASSASSINAT DANS LA RUE MORGUE. 87 par les escaliers, et, profitant d'une fenêtre ouverte par malheur, il se jeta dans la rue. Le Français, désespéré, poursuivit le singe; celui-ci, tenant toujours son rasoir d'une main, s'arrêtait de temps en temps, se retournait, et faisait des grimaces à l'homme qui le poursuivait, jusqu'à ce qu'il se vît près d'être atteint, puis il reprenait sa course. Cette chasse dura ainsi un bon bout de temps. Les profondément tranquilles, étaient trois heures du matin. rière la rue et il En traversant un passage Morgue, l'attention du rues pouvait être der- fugitif fut attirée par une lumière qui partait de la fenêtre de madame l'Espanaye', au quatrième étage de sa maison. précipita vers le mur, il_ Il se aperçut la chaîne du paraton- nerre, y grimpa avec une inconcevable agilité, saisit le volet, et, qui était complètement rabattu contre le mur, en s' appuyant dessus, il s'élança droit sur le chevet du lit. Toute cette gymnastique ne dura pas une minute. mur par le bond Le volet avait été repoussé contre le que l'orang-outang avait fait en se jetant dans la chambre. Cependant, le matelot quiet. Il était à la fois joyeux et in- avait donc bonne espérance de ressaisir l'ani- mal, qui pouvait difficilement s'échapper de la trappe où il s'était aventuré, et d'où on pouvait lui barrer la fuite. D'un autre côté il y avait lieu d'être fort inquiet de ce qu'il pouvait faire dans la maison. Cette dernière réflexion incita l'homme à se remettre à la poursuite HISTOIRES EXTRAORDINAIRES. 88 de son fugitif. Il n'est pas difïicile pour un marin de grimper à une chaîne de paratonnerre; mais, quand il fut arrivé à la hauteur de la fenêtre, située assez loin sur sa gauche, il se trouva fort qu'il put faire jeter empêché; tout ce de mieux fut de se dresser de manière à un coup d'œil dans l'intérieur de la chambre. Mais ce qu'il vit lui lit presque lâcher prise dans l'excès de sa terreur. C'était alors que s'élevaient les horribles cris qui, à travers le silence de la nuit, réveillèrent en sursaut les habitants de la rue Morgue. Madame l'Espanaye et sa fille, vêtues de leurs toilettes de nuit, étaient sans doute occupées à ranger quelques papiers dans le coffret de fer dont il a été fait mention, et qui avait été traîné au milieu de la chambre. Il était ouvert, et tout son contenu était éparpillé sur le parquet. Les victimes avaient sans doute la fenêtre; et, à en juger par le le dos tourné à temps qui s'écoula entre l'invasion de la bête et les premiers cris, il est probable qu'elles ne l'aperçurent pas tout de suite. Le claquement du volet a pu être vraisemblablement attribué au vent. Quand le matelot regarda dans la chambre, le terrible animal avait empoigné madame l'Espanaye par ses cheveux qui étaient épars et qu'elle peignait, et il agitait le rasoir autour de sa ligure, en imitant les gestes d'un barbier. La fdle était par terre, immobile; s'était évanouie. Les cris et les efforts de la elle vieille dame, pendant lesquels les cheveux lui furent arrachés de la tête, eurent pour eifet de changer en fureur les DOUBLE ASSASSINAT DANS LA RUE MORGUE. 89 dispositions probablement pacifiques de l'orang-outang. D'un coup rapide de son bras miisculeux, il sépara presque la tête du corps. La vue du sang transforma sa fureur en frénésie. Il grinçait des dents, il lançait du feu par les yeux. Il se jeta sur le corps de la jeune personne, il lui ensevelit ses griffes dans la gorge, et les y laissa jusqu'à ce qu'elle fût morte. Ses yeux éga- rés et sauvages tombèrent en ce moment sur le chevet du lit, au-dessus duquel put apercevoir la face de il son maître, paralysée par l'horreur. La furie de la bête, qui sans aucun doute se souvenait du terrible fouet, se frayeur. changea immédiatement en Sachant bien qu'elle avait mérité un • châti- ment, elle semblait vouloir cacher les traces sanglantes de son action, et bondissait à travers la chambre dans un accès d'agitation nerveuse, bousculant et brisant les meubles à chacun de ses mouvements, et arrachant les matelas du lit. Finalement, elle s'empara du corps de et la fille, le poussa dans posture où elle fut trouvée , la cheminée, dans la puis de celui de là vieille dame qu'elle précipita la tête la première à travers la fenêtre. Comme le singe s'approchait de la fenêtre avec son fardeau tout mutilé, le matelot épouvanté se baissa, et, se laissant couler le long de la chaîne sans précautions, il s'enfuit tout les d'un trait jusque chez sa terreur, abandonnant redoutant lui, conséquences de cette atroce boucherie , et , dans volontiers tout souci de la destinée de son orang-outang. Les voix entendues par ^ , m HISTOIRES EXTRAORDINAIRES. les gens de Tescalier étaient ses exclamations d'hor- reur et d'effroi mêlées aux glapissements diaboliques de la bête. Je n'ai presque rien à ajouter. L'orang-outang s'était sans doute échappé de la chambre par la chauie du paratonnerre, juste avant que la porte fût enfoncée. En passant par la fenêtre, il l'avait évidemment refermée. Il fut rattrapé plus tard par le propriétaire lui-même qui le vendit pour un bon prix au Jardin des plantes. Lebon fut immédiatement relâché, après que nous eûmes raconté toutes les circonstances de l'affaire , saisonnées de quelques commentaires de Dupin dans le , as- cabinet même du préfet de police. Ce fonctionnaire, quelque bien disposé qu'il fût envers mon ami, ne pouvait pas absolument déguiser sa mauvaise humeur en voyant l'affaire prendre cette tournure, et se laissa aller à un ou deux sarcasmes sur la manie des personnes qui se mêlaient de ses fonctions. — Laissez-le parler, dit Dupin, qui n'avait pas jugé à propos de répliquer. Laissez-le jaser, cela allégera sa conscience. Je suis content de l'avoir battu sur son propre terrain. Néanmoins, qu'il n'ait pas pu débrouiller ce mystère, il n'y a et cela est nullement lieu de s'en étonner, moins singulier qu'il ne le croit; car, en vérité, notre ami le préfet est un peu trop fin pour être profond. Sa science n'a pas de base. Elle est tout en tête et n'a pas de corps, comme les portraits de la déesse Laverna, — ou, vous aimez mieux, tout en si tête et en épaules, comme une morue. Mais, après tout, DOUBLE ASSASSINAT DANS LA RUE MORGUE. 01 c'est un brave homme. Je l'adore particulièrement pour un merveilleux genre de cant auquel il doit sa réputation de génie. Je veux parler de sa manie de nier ce qui est, et cV expliquer ce 1. qui n'est pas ^ Rousseau, Nouvelle Héloïsé. — E. A. P. LA LETTRE VOLEE Nil sapientiaî odiosius acunjine lumio. SÉNÈQUE. J'étais à Paris en 18... Après une sombre et ora- geuse soirée d'automne, je jouissais de la double vo- lupté de la méditation et d'une pipe d'écume de mer, en compagnie de mon ami Dupin, dans sa petite biblio- thèque ou cabinet d'étude, rue Dunot, n° 33, au troisième , faubourg Saint-Germain. Pendant une bonne heure, nous avions gardé le silence; chacun de nous, pour le premier observateur venu, aurait paru profon- dément et exclusivement occupé des tourbillons frisés de fumée qui chargeaient l'atmosphère de la chambre. Pour mon compte , je discutais points qui avaient été dans en moi-même certains la première partie de la soirée l'objet de notre conversation; je veux parler de l'affaire de la rue Morgue, et du mystère relatif à l'as- sassinat de 1. Marie Roget ^ Je rêvais _donc à l'espèce Encore un meurtre dont Dupin refait l'instruction. — Le Double Assassinat dans la rue Morgue, le Mystère de Marie lioget e la Lettre volée font une espèce de trilogie. — G. B. HISTOIRES EXTRAORDINAIRES. 94 d'analogie qui reliait ces deux affaires , quand la porte de notre appartement s'ouvrit et donna passage à notre vieille connaissance, à M. G de police de le préfet , Paris. Nous car souhaitâmes cordialement lui la bienvenue; l'homme avait son côté charmant comme son coté méprisable, et nous ne l'avions pas vu depuis quelques années. Comme nous étions assis dans les ténèbres, Dirpin se lova pour allumer une lampe; mais il se rassit et n'en lit rien, en entendant G dire qu'il était venu pour nous consulter, ou plutôt pour demander mon ami relativement à une affaire qui lui l'opinion de avait causé une masse d'embarras. — Si c'est un cas qui demande de Dupin, s'abstenant d'allumer serva la réflexion la , ob- mèche, nous l'examinerons plus convenablement dans les ténèbres. — Voilà encore une de vos idées bizarres , dit lé préfet, qui avait la manie d'appeler bizarres toutes les choses situées au delà de sa compréhension vait ainsi , et qui vi- au milieu d'une immense légion de bizar- reries. — C'est, ma foi, vrai! dit Dupin en présentant une pipe à notre visiteur, et roulant vers lui un excellent fauteuil. — Et maintenant, quel est le cas embarrassant? de- mandai-je; j'espère bien que ce n'est pas encore dans le genre assassinat. — Oh! non. Rien de pareil. est vraiment très-simple , et je Le fait est que l'affaire ne doute pas que nous LA LETTRE VOLÉE. 95 ne puissions nous en tirer fort bien nous-mêmes j'ai mais ; pensé que Dupin ne serait pas fâché d'apprendre les détails de cette affaire, parce qu'elle est excessive- ment bizarre. — Simple et bizarre, dit Dupin. — Mais oui ; et cette expression n'est pourtant pas exacte; l'un ou l'autre, si vous aimez mieux. Le est fait, que nous avons été tous là-bas fortement embar- rassés par cette affaire; car, toute simple qu'elle est, elle nous déroute complètement. — Peut-être est-ce la simplicité même de la chose qui vous induit en erreur, dit mon ami. — Quel non-sens nous dites-vous là! répliqua le préfet, en riant de bon cœur. — Peut-être mystère est-il un peu trop clair, dit le Dupîn. — Oh! bonté du ciel! qui a jamais ouï parler d'une idée pareille. — Un peu trop évident. — Ah ah ah ah oh oh ! ! ! ! ! ! criait notre hôte , qui se divertissait profondément. Oh! Dupin, vous me ferez mourir de joie, voyez-vous. — Etenfm, demandai-je, quelle est la chose en ques- tion? — Mais, je vous la dirai, répliqua le préfet, en lâ- chant une longue, solide et contemplative bouffée de fumée, et s'établissant dans son fauteuil. Je vous dirai en peu la de mots. Mais, avant de commencer, lais- sez-moi vous avertir que c'est une affaire qui demande m HISTOIRES EXTRAORDINAIRES. le plus grand secret, et que je perdrais très-probable- ment le poste que j'occupe, si l'on savait que je l'ai confiée à qui que ce soit. — Commencez, — Ou ne commencez pas, — C'est bien; commence. dis-je. dit Dupin. je J'ai été informé person- nellement, et en très-haut lieu, qu'un certain docu- ment de la plus grande importance avait été soustrait dans les appartements royaux. On sait quel est l'individu qui l'a volé; cela est hors de doute; on l'a vu s'en emparer. On sait aussi que ce document est tou- jours en sa possession. — Comment sait-on cela? demanda Dupin. — Cela clairement déduit de nature du docula est ment et de la non-apparition de certains résultats qui surgiraient immédiatement s'il sortait des mains du oleur; en d'autres termes, vue du but que celui-ci doit s'il était employé en évidemment se propo- ser. — Veuillez — Eh bien, être un peu plus clair, dis-je. j'irai jusqu'à dire que ce papier confère à son détenteur un certain pouvoir dans un certain lieu où ce pouvoir est d'une valeur inappréciable. Le préfet raffolait du cant diplomatique. — continue à ne rien comprendre, Dupin. — Rien, vraiment? Allons! Ce document, révélé Je dit à un troisième personnage, dont je tairai le nom, mettrait en question l'honneur d'une personne du plus haut rang; et voilà ce qui donne au détenteur du document LA LETTRE VOLÉE. 97 un ascendant sur l'illustre personne dont l'honneur et mis en péril. la sécurité sont ainsi — Mais cet ascendant, interrompis-je, dépend de ceci : voleur sait-il que la personne volée connaît le son voleur? Qui oserait — Le voleur, ? G c'est D..., qui ose tout ce qui est indigne d'un homme, aussi bien que ce qui est digne de lui. dit , Le procédé du vol a été aussi ingénieux que hardi. Le document en question, une lettre, pour être franc, a été reçue par la personne volée qu'elle était seule dans le boudoir royal. pendant Pendant qu'elle le lisait, elle fut soudainement interrompue par l'entrée rait de l'autre illustre personnage à qui elle dési- particulièrement vain de le jeter obligée de le cacher. Après avoir essayé en le rapidement dans un fut tiroir, elle déposer tout ouvert sur une table. La lettre, toutefois, était retournée, la suscription en des- sus, et, le contenu étant ainsi caché, elle n'attira pas l'attention. Sur ces entrefaites arriva le ministre D.... Son œil de lynx perçoit immédiatement le papier connaît l'écriture de la suscription, , re- remarque l'em- barras de la personne à qui elle était adressée, et pénètre son secret. )) Après avoir traité quelques affaires, expédiées tam- bour battant, à sa manière habituelle, poche une question, il tire de sa lettre à peu près semblable à la lettre en l'ouvre, fait semblant de la lire, et la place juste à côté de l'autre. Il se remet à causer, pendant un quart d'heure environ, des affaires publiques. A la HISTOIRES EXTRAORDINAIRES. 98 longue, il prend congé, et met la main sur la lettre à laquelle il n'a aucun droit. La personne volée le vit, mais, naturellement, n'osa pas attirer l'attention sur ce en présence du troisième personnage qui fait, était à son côté. Le ministre décampa, laissant sur la table sa propre lettre, — une lettre sans importance. Ainsi, dit Dupin en se tournant à moitié vers moi, voilà précisément le cas demandé pour rendre l'ascen- dant complet : le voleur sait que la personne volée connaît son voleur. — Oui, répliqua il le préfet, et, depuis quelques mois, a été largement usé, dans un but politique, de l'em- pire conquis fort par ce stratagème, et jusqu'à un point dangereux. La personne volée est de jour en jour plus convaincue de la nécessité de retirer sa lettre. Mais, naturellement, cela ne peut pas se faire ouver- tement. Enfin, poussée au désespoir, elle m'a chargé de la commission. — Il n'était pas possible, je suppose, dit Dupin dans une auréole de fumée, de choisir ou même d'imaginer un agent plus sagace. — Vous me flattez, bien possible qu'on ait répliqua le préfet; mais il est conçu de moi quelque opinion de ce genre. — 11 est clair, dis-je, comme vous l'avez remarqué, que la lettre est toujours entre les mains du ministre; puisque c'est le fait de la possession et non l'usage de la lettre qui crée l'ascendant. Avec l'usage, l'ascendant s'évanouit. LA LETTRE VOLÉE. — C'e§t vrai, dit G tion que j'ai , 00 et c'est d'après cette convic- marché. Mon premier soin a été de faire une recherche minutieuse à l'hôtel du ministre; et, là, mon principal embarras fut de chercher à son insu. Par-dessus tout, j'étais en garde contre le danger qu'il y aurait eu à lui donner un motif de soupçonner notre dessein. — Mais, dis-je, vous êtes tout à fait à votre affaire, dans ces espèces d'investigations. La police parisienne a pratiqué la chose plus d'une fois. — Oh sans doute; — ! et c'est pourquoi j'avais bonne espérance. Les habitudes du ministre d'ailleurs un me donnaient grand avantage. Il est souvent absent de chez lui toute la nuit. Ses domestiques ne sont pas nombreux. Ils couchent à une certaine distance de l'apparte- ment de leur maître, et, comme ils sont Napolitains avant tout, ils mettent de la bonne volonté à se laisser enivrer. comme vous savez, des clefs avec lesquelles je puis J'ai, ouvrir toutes les chambres et tous les cabinets de Paris. Pendant trois mois, il ne s'est pas passé une nuit dont je n'aie employé la plus grande partie à fouiller, en personne, l'hôtel D... Mon honneur y est intéressé, et, pour vous confier un grand secret, la récompense est énorme. Aussi je n'ai abandonné les recherches que lorsque était j'ai été pleinement convaincu que le voleur encore plus fin que moi. Je crois que j'ai scruté tous les coins et recoins de la maison dans lesquels il était possible de cacher un papier — Mais ne serait-il pas possible, insinuai-je, que HISTOIRES EXTRAORDINAIRES. 100 bien que la lettre fût au pouvoir du ministre, est indubitablement, — il l'eût — elle y cachée ailleurs que dans sa propre maison. — Gela n'est guère La situation possible, dit Dupin. particulière, actuelle, des affaires de la cour, spéciale- ment la nature de l'intrigue dans laquelle D... a pénétré, comme on sait, font de l'efficacité immédiate du — de de produire à mi— un point d'une importance presque égale à sa nute, document, la possibilité la le possession. — La — Ou, de le produire? dis-je possibilité si vous aimez . mieux, de l'annihiler, dit Dupin. — C'est vrai, remarquai-je. demment dans la personne l'hôtel. Le papier est donc évi- Quant au cas où il serait sur même du ministre, nous le considérons comme tout à fait hors de la question. — Absolument, fois dit le préfet. Je l'ai fait arrêter deux par de faux voleurs, et sa personne a été scrupu- leusement fouillée sous mes propres yeux. — Vous auriez pu vous épargner cette peine, dit Dupin, — D... n'est pas absolument fou, je présume, et dès lors il a dû prévoir ces guet-apens comme choses naturelles. — Pas absolument fou, c'est vrai, dit G tefois, c'est un poëte, ce qui, je crois, , — tou- n'en est pas fort éloigné. — G'est vrai, dit Dupin après avoir longuement et pensivement poussé la fumée de sa pipe d'écume, bien LA LETTRE VOLEE. 101 que je me sois rendu moi-même coupable de certaine rapsodie. — Voyons, dis-je, racontez-nous les détails précis de votre recherche. — Le fait est que nous avons pris notre temps, et que nous avons cherché j^artout. J'ai une vieille expé- rience de ces sortes d'affaires. Nous avons entrepris la maison de chambre en chambre à chacune les nuits de toute ; nous avons consacré une semaine. Nous avons d'abord examiné les meubles de chaque appartement. Nous avons ouvert tous les tiroirs possibles; et je pré- sume que vous n'ignorez pas que, pour un agent de police bien dressé, n'existe pas. un tiroir secret est une chose qui Tout homme qui, dans une perquisition de cette nature, permet à un tiroir secret de lui échapper, est une brute. La besogne est si facile! Il y a dans chaque pièce une certaine quantité de volumes surfaces dont on peut se rendre compte. et de Nous avons pour cela des règles exactes. La cinquantième partie d'une ligne ne peut pas nous échapper. » Après les chambres, nous avons pris les sièges. Les coussins ont été sondés avec ces longues et fines aiguilles que vous m'avez vu employer. Nous avons enlevé les dessus des tables. — Et pourquoi? — Quelquefois dessus d'une table ou de tout autre le pièce d'ameublement analogue est enlevé par une per- sonne qui désire cacher quelque chose; elle creuse le pied de la table ; l'objet est déposé dans la cavité, et le G. HISTOIRES EXTRAORDINAIRES. 102 dessus replacé. On se sert de la même manière des montants d'un lit. — Mais ne pourrait-on pas deviner la cavité par l'aus- cultation? demandai-je. — Pas le moins du monde, si, en déposant l'objot, on a eu soin de l'entourer d'une bourre de coton suffisante. D'ailleurs, dans notre cas, nous étions obligés de procéder sans bruit. — Mais vous n'avez pas pu défaire, — vous n'avez pas pu démonter toutes les pièces d'ameublement dans lesquelles on aurait pu cacher un dépôt de la façon dont vous parlez. Une lettre peut être roulée en une spirale très-mince, ressemblant et beaucoup par sa forme son volume à une grosse aiguille à tricoter, et être ainsi insérée dans un bâton de chaise, par exemple. Avez-vous démonté toutes les chaises ? — Non certainement, mais nous avons nous avons examiné les fait mieux, bâtons de toutes les chaises de l'hôtel, et même les jointures de toutes les pièces de l'ameublement, à l'aide d'un puissant microscope. S'il y avait eu la moindre trace d'un désordre récent, nous l'aurions infailliblement découvert à Tinstant. Un seul grain de poussière causée par la vrille, par ple, nous aurait sauté aux yeux exem- comme une pomme. La moindre altération dans la colle, — un simple bâil- lement dans les jointures aurait suffi pour nous révéler la cachette. — Je présume que vous avez examiné les glaces en- tre la glace et le planchéiage, et que vous avez fouillé LA LETTRE VOLEE. 103 les lits et les courtines des lits, aussi bien que les ri- deaux et les tapis. — Naturellement; et, quand nous eûmes absolument passé en revue tous les articles de ce genre, nous avons examiné la maison elle-même. Nous avons totalité divisé de sa surface en compartiments, que la nous avons numérotés, pour être sûrs de n'en omettre aucun; nous avons fait de chaque pouce carré l'objet d'un nouvel examen au microscope, et nous y avons compris les deux maisons adjacentes. — Les deux maisons adjacentes! m'écriai-je; vous avez dû vous donner bien du mal. — Oui, ma mais foi ! la récompense offerte est énorme. — Dans maisons, comprenez-vous sol? — Le est partout pavé en briques. Comparativeles le sol ment, cela ne nous a pas donné grand mal. Nous avons examiné la mousse entre les briques, elle était intacte. — Vous avez sans doute visité les papiers de D..., et les livres de la bibliothèque. — Certainement; nous avons ouvert chaque paquet et chaque article les livres, feuillet, ; nous n'avons pas seulement ouvert mais nous les avons parcourus feuillet par ne nous contentant pas de les secouer simple- ment comme font plusieurs de nos officiers de police. Nous avons aussi mesuré l'épaisseur de chaque reliure avec la plus exacte minutie, et nous avons appliqué à chacune avait la curiosité jalouse du microscope. Si Ton récemment inséré quelque chose dans une des — HISTOIRES EXTRAORDINAIRES. 104 reliures, eût été absolument impossible que le il fait six volumes qui échappât à notre observation. Cinq ou mains du relieur ont été soigneusement sortaient des sondés longitudinalement avec les aiguilles? — Vous avez exploré parquets, sous — Sans doute. Nous avons enlevé chaque les tapis. les tapis, et nous avons examiné les planches au microscope. — Et papiers des murs — Aussi. — Vous avez caves? — Nous avons caves. — fausse route, vous avez les ? visité les visité les Ainsi, dis-je, lettre n'est pas et fait dans l'hôtel, la comme vous le suppo- siez. — Je crains que vous n'ayez raison, dit le préfet. Et vous maintenant, Dupin, que me conseillez-vous de faire? — Faire une perquisition complète. — C'est absolument inutile! répliqua G sûr que je vis, la lettre n'est pas dans l'hôtel — Aussi ! Je n'ai pas de meilleur conseil à vous donner, dit Dupin. Vous avez, sans doute, un signalement exact de la lettre. — Oh! oui! Et lire ici, le préfet, tirant à haute voix un agenda, se mit à nous une description minutieuse du docu- ment perdu, de son aspect intérieur, et spécialement de Textérieur. Peu de temps après avoir fini la lecture de cette description, cet excellent homme prit congé de LA LETTRE VOLÉE. 105 nous, plus accablé et l'esprit plus complètement dé- couragé que je ne l'avais vu jusqu'alors. Environ un mois après, il nous fit une seconde visite, et nous trouva Il occupés à peu près de une pipe prit et un siège, et la même façon. causa de choses et d'autres. A la longue, je lui dis : — Eh bien, mais, G , et votre lettre volée? Je présume qu'à la fin vous vous êtes résigné à comprendre que ce n'est pas une petite besogne que d'enfoncer le ministre ? — Que le diable — l'emporte! J'ai pourtant recom- mencé cette perquisition, comme Dupin me l'avait conseillé; mais, comme je m'en doutais, c'a été peine perdue. — De combien est la récompense offerte? vous nous avez dit... demanda Dupin. — Mais... elle est très-forte... ment magnifique, — une récompense vrai- je ne veux pas vous dire au juste combien; mais une chose que je vous dirai, c'est que je m'engagerais bien à payer de ma bourse cinquante mille francs à celui qui pourrait me trouver cette lettre. Le fait est que la chose devient de jour en jour plus urgente , et la récompense a été doublée tout récem- ment. Mais, en vérité, on la triplerait, que je ne pourrais faire mon devoir mieux que je l'ai fait. — Mais... oui..., dit Dupin en traînant ses paroles au milieu des bouffées de sa pipe, je crois... réellement, G , que vous n'avez pas fait... tout votre possible... vous n'êtes pas allé au fond de la question. Vous HISTOIRES EXTRAORDINAIRES. 106 pourriez faire... un peu plus, je pense du moins, hein? — Comment? dans quel sens? — (une bouffée de fumée) vous pourriez... (bouffée sur bouffée) — prendre conseil en matière, hein? — bouffées de fumée.) — Vous rappelezMais... cette (Trois vous l'histoire qu'on raconte d'Abernethy ^ — Non au diable votre Abernethy — Assurément! au diable, cela vous amuse! — ! ! si Or donc, une fois, un certain riche, fort avare, conçut dessein de soutirer à Abernethy une consultation le médicale. Dans ce but, il entama avec lui, au milieu d'une société, une conversation ordinaire, à travers laquelle il insinua au médecin son propre cas, comme celui d'un individu imaginaire. — Nous supposeroné, dit l'avare, que les symptômes sont tels et tels; maintenant, docteur, que lui conseilleriez- vous de prendre? — Que prendre? seil, dit Abernethy, mais prendre con- à coup sûr. — Mais, dit le préfet, un peu décontenancé, je suis tout disposé à prendre conseil, et à payer pour cela. Je donnerais vraiment cinquante mille francs à qui- conque me tirerait d'affaire. — Dans ce cas, répliqua Dupin, ouvrant un tiroir et en tirant un livre de mandats, vous pouvez aussi bien me faire un bon pour la somme susdite.. Quand vous l'aurez signé, je vous remettrai votre lettre. 1. Médecin anglais très-célèbre et très-excentrique. — C. B. LA LETTRE VOLEE. Je fus stupéfié. 107 Quant au préfet, il semblait absolu- ment foudroyé. Pendant quelques minutes, muet et immobile, regardant mon ami, la il resta bouche béante, avec un air incrédule et des yeux qui semblaient lui sortir de la tête; enfin, lui, le il saisit parut revenir un peu il à une plume, et, après quelques hésitations, regard ébahi et vide, il remplit et signa un bon de cinquante mille francs, et le tendit à Dupin par-dessus la table. Ce dernier l'examina soigneusement et le serra dans son portefeuille; puis, ouvrant un pupitre^ il en tira une lettre et la donna au préfet. Notre fonc- tionnaire Tagrippa dans une parfaite agonie de joie, l'ouvrit d'une main tremblante, jeta un coup d'oeil sur son contenu, puis, attrapant précipitamment la porte, se rua sans plus de cérémonie hors de la chambre et de la maison, sans avoir prononcé une syllabe depuis le moment où Dupin l'avait prié de remplir le mandat. Quand il fut parti, mon ami entra dans quelques explications. — La police parisienne, dit-il, est excessivement habile dans son métier. Ses agents sont persévérants, ingénieux, rusés, et possèdent à fond toutes les connais- sances que requièrent spécialement Aussi, quand G nous détaillait leurs son fonctions. mode de per- quisition dans l'hôtel D..., j'avais une entière confiance dans ses talents, et j'étais sûr qu'il avait fait une investigation pleinement suffisante, dans le cercle de sa spé- cialité. — Dans le cercle de sa spécialité? dis-je. HISTOIRES EXTRAORDINAIRES. 108 — Oui, dit Dupin; les mesures adoptées n'étaient pas seulement dans l'espèce, les meilleures elles aussi poussées à une absolue perfection. Si avait été cachée dans le rayon la furent lettre de leur investigation, ces gaillards l'auraient trouvée, cela ne fait pas pour moi l'ombre d'un doute. Je me contentai de rire mais Dupin semblait avoir ; dit cela fort sérieusement. — Donc, les mesures, continua-t-il, étaient bonnes dans l'espèce et admirablement exécutées ; elles avaient pour défaut d'être inapplicables au cas et à l'homme en question. 11 y a tout un ordre de moyens singuliè- rement ingénieux qui sont pour le préfet une sorte de lit de Procruste, sur lequel il adapte et garrotte tous ses plans. Mais il erre sans cesse par trop de profondeur ou par trop de superficialité pour le cas en question, et plus d'un écolier raisonnerait )) J'ai connu au jeu de pair ou impair selle. mieux que lui. un enfant de huit ans, dont l'infaillibilité faisait l'admiration univer- Ce jeu est simple, on y joue avec des billes. L'un des joueurs tient dans sa main un certain nombre de ses billes, et demande à l'autre ci devine juste, il : « Pair ou non? » Si celui- gagne une bille; s'il se trompe, il en perd une. L'enfant dont je parle gagnait toutes billes de l'école. Naturellement, il avait les un mode de divination, lequel consistait dans la simple observation et dans l'appréciation de la finesse de ses adversaires. Supposons que son adversaire soit un parfait nigaud, et, levant sa main fermée, lui demande : «Pair ou im- LA LETTRE VOLEE. pair? » Notre écolier répond : Mais, à la seconde épreuve, lui-même « Le : 109 «Impair! » et il a perdu. il gagne, car il se dit en niais avait mis pair la première fois, et toute sa ruse ne va qu'à lui faire mettre impair à la seconde; je dirai donc : « Impair! » dit: «Impair, » 11 gagne. et il Maintenant, avec un adversaire un peu moins sim- » ple, il aurait raisonné ainsi : « Ce garçon voit que, dans le premier cas, j'ai dit impair, et, proposera, — dans le second, il se c'est la première idée qui se présentera à — une simple variation de pair à impair comme a lui, fait le premi(îr bêta; mais une seconde réflexion dira que c'est là un changement trop simple, et lui fina- lement il se décidera à mettre pair comme la première — fois. Je dirai donc « : Pair! » Il dit pair, et gagne. Maintenant, ce mode de raisonnement de notre écolier, que ses camarades appellent la chance, — en dernière analyse, qu'est-ce que c'est? — C'est simplement, l'intellect dis-je, une identification de de notre raisonneur avec celui de son ad- versaire. — C'est cela même, ditDupin; à ce petit garçon par quel et, quand je demandai moyen il effectuait cette parfaite identification qui faisait tout son succès, il me fit la » réponse suivante : —Quand je veux savoir jusqu'à quel point quelqu'un est circonspect ou stupide, jusqu'à quel point il est bon ou méchant, ou quelles sont actuellement ses pensées, je compose mon visage d'après le sien, aussi exacte7 HISTOIRES EXTRAORDINAIRES. 110 ment que possible, et j'attends alors pour savoir quels pensers ou quels sentiments naîtront dans mon esprit ou dans mon cœur, comme pour s'appareiller et cor- respondre avec ma physionomie. )) Cette réponse de l'écolier enfonce de beaucoup toute la profondeur sophistique attribuée à la Rochefoucauld, à la Bruyère, à Machiavel et à Gampanella. — Et l'identification de l'intellect du raisonneur avec celui de son adversaire dépend, si je vous comprends bien, de l'exactitude avec laquelle l'intellect de l'adversaire est apprécié. — Pour la valeur pratique, c'est en effet la condition, répliqua Dupin, et, si le préfet et toute sa bande se sont trompés si souvent, c'est, d'abord, faute de cette identification, en second lieu, par une appréciation inexacte, ou plutôt par la non-appréciation de l'intel- ligence avec laquelle ils se mesurent. Ils ne voient que leurs propres idées ingénieuses; et, quand ils cherchent quelque chose de caché, ils ne pensent qu'aux moyens dont ils se seraient servis pour le cacher. Ils ont for- tement raison en cela que leur propre ingéniosité est une représentation fidèle de celle de la foule; mais, quand il se trouve diffère, un malfaiteur particulier dont la finesse en espèce, de la leur, ce malfaiteur, naturel- lement, les roule. )) Gela ne manque jamais quand son astuce est au- dessus de la leur, et cela arrive très-fréquemment même quand elle est au-dessous. Ils ne varient pas leur système d'investigation; tout au plus, quand ils LA LETTRE VOLÉE. 111 sont incités par quelque cas insolite, — par quelque récompense extraordinaire, — ils exagèrent et poussent à outrance leurs vieilles routines ; mais ils ne changent rien à leurs principes. » Dans le cas de D..., par exemple, qu'a-t-on fait pour changer le système d'opération? Qu'est-ce que c'est que toutes ces perforations, ces fouilles, ces sondes, cet examen au microscope, cette division des surfaces en qu'est-ce que tout cela, si pouces carrés numérotés? — ce n'est Texagération, dans son application, principes ou de plusieurs principes d'un des d'investigation, qui sont basés sur un ordre- d'idées relatif à l'ingéniosité humaine, l'habitude et dont le préfet a pris dans la longue routine de ses fonctions? )) Ne voyez-vous pas qu'il considère comme chose démontrée que tousles hommes qui veulent cacher une lettre se servent, — si ce n'est précisément d'un trou fairà la vrille dans le pied d'une chaise, — au moins de quelque trou, de quelque coin tout à fait singulier dont ils ont puisé l'invention dans le même registre d'idées que le troujait avec une vrille? )) Et ne voyez-vous pas aussi que des cachettes aussi originales ne sont employées que dans des occasions ordinaires, et ne sont adoptées que par des intelligences ordinaires; car, dans tous les cas d'objets cachés, cette manière ambitieuse et torturée de cacher dans le principe, présumable et l'objet est, présumée; ainsi, la découverte ne dépend nullement de la perspicacité, mais simplement du soin, de la patience et de la réso- HISTOIRES EXTUAORDINAIUES. 112 liition des chercheurs. Mais, quand le cas est important, ou, ce qui revient au même aux yeux de la police, quand récompense est considérable, la on voit toutes ces belles quaUtés échouer infailliblement. Vous comprenez maintenant ce que je voulais dire en affirmant que, si la lettre volée avait été cachée dans le rayon de la per- quisition de notre préfet, — en d'autres termes, si le principe inspirateur de la cachette avait été compris dans les principes du préfet, — il l'eût infailliblement découverte. Cependant, ce fonctionnaire a été complè- tement mystifié ; et la cause première, originelle, dans la supposition que le ministre est un défaite, gît fou, parce qu'il s'est fait une réputation de poëte. les fous sont poètes, préfet, — de sa — c'est la Tous manière de voir du et il n'est coupable que d'une fausse distri- bution du terme moyen, en inférant de là que tous les poètes sont fous. — Mais est-ce vraiment le poëte? demandai-je. Je sais qu'ils sont deux frères, et ils se sont fait tous deux une réputation dans les lettres. Le ministre, je crois, a écrit un livre fort remarquable sur le calcul différentiel et intégral. 11 est le mathématicien, et non pas le poëte. — Vous vous trompez ; je le connais fort bien; il est Gomme poëte et mathématicien, il a dfi raisonner juste; comme simple mathé- poëte et mathématicien. maticien, il n'aurait pas raisonné du tout, et se serait ainsi mis à la merci du préfet. — Une pareille opinion, dis-je, est faite pour m'é- LA LETTRE VOLÉE. ;1J3 tonner; elle est démentie par la voix du monde entier. Vous n'avez pas l'intention de mettre à néant mûrie par plusieurs est depuis siècles. l'idée La raison mathématique comme la raison par longtemps regardée excellence. — fort, Il y a à parier, répliqua Dupin, en citant Cham- que toute idée publique, est une sottise, Les mathématiciens, fait toute convention reçue car elle a convenu au plus grand nombre. — je vous accorde cela, — ont de leur mieux pour propager l'erreur populaire dont vous parlez, et qui, bien qu'elle propagée ait été comme vérité, n'en est pas moins une parfaite erreur. Par exemple, ils nous ont, avec un art digne d'une meilleure cause, accoutumés à appliquer le terme aiialyse aux opérations algébriques. Les Français sont les premiers coupables de cette tricherie scientifique; mais, si l'on reconnaît que les termes de la langue ont — — oh! une réelle importance, de leur application, lyse traduit algèbre, si les — Je concède qu'ana- comme en latin am- à peu près bitus signifie ambition; religio, honesti, la classe des mots tirent leur valeur alors, je religion; ou homines gens honorables. vois, dis-je, que vous allez vous faire une que- relle avec un bon nombre d'algébristes de Paris ; continuez. — ,.. — mais , , ,., Je conteste la validité, et conséquemment les ré- sultats d'une raison cultivée par tout procédé spécial autre que la logique .abstraite. Je conteste particuliè- rement le raisonnement tiré de l'étude des mathéma- 114 HISTOIRES EXTRAORDINAIRES. tiques. Les mathématiques sont la science des formes et des quantités ; le raisonnement mathématique n'est autre que la simple logique appliquée à la forme et à la quantité. La grande erreur consiste à supposer que les vérités qu'on nomme i:)urement algébriques sont des vérités abstraites ou générales. Et cette erreur est si énorme, que je suis émerveillé de l'unanimité avec laquelle elle est accueillie. Les axiomes mathématiques ne sont pas des axiomes d'une vérité générale. Ce qui est vrai d'un rapport de forme ou de quantité est sou- vent une grossière erreur relativement à la morale, par exemple. Dans cette dernière science, il est très-com- munément faux que la somme des fractions soit égale au tout. De même en chimie, l'axiome a tort. Dans l'appréciation d'une force motrice, il a également tort; car deux moteurs, chacun étant d'une puissance donnée, n'ont pas, nécessairement, quand ils sont associés, une puissance égale à la somme de leurs puissances prises séparément. Il y a une foule d'autres vérités mathématiques qui ne sont des vérités que dans des limites de rapport. Mais le mathématicien argumente incorrigi- blement d'après ses vérités finies, comme étaient d'une application générale et absolue, si elles — valeur que d'ailleurs le monde leur attribue. Bryant, dans sa très-remarquable Mythologie, mentionne une source analogue d'erreurs, quand il dit que, bien que personne ne croie aux fables du paganisme, cependant nous nous oublions nous-mêmes sans cesse au point d'en tirer des déductions, comme si elles étaient des réalités LA LETTRE VOLEE. vivantes. Il 115 y a d'ailleurs chez nos algébristes, qui sont eux-mêmes des païens, de certaines auxquelles on ajoute foi, et fables païennes dont on a tiré des consé- quences, non pas tant par une absence de mémoire que par un incompréhensible trouble du cerveau. Bref, je n'ai jamais rencontré de pur mathématicien en qui on pût avoir confiance en dehors de ses racines et de ses équations; je n'en ai pas connu un seul qui ne tînt pas clandestinement pour article de foi que x^-\-yx est absolument et inconditionnellement égal à q. Dites à l'un de ces messieurs, en matière d'expérience, si cela vous amuse, que vous croyez à la possibilité de cas oii a?^ -f iix ne serait pas absolument égal à q ; vous lui aurez fait et, quand comprendre ce que vous voulez dire, mettez-vous hors de sa portée et le plus lestement possible; car, sans aucun doute, il essayera de vous assommer. )) Je veux dire, continua Dupin, pendant que je me contentais de rire de ses dernières observations, que, si le ministre n'avait été qu'un mathématicien, le pré- fet n'aurait pas été dans la nécessité de me souscrire ce billet. Je le connaissais pour un mathématicien et un poëte, et j'avais pris mes mesures en raison de sa capacité, et en tenant compte des circonstances où il se trouvait placé. Je savais que c'était un homme do cour et un intrigant déterminé. Je réfléchis qu'un pareil homme devait indubitablement être au courant des pratiques de la police. Évidemment, il devait avoir prévu — et l'événement Ta prouvé — les guet-apens HISTOIRES EXTRAORDINAIRES. iI6 qui lui ont été préparés. Je me dis qu'il avait prévu les perquisitions secrètes dans son hôtel. Ces fréquentes absences nocturnes que notre bon préfet avait saluées comme des adjuvants positifs de son futur succès, je les regardais simplement comme des ruses pour faciliter les libres recherches de la police et lui persuader plus facilement que la lettre n'était pas dans l'hôtel. Je sentais aussi que toute la série d'idées relatives aux principes invariables de l'action policière dans les cas de perquisition, l'heure, — idées que je non sans quelque peine, que toute cette vous expliquai tout à — je sentais, dis-je, série d'idées avait dCi nécessairement se dérouler dans l'esprit du ministre. )) Cela devait impérativement le conduire à dédai- gner toutes les cachettes vulgaires. Cet homme-là ne pouvait pas être assez faible pour ne pas deviner que la cachette la plus compliquée, la plus profonde de son hôtel serait aussi peu secrète qu'une antichambre ou une armoire pour les yeux, microscopes du les sondes, les vrilles et les préfet. Enfin je voyais qu'il avait dû viser nécessairement à la simplicité, s'il n'y avait pas été induit par un goût naturel. Vous vous rappelez sans doute avec quels éclats de rire le préfet accueillit l'idée que j'exprimai dans notre première entrevue, à savoir que, si le mystère l'embarrassait si fort, c'était peut-être en raison de son absolue simplicité. — Oui, dis-je, je rité. Je croyais me rappelle parfaitement son hila- vraiment qu'il allait tomber dans des attaques de nerfs. • . LA LETTRE VOLEE. tl7 — Le monde matériel, continua Diipin, est plein d'analogies exactes avec l'immatériel, et c'est ce qui donne une couleur de vérité à ce dogme de rhétorique, qu'une métaphore ou tifier un argument une aussi comparaison peut for- bien qu'embellir une des- cription. )) Le principe de par exemple, la force d'inertie, semble identique dans les deux natures, physique et métaphysique; un gros corps mâs en mouvement qu'un est plus difficilement sa quantité de petit, et mouvement est en proportion de cette difficulté; voilà qui est aussi positif que cette proposition analogue d'une intellects vaste capacité, qui en sont : les même temps plus impétueux, plus constants et plus accidentés dans leur mouvement que ceux d'un degré infé- rieur, sont ceux qui se meuvent le moins aisément, et qui sont le plus embarrassés d'hésitation se mettent en marche. Autre exemple : quand ils avez-vous ja- mais remarqué quelles sont les enseignes de boutique qui attirent le plus l'attention ? — Je — 11 n'ai jamais songé à cela, dis-je. existe, qu'on joue reprit avec une Dupin, un jeu de carte ''^ ' divination, Un géographique. joueurs prie quelqu'un de deviner un mot donné, un nom de ville, de rivière, d'état -. ou d'empire, des — — un mot quelconque compris dans l'étendue bigarrée et embrouillée de la carte. Une personne novice enfin dans le jeu cherche en général à embarrasser ses adversaires en leur donnant à doviner des noms écrits en 7. HISTOIRES EXTRAORDINAIRES. 118 caractères imperceptibles; mais les adeptes du jeu choisissent des mots en gros caractères qui s'étendent d'un bout de la carte à l'autre. Ces mots-là, comme les enseignes' et les affiches à lettres énormes, échappent à l'observateur par évidence; et, le fait Toubli ici, même de leur excessive matériel est précisément analogue à l'inattention morale d'un esprit qui laisse échapper les considérations trop palpables, évidentes jusqu'à la banalité et l'importunité. Mais c'est là un cas, à ce qu'il semble, de l'intelligence du un peu au-dessus ou au-dessous préfet. Il n'a jamais cru pro- bable ou possible que le ministre eût déposé sa lettre juste sous le nez du monde comme pour entier, mieux empêcher un individu quelconque de l'apercevoir. » Mais plus je réfléchissais à l'audacieux, au distinctif et brillant esprit de D..., — à ce fait qu'il avait dû toujours avoir le document sous la main, pour en faire immédiatement usage, si besoin était, fait — et à cet autre que, d'après la démonstration décisive fournie par le préfet, ce document n'était pas caché dans les limites d'une perquisition ordinaire et en règle, je me sentais — plus convaincu que le ministre, pour cacher sa lettre, avait eu recours à l'expédient le plus ingé- nieux du monde, le plus large, qui était de ne pas même essayer de la cacher. » Pénétré de ces idées, j'ajustai sur mes yeux une paire de lunettes vertes, et je me présentai un beau matin, comme par hasard, à l'hôtel du ministre. Je trouve LA LETTRE VOLEE. D... chez lui, bâillant, flânant, lU) musant, et se prétendant accablé d'un suprême ennui. D... est peut-être l'homme plus le mais énergique qui réellement c'est seulement soit aujourd'hui, quand il est sûr de n'être vu de personne. )) Pour n'être pas en reste avec lui, je me plaignis de la faiblesse de mes yeux et de la nécessité de porter des lunettes. Mais, derrière ces lunettes, j'inspectais soigneusement et minutieusement tout l'appartement, en faisant semblant d'être tout à la conversation de mon hôte. )) Je donnai une attention spéciale à un vaste bureau auprès duquel il était assis, et sur lequel gisaient pêle- mêle des lettres diverses et d'autres papiers, avec un ou deux instruments de musique et quelques livres. Après un long examen, fait à loisir, je n'y vis rien qui pût exciter particulièrement mes soupçons. )) A la longue, mes yeux, en chambre, tombèrent sur un faisant le tour de la misérable porte-cartes, orné de clinquant, et suspendu par un ruban bleu crasseux à un petit bouton de manteau de trois la cuivre au-dessus cheminée. Ce porte-cartes, ou quatre compartiments, contenait cinq ou cartes de visite et une lettre unique. Cette était fortement salie et du qui avait six dernière chiffonnée. Elle était presque déchirée en deux par le milieu, comme si on avait eu d'abord l'intention de la déchirer entièrement, ainsi qu'on fait d'un objet sans valeur semblablement changé ; d'idée. Elle mais on avait vraiportait un large HISTOIRES EXTRAORDINAIRES. 120 sceau noir avec le chiffre de D... très en évidence, et au ministre lui-même. La suscription était d'une écriture de femme très-fme. On l'avait jetée était adressée négligemment, et même, à ce qu'il semblait, assez dé- daigneusement dans l'un des compartiments supérieurs du porte-cartes. )) A peine eus-je jeté un coup d'œil sur cette lettre, que je conclus que dont j'étais en quête. c'était celle Évidemment elle était, par son aspect, absolument différente de celle dont le préfet nous avait lu une description si minutieuse. le chiffre Ici, le sceau était large et noir avec de D...; dans l'autre, il était petit et rouge, avec les armes ducales de la famille S... Ici, la suscription était l'autre, d'une écriture l'adresse, menue portant le et nom féminine ; dans d'une personne royale, était d'une écriture hardie, décidée et caractérisée; les deux lettres point, ces la dimension. différences, l'état ne se ressemblaient qu'en un Mais le caractère excessif fondamentales en somme, la de saleté, déplorable du papier, fripé et déchiré, qui con- tredisaient les véritables habitudes de D..., si métho- diques, et qui dénonçaient l'intention de dérouter un indiscret en lui offrant toutes les apparences d'un do- cument sans valeur, — tout cela, en y ajoutant la si- tuation impudente du document mis en plein sous les yeux de tous les visiteurs et concordant ainsi exacte- ment avec mes conclusions antérieures, — tout dis-je, était fait pour corroborer décidément les cela, soup- çons de quelqu'un venu avec le parti pris du soupçon. LA LETTRE VOLEE. » Je prolongeai ble, et, tout en le ministre ma visite aussi longtemps que possi- soutenant une discussion très-vive avec sur un point que je savais être pour lui d'un toujours nouveau, je intérêt l'21 gardais invariablement mon attention braquée sur la lettre. Tout en faisant cet examen, je réfléchissais sur son aspect extérieur et sur la manière dont elle était arrangée dans le porte- cartes, et à la longue je tombai sur une découverte qui mit à néant le léger doute qui me rester en- pouvait En analysant les bords du papier, core. qu'ils étaient plus éraillés remarquai je que nature. Ils présentaient l'aspect cassé d'un papier dur, qui, ayant été plié et foulé par le couteau à papier, a été replié dans le sens inverse, mais dans mêmes -plis qui constituaient les sa forme première. Cette était clair pour moi découverte que la lettre me suffisait. 11 avait été retournée comme un gant, repliée et recachetée. Je souhaitai le bonjour au ministre, et je pris soudainement congé de lui, en oubliant une tabatière en or sur son bureau. » Le matin suivant, je vins pour chercher ma tabatière, et nous reprîmes très-vivement la conversation de la veille. Mais, pendant s'engageait, une détonation coup de pistolet, se de l'hôtel, et fut d'une foule lit suivie des et cris et les fenêtres des vociférations se précipita regarda dans loinps, j'allai droit au comme un très-forte, entendre sous épouvantée. D... fenêtre, l'ouvrit, que la discussion la rue. vers une En même porte-cartes, je pris la lettre, HISTOIRES EXTRAORDINAIRES. 122 je la mis dans ma poche, et je la remplaçai par une antre, une espèce de fac-similé (quant à l'extérieur), que j'avais soigneusement préparé chez moi, — en contrefaisant le chiffre de D... à l'aide d'un sceau de mie de pain. )) Le tumulte de la rue avait été causé par le caprice insensé d'un homme armé d'un fusil. Il avait déchargé son arme au milieu d'une foule de femmes et d'enfants. Mais comme elle n'était pas chargée à balle, on prit ce drôle pour un lunatique ou un ivrogne, et on lui per- mit de continuer son chemin. Quand il fut parti, D... se retira de la fenêtre, où je l'avais suivi immédiate- ment après m'être assuré de la précieuse lettre. Peu d'instants après, je lui dis adieu. Le prétendu fou était un homme payé par moi. — Mais quel était votre ami, en remplaçant la but, lettre demandai-je à mon par une contrefaçon? N'eût-il pas été plus simple, dès votre première visite, de 'VOUS en emparer, sans autres précautions, et de vous en aller ? — D..., répliqua Dupin, est plus, c'est un homme solide. capable de tout, et, de D'ailleurs, il a dans son hôtel des serviteurs à sa dévotion. Si j'avais fait l'extra- vagante tentative dont vous parlez, je ne serais pas sorti vivant de chez lui. Le bon peuple de Paris n'aurait plus entendu parler de moi. Mais, à part ces considérations, j'avais un but particulier. Vous connaissez mes sym- pathies politiques. Dans cette affaire, j'agis comme partisan de la dame en question. Voilà dix-huit mois que LA LETTRE VOLEE. 123 le ministre la tient en son pouvoir. C'est nant qui le puisqu'il ignore que la lettre n'est tient, mainte- elle plus chez lui, et qu'il va vouloir procéder à son chantage habituel. Il va donc infailliblement opérer lui- même et du premier coup sa ruine politique. Sa chute ne sera pas moins précipitée que ridicule. On parle lestement du fort facilis descensus Averni ; mais, matière d'escalades, on peut dire ce que disait du chant : que de descendre. plus est il Dans le facile aucune sympathie, pas même de Catalani la monter de présent, cas pitié pour en je n'ai celui qui va descendre. D..., c'est le vrai monstrum horrendum, — un homme de génie sans principes. Je vous avoue, cependant, que je ne serais pas fâché de connaître le caractère exact de ses pensées, quand, mis au défi par celle que le préfet appelle U7ie certaine personne, sera réduit à ouvrir la lettre que j'ai laissée il pour lui dans son porte-cartes. — Comment ! est-ce que vous y avez mis quelque chose de particulier? — Eh mais ! il ne m'a pas semblé tout à fait conve- nable de laisser l'intérieur en blanc, l'air d'une insulte. Une — cela aurait eu fois, à Vienne, D... m'a joué un vilain tour, et je lui dis d'un ton tout à fait gai que je m'en souviendrais. Aussi, comme je savais éprouverait une certaine curiosité relativement personne par qui il Il à la se trouvait joué, je pensai que ce serait vraiment dommage dice quelconque. qu'il de ne pas lui laisser un in- connaît fort bien mon écriture, et HISTOIRES EXTRAORDINAIRES. l'ii j'ai copié mots tout au beau milieu de la page blanche ces : Un dessein si funeste, • • • S'il n'est digne d'Atrée, est digne de Thyeste. Vous trouverez cela dans VAtrèe de Crébillon. i;i:v; LE SCARABEE D'OR Oh! oh! qu'est-ce que cela? Ce garçon a une dans les jambes! Il a été mordu par la folie tarentule. [Tout de travers.) Il y a quelques années, je me liai intimement avec un M. William Legrand. Il était d'une ancienne fa- mille protestante, et jadis il avait été riche; mais une à la misère. Pour éviter l'humiliation de ses désastres, il quitta la Nou- série de malheurs l'avait velle-Orléans, la ville de réduit ses aïeux, et établit sa de- meure dans l'île de Sullivan, près Gharleston, dans la Caroline du Sud. Cette île est des plus singulières. Elle n'est guère composée que de sable de mer et a environ trois milles de long. En largeur, elle n'a jamais plus d'un quart de mille. Elle est séparée du continent par une crique à peine visible, qui filtre à travers une masse de roseaux et de vase, rendez-vous habituel des poules d'eau. La végétation, comme on peut le supposer, est pauvre, ou, pour ainsi dire, naine. On n'y trouve pas d'arbres d'une certaine dimension. Vers l'extrémité occidentale, à l'endroit où s'élève le fort Moultrie et quelques misé- HISTOIRES EXTRAORDINAIRES. 126 rables bâtisses de bois habitées pendant l'été par les gens qui fuient les poussières et les fièvres de Charleston, on rencontre, il est vrai, le palmier nain sétigère; mais toute l'île, à l'exception de ce point occidental et d'un espace triste et blanchâtre qui borde la mer, est couverte d'épaisses broussailles de myrte odoriférant, si estimé par horticulteurs les anglais. L'arbuste y monte souvent à une hauteur de quinze ou vingt pieds; il y forme un taillis presque impénétrable et charge l'atmosphère de ses parfums. Au plus profond de ce taillis, non loin de l'extrémité orientale de c'est-à-dire de la plus éloignée, Le- l'île, grand s'était bâti lui-même une petite hutte, qu'il occupait quand, pour la première fois et par hasard, je fis sa connaissance. Cette connaissance mûrit bien vite en amitié, — car il y avait, certes, dans le cher reclus de quoi exciter l'intérêt et l'estime. Je vis qu'il avait reçu une forte éducation, heureusement servie par des facultés spirituelles peu communes, mais qu'il était infecté de misanthropie et sujet à de malheureuses al- ternatives d'enthousiasme et de mélancolie. Bien qu'il eût chez lui beaucoup de livres, il s'en servait rarement. Ses principaux amusements consistaient à chasser et à pêcher, ou à flâner sur la plage et à travers les myrtes, en quête de coquillages giques; — sa et d'échantillons entomolo- collection aurait pu faire envie à un Swammerdam. Dans ces excursions, il était ordinairement accompagné par un vieux nègre nommé* Jupiter, qui avait été affranchi avant les revers de la famille. LE SCARABÉE D'OR. 127 mais qu'on n'avait pu décider, ni par menaces ni par promesses, à abandonner son jeune massa Will; il considérait comme son droit de le suivre partout. Il n'est pas improbable que les parents de Legrand, jugeant que celui-ci avait la tête un peu dérangée, se soient appliqués à confirmer Jupiter dans son obstination, dans le but de mettre une espèce de gardien et de surveillant auprès du fugitif. Sous la latitude de l'île de Sullivan, les hivers sont rarement rigoureux, et c'est un événement quand, au déclin de l'année, le feu devient indispensable. Cepen- dant, vers le milieu d'octobre 18.., ily eut une journée d'un froid remarquable. Juste avant le coucher du soleil, je me frayais un chemin à travers les taillis vers de mon ami, que je n'avais pas vu depuis la hutte quelques semaines; je demeurais alors à Charleston, à une distance de neuf milles de l'île, et les facilités pour aller et revenir étaient bien moins grandes qu'aujour- d'hui. En arrivant à la hutte, je frappai selon mon ha- bitude, et, ne recevant pas de réponse, je cherchai la clef où je savais qu'elle était cachée, j'ouvris la porte et j'entrai. Un beau feu flambait dans le foyer. C'était une surprise, et, à coup sûr, une des plus agréables. Je me débarrassai de mon paletot, je traînai un fauteuil auprès des bûches pétillantes, et j'attendis patiemment l'arrivée de mes hôtes. Peu après la tombée de la nuit, ils arrivèrent et me firent un accueil tout à fait cordial. Jupiter, tout en riant d'une oreille à l'autre, se donnait du mouvement HISTOIRES EXTRAORDINAIRES. 128 "et préparait quelques poules d'eau pour le souper. Legrand était dans une de ses crises d'enthousiasme; — car de quel autre nom appeler cela? Il avait trouvé un bivalve inconnu, formant un genre nouveau, et, mieux encore, il avait chassé et attrapé, avec l'assistance de Jupiter, un scarabée qu'il croyait tout à fait nouveau, et sur lequel il désirait avoir mon opinion le lendemain matin. — Et pourquoi pas ce soir? demandai-je en me frot- tant les mains devant la flamme, et envoyant mentale- ment au diable toute la race des scarabées. Ah si j'avais seulement su que vous étiez ici — ! ! dit Legrand; mais il y a si longtemps que je ne vous ai vul Et comment pouvais-je deviner que vous me rendriez visite justement cette nuit? En revenant au logis, j'ai rencontré le lieutenant G..., du fort, et très-étourdi- ment je lui ai prêté le scarabée; de sorte qu'il vous sera impossible de le voir avant demain matin. Restez ici cette nuit, et j'enverrai Jupiter le chercher au lever du soleil. C'est bien la plus ravissante chose de la création ! — Quoi? lever du — Eh non que diable! — scarabée. d'une — gros à peu près comme une brillante couleur grosse noix, — avec deux taches d'un noir de soleil? le le ! Il est d'or, jais à une extrémité du dos, et une troisième, un peu plus allongée, à l'autre. Les antennes sont... — Il n'y a pas du tout d'étain sur lui\ massa Will, 1. La prononciation du mot antennœ une méDey aint no fait commettre prise au nègre, qui croit qu'il est question d'étain : LE SCARABEE D'OR. interrompit Jupiter je vous un scarabée d'or, d'or massif, d'un le parie, dans et partout, excepté les ailes ; ; \i\) le scarabée est bout à l'autre, de- — je n'ai jamais vu de ma vie un scarabée à moitié aussi lourd. — C'est bien, mettons que vous ayez raison, Jup, répliqua Legrand un peu plus vivement, à ce qu'il me sembla, que ne le comportait la situation, est-ce une raison pour laisser brûler les poules? l'insecte, — et il se tourna vers moi, La couleur de — suffirait en vérité à rendre plausible l'idée de Jupiter. Vous n'avez jamais vu un éclat métallique plus brillant que celui de ses élytres ; mais vous ne pourrez en juger que demain matin. En attendant, j'essayerai de vous donner une idée de sa forme. Tout en parlant, il s'assit à une petite table sur la- quelle il y avait une plume et de l'encre, mais pas de papier. Il chercha dans un tiroir, mais n'en trouva pas. — N'importe, dit-il à la fm, cela suffira. Et il tira de la poche de son gilet quelque chose qui me fit l'effet d'un morceau de vieux vélin fort sale, et il fit dessus une espèce de croquis à la plume. Pendant ce temps, j'avais gardé ma place auprès du feu, car him. Calembour intraduisible. Le nègre parlera toujours dans une espèce de patois anglais, que le patois nègre français tin in normand ou le breton ne traduiEn se rappelant les orthographes figuratives de Balzac, on se fera une idée de ce que ce moyen un peu physique peut ajouter de pittoresque et de comique, mais j'ai dû renoncer n'imiterait pas mieux que le bas rait l'irlandais. à m'en servir, faute d'équivalent. — C. B. HISTOIRES EXTRAORDINAIRES. 130 j'avais toujours très-froid. Quand son dessin fut achevé, il me le passa, sans se lever. Comme je le recevais de sa main, un fort grognement se fit entendre, suivi d'un grattement à la porte. Jupiter ouvrit, et un énorme terre-neuve, appartenant à Legrand, se précipita dans la chambre, sauta sur mes épaules et m'accabla de ca- resses; dans mes Quand il eut fini ses gambades, je car je m'étais fort occupé de lui visites précédentes. regardai le papier, et, pour dire la vérité, je me trouvai passablement intrigué par le dessin de mon ami. — Oui! dis-je après l'avoir contemplé quelques mi- nutes, c'est là un étrange scarabée, je le confesse; il est nouveau pour moi; je n'ai jamais rien vu d'appro- chant, à moins que ce ne soit un crâne ou une tête de mort, à quoi il ressemble plus qu'aucune autre chose qu'il m'ait jamais été donné d'examiner. — Une tête de mort! répéta Legrand. Ah ! oui, il y a un peu de cela sur le papier, je comprends. Les deux taches noires supérieures font les yeux, et la plus longue qui est plus bas figure une bouche, n'est-ce pas? D'ailleurs, la forme générale est ovale... — C'est peut-être cela, dis-je; mais je crains, Le- grand, que vous ne soyez pas très-artiste. J'attendrai que j'aie vu la bête elle-même, pour me faire une idée quelconque de sa physionomie. — Fort bien! Je ne sais comment cela se fait, dit-il, un peu piqué, je dessine assez joliment, ou du moins je le devrais, — car j'ai eu de bons maîtres, et je me flatte de n'être pas tout à fait une brute. LE SCARABEE D'OR. — Mais santez ; 131 mon cher camarade, dis-je, vous plaiceci est un crâne fort passable, je puis même alors, dire que c'est un crâne parfait, d'après toutes les idées reçues relativement à cette partie de l'ostéologie, et votre scarabée serait le plus étrange de tous les sca- rabées du monde, s'il ressemblait à ceci. Nous pourrions établir là-dessus quelque petite superstition saisissante. Je présume que vous nommerez votre insecte scarabœus capitt hominis, ou quelque chose d'approchant; il y a dans les livres d'histoire naturelle beaucoup d'appellations — Mais où sont de ce genre. les antennes dont vous parliez? — Les antennes ! dit Legrand, qui s'échauffait inex- plicablement; vous devez voir les antennes, j'en suis sur. Je les ai faites aussi distinctes qu'elles le sont dans l'original, et je —A la présume que cela est bien suffisant. bonne heure, ayez faites; toujours dis-je; est-il vrai mettons que vous les que je ne les vois pas. Et je lui tendis le papier, sans ajouter aucune re- marque, ne voulant pas le pousser à bout; mais j'étais fort étonné de la tournure que l'affaire avait prise; sa mauvaise humeur m'intriguait, — et, quant au croquis de l'insecte, il n'y avait positivement pas d'antennes visibles, et l'ensemble ressemblait, à s'y méprendre, à l'image ordinaire d'une tête de mort. Il reprit son papier d'un air maussade, et il était au moment de le froisser, sans doute pour le jeter dans le feu, quand, son regard étant tombé par hasard sur le dessin, toute son attention y parut enchaînée. En un HISTOIRES EXTRAORDINAIRES. 132 instant, son visage devint d'un rouge intense, puis ex- cessivement pâle. Pendant quelques minutes, sans bouger de sa place, il continua à examiner minutieuse- ment le dessin. A la longue, il se leva, prit une chans'asseoir sur un à delle sur la table, et alla l'autre extrémité de la chambre. Là, il recommença à coffre, examiner curieusement le papier, le tournant dans tous 'les sens. Néanmoins, il ne dit rien, et sa conduite me causait un étonnement extrême; mais je jugeai prudent de n'exaspérer par aucun commentaire sa mauvaise humeur croissante. Enfin, il tira de la poche de son habit un portefeuille, y serra soigneusement le papier, et déposa le tout dans un pupitre qu'il ferma à clef. Il revint dès lors à des allures plus calmes, mais son pre- mier enthousiasme avait totalement disparu. l'air plutôt concentré Il avait que boudeur. A mesure que la soirée s'avançait, il s'absorbait de plus en plus dans sa rêverie, et aucune de mes saillies ne put l'en arracher. Primitivement, j'avais eu l'intention de passer la nuit dans la cabane, comme j'avais déjà fait plus d'une fois; mais, en voyant l'humeur de mon hôte, je jugeai plus convenable de prendre congé. Il ne fit aucun effort pour me retenir; mais, quand je partis, il me serra la main avec une cordialité encore plus vive que de coutume. Un mois environ après cette aventure, — et durant cet intervalle je n'avais pas entendu parler de Legrand, — je reçus à Charleston une visite de son serviteur Jupiter. Je n'avais jamais vu le bon vieux nègre si LE SGAHABEE D'OU. 133 complètement abattu, et je fus pris de la crainte qu'il ne fût arrivé à mon ami quelque sérieux malheur. — Eh bien, Jup, quoi de neuf? Gomment va dis-je, ton maître? — Dame î pour dire massa, il ne va pas vérité, la aussi bien qu'il devrait. — Pas bien! vraiment je d'apprendre navré suis cela. Mais de quoi se plaint-il? — Ah! voilà — question! la ne se plaint jamais il de rien, mais il est tout de même bien malade. — Bien malade, Jupiter! — Eh! que ne disais-tu cela tout de suite? Est-il au lit? — Non, non, n'est pas au bien nulle — voilà justement soulier me blesse; — lit! il Il n'est part; oii j'ai l'esprit très-inquiet au sujet du pauvre massa Will. — Jupiter, je voudrais le bien comprendre quelque chose à tout ce que tu me racontes là. Tu dis que ton maître est malade. Ne t'a-t-il pas dit de quoi il souffre? — Oh! massa, — Massa Will c'est tête. bien inutile de se creuser la dit qu'il n'a mais, alors, pourquoi donc s'en absolument rien va-t-il, ; — deçà et delà, tout pensif, les regards sur son chemin, la tête basse, les épaules voûtées, et pâle quoi donc — — 11 doise, Il fait-il toujours fait quoi, fait — comme une oie? Et pour- et toujours des chiffres? Jupiter? des chiffres avec des signes sur une ar- les signes les plus bizarres que j'aie jamais vus. Je commence à avoir peur, tojt de même. II faut que j'aie toujours un œil braqué sur lui, rien que sur ***** g HISTOIRES EXTRAORDINAIRES. 134 lui. L'autre jour, il m'a échappé avant leil, et a décampé il pour toute J'avais coupé un bon bâton exprès le lever du so- sainte la journée. pour lui administrer une correction de tous les diables quand il reviendrait; — mais rage — ; que je n'en ai pas eu le cou- je suis si bête, il a l'air si malheureux! — Ahî vraiment! — Eh bien, après que tu as mieux garçon. — il — Il fait d'être tout, je crois indulgent pour le pauvre ne faut pas lui donner le fouet, Jupiter; en état de n'est peut-être pas le supporter. — Mais ne peux-tu pas te faire une idée de ce qui a occasionné cette maladie, ou plutôt ce changement de conduite? Lui est-il arrivé quelque chose de fâcheux depuis que je vous ai vus? — Non, massa, — mais avant il lors, n'est rien arrivé de fâcheux depuis cela, — oui, — j'en ai peur, — c'était le jour même que vous étiez là-bas. — Comment? que veux-tu dire? — Eh! massa, veux parler du scarabée, je voilà tout. — Du quoi — Du scarabée... — ? été mordu quelque Je suis sûr que part massa Will a à la tête par ce scarabée d'or. — Et quelle raison as-tu, Jupiter, pour faire une pareille supposition? — li a bien assez de pinces pour cela, massa, et une bouche aussi. Je n'ai jamais vu un scarabée aussi endiablé ; — il attrape et il mord tout ce qui l'ap- LE SCARABEE D'OR. 135 proche. Massa Will l'avait d'abord attrapé, mais il' Ta bien vite lâché, je vous assure; — sans c'est alors, doute, qu'il a été mordu. La mine de ce scarabée et — aussi sa bouche ne me plaisaient guère, certes ; ne voulus pas pris un le prendre avec mes doigts ; je mais je morceau de papier, et j'empoignai le scarabée dans le papier; je l'enveloppai donc dans le papier, avec un petit morceau de papier dans la bouche ; — voilà comment je m'y pris. — Et tu penses donc que ton maître a été réelle- ment mordu par le scarabée, et que cette morsure l'a rendu malade? — Je ne pense rien du — tout, je le sais^ Pour- quoi donc rêve-t-il toujours d'or, si ce n'est parce qu'il a été mordu par le scarabée d'or ? J'en ai déjà entendu parler, de ces scarabées d'or. — Mais comment sais-tu rêve d'or — Comment sais? parce en parle, même en dormant; — voilà comment — Au Jupiter, tu as peut-être raison; mais à qu'il ? qu'il je le je le sais. fait, quelle bienheureuse circonstance dois-je l'honneur de ta visite aujourd'hui? — Que voulez-vous massa? — M'apportes-tu un message de M. Legrand? — Non, massa, vous apporte une que dire, lettre je Et Jupiter me tendit un papier où je lus 1. sni<;. Calembour. / nose pour / know. — C. B. — Je le voici. : sens pour Je le HISTOIRES EXTRAORDINAIRES. 136 -: (( . , Mon cher, » Pourquoi donc ne vous ai-je pas vu depuis si longtemps? J'espère que vous n'avez pas été assez enfant pour vous formaliser d'une petite brusquerie de part mais non, — cela » ; est par trop Depuis que je vous ai vu, ma improbable. eu un grand sujet j'ai d'inquiétude. J'ai quelque chose à vous dire, mais à peine sais-je comment vous le dire. Sais-je même si je vous le dirai? )) Je n'ai pas été tout à fait bien depuis quelques jours, et le pauvre vieux Jupiter m'ennuie insupporta- blement par toutes ses bonnes intentions et attentions. Le croiriez-vous? Il avait, l'autre jour, préparé un gros bâton à l'effet de me châtier, pour et avoir passé la journée, seul, lui avoir échappé au milieu des collines, sur le continent. Je crois vraiment que ma mauvaise mine m'a seule sauvé de la bastonnade. )) Je n'ai rien ajouté à ma collection depuis que nous nous sommes vus. » Revenez avec Jupiter si vous le pouvez sans trop d'inconvénients. Venez, venez. Jedésirevous voircesoir pour affaire grave. Je vous assure que c'est de la plus haute importance. )) Votre tout dévoué, » Il WILLIAM LEGRAND. » y avait dans le ton de cette lettre quelque chose qui me causa une forte inquiétude. Ce style différait LE SCARABÉE D'OR. absolument du style habituel dia])le rêvait-il? 137 de Legrand. Quelle nouvelle lubie A quoi avait pris pos- session de sa trop excitable cervelle? Quelle affaire de si haute importance pouvait-il avoir à accomplir? Le rapport de Jupiter ne présageait rien de bon; — je tremblais que la pression continue de l'infortune n'eût, à la longue, singulièrement dérangé la raison de mon ami. Sans hésiter un instant, je me préparai donc à accompagner le nègre. En arrivant au quai, je remarquai une faux et trois bêches, toutes également neuves, qui gisaient au fond du bateau dans lequel nous allions nous embarquer. — Qu'est-ce que tout cela signifie, Jupiter? deman- dai-je. — Ça, une faux, massa, des bêches. — vois bien mais qu'est-ce que tout cela c'est et Je le ; fait ici? — Massa Will m'a dit d'acheter pour lui cette et ces bêches à la ville, et je les ai faux payées bien cher; cela nous coûte un argent de tous les diables. — Mais, au nom de tout ce qu'est-ce que ton qu'il y a de mystérieux, massa Will a à faire de faux et de bêches? — Vous m'en demandez plus que même, massa, je ne sais; lui- n'en sait pas davantage; le m'emporte si je n'en suis pas convaincu. Mais tou diable cela vient du scarabée. Voyant que je ne pouvais tirer aucun éclaircissement de Jupiter dont tout l'entendement paraissait absorbé V 8. HISTOIRES EXTRAORDINAIRES. 138 par le scarabée, je descendis dans le bateau et je déployai la voile. Une belle et forte brise nous poussa bien vite dans la petite anse au nord du fort Moultrie, et, après une promenade de deux milles environ, nous arrivâmes à la hutte. Il était à peu près trois heures de l'après-midi. Legrand nous attendait avec une vive impatience. 11 me serra la main avec un empressement nerveux qui m'alarma et renforça mes soupçons naissants. Son visage était d'une pâleur spectrale, ses et yeux, naturellement fort enfoncés, brillaient d'un éclat surnaturel. Après quelques questionsrelatives à sa santé, je lui demandai, ne trouvant rien de mieux à dire, si le lieutenant G... lui avait enfin rendu son scarabée. — Oh ! oui, répliqua-t-il en rougissant beaucoup; je le lui ai repris le — lendemain matin. Pour rien au monde je ne me séparerais de ce scarabée. Savez-vous bien que Jupiter a tout à fait raison à son égard? — En quoi? demandai-je avec un triste pressenti- ment dans le cœur. — En supposant que Il dit cela c'est un scarabée d'or véritable. avec un sérieux profond, qui me fit indici- blement mal. — Ce scarabée tinua-t-il est destiné à faire ma fortune, con- avec un sourire de triomphe, à me réintégrer dans mes possessions de famille. Est-il donc étonnant que je le tienne en si haut prix ? Puisque la Fortune a jugé bon de me l'octroyer, je n'ai qu'à en user conve- nablement, et j'arriverai jusqu'à For dont dice. — Jupiter, apporte-le-moi. il est l'in- LE SCARABEE D'OR. — Quoi scarabée, massa ? J'aime ? le rien à démêler avec le scarabée ; 13 mieux n'avoir — vous saurez bien le prendre vous-même. Là-dessus, Legrand se leva avec un air grave et imposant, et alla me chercher Tinsecte sous un globe de verre où il était déposé. C'était un superbe scarabée, inconnu à cette époque aux naturalistes, et qui devait avoir un grand prix au point de vue scientifique. à portait l'une des extrémités 11 du dos deux taches noires et rondes, et à l'autre une tache de forme allongée. Les élytres étaient excessivement dures et lui- santes et avaient positivement l'aspect de l'or bruni. L'insecte était remarquablement lourd, et, tout bien considéré, je ne pouvais pas trop blâmer Jupiter de son opinion ; mais que Legrand s'entendît avec lui sur ce sujet, voilà ce qu'il m'était impossible de com- prendre, et, quand il se serait agi de ma vie, je n'aurais pas trouvé le mot de l'énigme. — Je vous gnifique, ai envoyé chercher, dit-il d'un ton ma- quand j'eus achevé d'examiner l'insecte, je vous ai envoyé chercher pour vous deniander conseil et assistance dans l'accomplissement des vues de la Destinée et du scarabée... — Mon cher Legrand, m'écriai -je en l'interrompant, vous n'êtes feriez beaucoup mieux de prendre quelques précau- tions. Vous allez certainement pas bien, et vous vous mettre au lit, et je resterai au- près de vous quelques jours, jusqu'à ce que vous soyez rétabli. Vous avez la fièvre, et... HISTOIRES EXTRAORDINAIRES. 140 — Tâtez mon pouls, Je le tâtai, et, le dit-il. pour dire la vérité, je ne trouvai pas plus léger symptôme de fièvre. — Mais vous pourriez bien être malade sans avoir la fièvre. Permettez-moi, pour cette fois seulement, de faire le médecin avec vous. Avant toute chose, allez vous mettre au lit. Ensuite... — Vous vous trompez, interrompit-il bien que je puis espérer de tion que j'endure. ; je suis aussi dans l'état d'excita- l'être réellement vous voulez Si me voir tout à fait bien, vous soulagerez cette excitation. — Et que faut-il faire pour cela? — C'est Jupiter très-facile. et moi, nous partons pour une expédition dans les collines, sur le continent, et nous avons besoin de l'aide d'une personne en qui nous puissions absolument nous personne unique. Que notre fier. Vous êtes cette entreprise échoue ou moi mainte- réussisse, l'excitation que vous voyez en nant sera également apaisée. — J'ai le vif désir de vous servir en toute chose, répliquai-je ; mais prétendez-vous dire que cet infernal scarabée ait quelque rapport avec votre expédition dans les collines? •— Oui, certes. — Alors, Legrand, •• . il :. m'est impossible de coopérer à une entreprise aussi parfaitement absurde. — J'en suis fâché, — très-fâché, — car il nous fau- dra tenter l'affaire à nous seuls. — A vous seuls! Ahî le malheureux est fou, à coup LE SGARABEK D'OR. sûr! 141 — Mais, voyons, combien de temps durera votre absence? — Probablement toute la nuit. Nous allons partir immédiatement, et, dans tous les cas, nous serons de retour au lever du soleil. — Et vous me promettez, sur votre honneur, que ce caprice passé, du scarabée — bon Dieu — et l'affaire ! vidée à votre satisfaction, vous rentrerez au logis, et mes que vous y suivrez exactement prescriptions, comme celles de votre médecin? — Oui, je vous le promets; et maintenant partons, car nous n'avons pas de temps à perdre. mon J'accompagnai le chien et ami, le cœur gros. A quatre mîmes en route, Legrand, Jupiter, heures, nous nous moi. Jupiter prit la faux et insista pour s'en les bêches ; il charger, plutôt, à ce qu'il me parut, par crainte de laisser un de ces instruments dans la main de son maître que par excès de zèle et de complaisance. ces mots Il : était d'ailleurs d'une humeur de chien, et Damné scarabée ! furent les seuls qui échappèrent tout le long du voyage. part, la J'avais, charge de deux lanternes sourdes Legrand, il s'était ; lui pour ma quant à contenté du scarabée, qu'il portait attaché au bout d'un morceau de ficelle, et qu'il faisait tourner autour de lui, tout en marchant, avec des airs de magicien. Quand j'observais ce symptôme suprême de démence dans mon pauvre ami, je pouvais à peine retenir mes larmes. Je pensai toutefois qu'il valait mieux épouser sa fantaisie, au moins pour le moment, HISTOIRES EXTRAORDINAIRES. 142 OLi jusqu'à ce que je pusse prendre quelques mesures énergiques avec chance de succès. Cependant, sayais, mais fort inutilement, de ment au but de l'expédition. Il le j'es- sonder relative- me réussi à avait persuader de l'accompagner, et semblait désormais peu disposé à lier conversation sur un sujet d'une A maigre importance. si ne daignait répondre toutes que par mes questions, un a il Nous verrons bien! » Nous traversâmes dans un esquif la crique à la pointe de l'île, et, grimpant sur les terrains montueux de la rive opposée, nous nous dirigeâmes vers le nord-ouest, à travers un pays horriblement sauvage et désolé, où il était impossible de découvrir la trace d'un pied hu- main. Legrand suivait sa route avec décision, s' arrêtant seulement de temps en temps pour consulter certaines indications qu'il paraissait avoir laissées lui-même dans une occasion précédente. Nous marchâmes soleil était ainsi deux heures environ, et le au moment de se coucher quand nous en- trâmes dans une région infiniment plus sin^'stre que que nous avions vu jusqu'alors. C'était une tout ce espèce de plateau près du sommet d'une montagne affreusement escarpée, couverte de bois de la base au sommet, et semée d'énormes blocs de pierre qui semblent éparpillés pêle-mêle sur le sol, et dont plusieurs se seraient infailliblement précipités dans les vallées inférieures sans le secours des arbres contre lesquels ils s'appuyaient. De profondes ravines irradiaient dans , LE SCARABÉE D'OR. 143 diverses directions et donnaient à la scène un caractère de solennité plus lugubre. La plate-forme naturelle sur laquelle nous étions grimpés était si profondément encombrée de ronces, que nous vîmes bien que, sans la faux, il nous eût été impossible de nous frayer un passage. Jupiter, d'après les ordres de son maître, commença à nous éclaircir un chemin jusqu'au pied d'un tulipier gigantesque qui se dressait, la en compagnie de huit ou dix chênes, sur plate-forme, les arbres et les surpassait tous, ainsi que tous que j'avais vus jusqu'alors, par la beauté de sa forme et de son feuillage, par l'immense développe- ment de son branchage et par la majesté générale de son aspect. Quand nous eûmes atteint cet arbre, Legrand se tourna vers Jupiter, et lui demanda s'il se croyait capable d'y grimper. Le pauvre vieux parut légèrement étourdi par cette question, et resta quelques instants sans répondre. Cependant, tronc, en fit il s'approcha de l'énorme lentement le tour et l'examina avec une attention minutieuse. Quand il eut achevé son examen, il dit simplement : — Oui, massa; Jup n'a pas vu d'arbre où il ne puisse grimper. — Alors, monte; il allons, allons! et rondement! car fera bientôt trop noir pour voir ce que nous faisons. — Jusqu'où monter, massa? demanda — Grimpe d'abord sur puis quel chemin tu dois suivre. — Ah! un instant! — prends faut-il Jupiter. le ce scarabée avec toi. tronc, et je te dirai IIJSTOIHES EXT11A0MDIJNAI1U":S. 144 — Le scarabée, massa Wiil! — cria nègre le aut-il de reculant le scarabée d'or! frayeur; pourquoi donc que je porte avec moi ce scarabée sur l'arbre? Que je sois damné si je le fais — Jup, ! si vous avez peur, vous, un grand nègre, un gros et fort nègre, de loucher à un petit insecte mort inoffensif, et cette ficelle; eli bien, vous pouvez l'emporter avec — mais, vous ne l'emportez pas avec si vous d'une manière ou d'une autre, je serai dans la cruelle nécessité de vous fendre la tête avec cette bêche. — Mon Dieu! qu'est-ce Jup, que la honte rendait qu'il y a donc, massa? dit évidemment plus complai- sant; il faut toujours que vous cherchiez noise à votre vieux nègre. C'est une farce, voilà tout. Moi, avoir peur du scarabée! je m'en soucie bien du scarabée! Et il prit avec précaution l'extrême bout de la corde, et, maintenant l'insecte aussi loin de sa personne que les circonstances le permettaient, il se mit en devoir de grimper à l'arbre. Dans sa jeunesse, le tulipier, ou liriodendron tull- piferam, le plus magnifique des forestiers américains, a un tronc singulièrement lisse et s'élève souvent à une grande hauteur, sans pousser de branches latérales mais ; quand il arrive à sa maturité, l'écorce devient rugueuse et inégale, et de petits rudiments de branches se ma- nifestent en grand nombre sur le tronc. Aussi l'escalade, dans le cas actuel, apparence qu'en l'énorme était réalité. beaucoup plus dillicile Embrassant de son en mieux cvlindie avec ses bras et ses genoax, empoi- LE SCARABÉE D'OR. 145 gnant avec les mains quelques-unes des pousses, ap- puyant ses pieds nus sur avoir failli tomber les autres, Jupiter, après une ou deux fois, se hissa à la lon- gue jusqu'à la première grande fourche, et sembla dès lors regarder la besogne comme virtuellement accom- En effet, le risque principal de l'entreprise avait plie. disparu, bien que que le brave nègre se trouvât à soixante et dix pieds du sol. — De quel côté faut-il que j'aille maintenant, massa Will? demanda-t-il. — Suis toujours la plus grosse branche, — celle de ce côté, dit Legrand. Le nègre lui obéit sans trop de peine ; promptement, il apparemment et monta, monta toujours plus haut, de sorte qu'à la fin sa personne rampante et ra- massée disparut dans l'épaisseur du feuillage; il était tout à fait invisible. Alors, sa voix lointaine se fit en- tendre; il criait : — Jusqu'où monter encore? — A quelle hauteur es-tu? demanda Legrand. — haut, haut, répliqua nègre, que peux faut-il Si si je le voir le ciel à travers le sommet de l'arbre. — Ne t'occupe pas du ciel, mais fais attention à ce que je te dis. Regarde le tronc, et compte les branches au-dessous de toi, de ce côté. Combien de branches astu passées? — Une, deux, trois, quatre, cinq; — j'ai grosses branches, massa, de ce côté-ci. — Alors, monte encore d'une braach3. passé cinq HISTOIRES EXTRAORDINAIRES. i4iB Au bout de quelques minutes, sa voix se fit entendre de nouveau. Il annonçait qu'il avait atteint la septième branche. — Maintenant, Jup, cria Legrand, en proie à une agitation manifeste, il faut que tu trouves le moyen de l'avancer sur cette branche aussi ras. Si tu vois loin que tu pour- quelque chose de singulier, tu me le diras. Dès lors, les quelques doutes que j'avais essayé de conserver relativement à la démence de mon pauvre ami disparurent complètement. Je ne pouvais plus ne pas le considérer comme frappé d'aliénation mentale, et je commençai m' inquiéter à sérieusement des moyens de le ramener au logis. Pendant que je méditais sur ce que j'avais de mieux à faire, la voix de Jupiter se fit entendre de nouveau. — J'ai bien peur de m' aventurer un peu loin sur cette branche; — c'est une branche morte presque dans toute sa longueur. — Tu dis bien que c'est une branche morte, Jupiter? cria Legrand d'une voix tremblante d'émotion. — Oui, massa, niorte comme un vieux clou déporte, — bien morte, tout une c'est elle affaire faite, est à fait sans vie. — Au nom du ciel, que faire? demanda Legrand, qui semblait en proie à — Que faire? dis-je, un vrai désespoir. heureux de pour placer un mot raisonnable : nous aller coucher.» Allons, venez saisir l'occasion retourner au logis et ! — Soyez gentil, mon LE SCARABEE D'OR. camarade. — Il 147 se fait tard, et puis souvenez-vous de votre promesse. — Jupiter, criait-il, sans m'écouter le moins du monde, m'entends-tu? — Oui, massa Will, vous entends parfaitement. — Entame donc bois avec ton couteau, dis-moi je et le si tu le trouves bien pourri. — Pourri, massa, assez pourri, répliqua bientôt nègre, mais pas aussi pourri qu'il pourrait l'être. le Je pourrais m' aventurer un peu plus sur la branche, mais moi seul. — Toi seul! — qu'est-ce que tu veux dire? — Je veux parler du scarabée. est bien lourd, 11 le scarabée. Si je le lâchais d'abord, la branche porterait bien, sans casser, le poids d'un nègre tout seul. — Infernal coquin! cria Legrand, qui avait l'air fort soulagé, quelles sottises me chantes-tu là? Si tu laisses tomber l'insecte, je te tords — tu m'entends, — Oui, massa, ce pas le cou. Fais-y attention, n'est-ce pas? Jupiter; n'est la peine de traiter comme ça un pauvre nègre. — Eh bien, écoute-moi, maintenant! — Si tu te ha- sardes sur la branche aussi loin que tu pourras le faire sans danger et sans lâcher le scarabée, je te ferai cadeau d'un dollar d'argent aussitôt que tu seras descendu. — J'y vais, ment le massa Will, — m'y voilà, répliqua leste- nègre, je suis presque au bout. — Au bout! cria Legrand, très-radouci. Veux-tu dire que tu es au bout de cette branche? HISTOIRES EXTRAORDINAIRES. 148 — Je suis bientôt — oh! oh! oh! au bout, massa; Seigneur Dieu! miséricorde! qu'y a-t-il sur l'arbre? — Eh bien, cria Legrand, au comble de la joie, qu'est-ce qu'il y a? — Eh! ce n'est rien qu'un laissé sa tête sur l'arbre, et les — quelqu'un crâne-, a corbeaux ont becqueté toute la viande. — Un crâne, dis-tu — Très-bien! — Comment est-il ? — qu'est-ce qui retient? — mais faut — Ah! une drôle de chose, sur ma parole — a un attaché à la branche? — Oh! c'est il le tient bien; voir. il il ; y gros clou dans le crâne, qui le retient à l'arbre. — Bien! maintenant, Jupiter, exactement ce que — m'entends? — Oui, massa. — Fais bien attention! — trouve gauche du fais je vais te dire; tu l'œil crâne. — Oh! oh! voilà qui est drôle! n'y a pas d'œil il gauche du tout. — Maudite stupidité! Sais-tu distinguer ta main droite de ta main gauche? — Oui, je sais, — je sais tout cela ; ma main gauche est celle avec laquelle je fends le bois. — Sans doute, tu es gaucher; et ton œil gauche est du même côté que ta main gauche. Maintenant, je suppose, tu peux trouver l'œil gauche du crâne, ou la place où était l'œil gauche. As-tu trouvé? Il y eut manda : ici une longue pause. Enfin, le nègre de- LE SCARABÉt: D'OU. — L'œil gauche du crâne est 149 le crâne n'a pas la fait voilà même côté du aussi main gauche du crâne? — Mais que rien! de mains du tout! — Gela ne gauche, — Tœil gauche Que j'ai trouvé l'œil faut-il faire, ! main- tenant? — Laisse filer le scarabée à travers, aussi loin que la ficelle peut aller; mais prends bien garde de lâcher le bout de la corde. — Voilà qui est fait, de faire passer le massa Will; c'était chose facile scarabée par le trou; — tenez, voyez-le descendre. Pendant tout ce dialogue, la personne de Jupiter était restée invisible; mais l'insecte qu'il laissait filer apparaissait maintenant au bout de la ficelle, et brillait comme une boule d'or brunie aux derniers rayons du soleil couchant, dont quelques-uns éclairaient encore faiblement l'éminence où nous étions placés. Le scarabée en descendant émergeait des branches, l'avait laissé et, si Jupiter tomber, il serait tombé à nos pieds. Le- grand prit immédiatement la faux et éclaircit un espace circulaire de trois ou quatre yards de diamètre, juste au-dessus de l'insecte, et, ayant achevé cette besogne, ordonna à Jupiter de lâcher la corde et, de descendre de l'arbre. Avec un soin scrupuleux, mon ami enfonça dans la terre une cheville, à fendroit précis où le scarabée était tombé, et tira l'attacha de sa poche un ruban à mesurer, 11 par un bout à l'endroit du tronc de l'arbre qui était le plus près de la cheville, le déroula jusqu'à HISTOIRES EXTRAORDINAIRES. 150 la cheville, et continua ainsi à le dérouler dans rection donnée par ces deux points, tronc, " — jusqu'à — la di- la cheville et le la distance de cinquante pieds. Pen- dant ce temps, Jupiter nettoyait les ronces avec la faux» Au point ainsi trouvé, il enfonça une seconde cheville, qu'il prit comme centre, et autour duquel il décrivit grossièrement un cercle de quatre pieds de diamètre environ. Il s'empara alors d'une bêche, en donna une à Jupiter, une à moi, et nous pria de creuser aussi vi- vement que possible. Pour parler franchement, je n'avais jamais eu beau- coup de goût pour un pareil amusement, et, dans le cas présent, je m'en serais bien volontiers passé; car la nuit s'avançait, et je me sentais passablement fatigué de l'exercice que j'avais déjà pris ; mais je ne voyais aucun moyen de m'y soustraire, et je tremblais de troubler , par un refus la prodigieuse sérénité de mon pauvre ami. Si j'avais pu compter sur l'aide de Jupiter, je n'aurais pas hésité à ramener par la force notre fou chez lui; mais je connaissais trop bien le caractère du vieux nègre pour espérer son assistance, dans le cas d'une lutte personnelle avec son maître et dans n'importe quelle circonstance. Je ne doutais pas que Le- grand n'eût le cerveau infecté de quelqu'une des innombrables superstitions du Sud relatives aux trésors enfouis, et que cette imagination n'eût été confirmée par la trouvaille du scarabée, ou peut-être l'obstination de Jupiter à soutenir même par que c'était un sca- rabée d'or véritable. Un esprit tourné à la folie pouvait LE SCARABÉE D'OR. 151 bien se laisser entraîner par de pareilles suggestions, surtout quand elles s'accordaient avec ses idées favo- rites préconçues ; puis je me rappelais le discours du pauvre garçon relativement au scarabée, indice de sa fortune! Par-dessus tout, j'étais cruellement tourmenté et embarrassé ; mais enfin je résolus de faire contre fortune bon cœur et de bêcher de bonne volonté, pour convaincre une mon visionnaire le plus tôt possible, par démonstration oculaire, de l'inanité de ses rê- veries. Nous allumâmes les lanternes, et nous attaquâmes notre besogne avec un ensemble et un zèle dignes d'une cause plus rationnelle; et, comme la lumière tombait sur nos personnes et nos outils, je ne pus m'empêcher de songer que nous composions un groupe vraiment pittoresque, et que, si quelque intrus était tombé par hasard au milieu de nous, nous lui aurions apparu comme faisant une besogne bien étrange et bien suspecte. Nous creusâmes ferme deux heures durant. Nous parlions peu. Notre principal embarras était causé par les aboiements du chien, qui prenait un intérêt exces- sif à nos travaux. A la longue, il devint tellement tur- bulent, que nous craignîmes qu'il ne donnât l'alarme — ou, grande appréhension de Legrand, — à quelques rôdeurs du voisinage, plutôt, c'était car, pour mon me serais réjoui de toute interruption qui m'aurait permis de ramener mon vagabond à la mai- la compte, son. je A la fin, le vacarme fut étouffé, grâce à Jupiter HISTOIRES EXTRAORDINx\IRES. 152 un air furieusement qui, s'élançant hors du trou avec décidé, musela la gueule de l'animal avec une de ses bretelles et puis retourna à sa tâche avec un petit rire de triomphe très-grave. Les deux heures écoulées, nous avions atteint une profondeur de cinq pieds, et aucun indice de trésor ne se montrait. Nous fîmes une pause générale, et je com- mençai à espérer que la farce touchait à sa fin. Cependant Legrand, quoique évidemment très-déconcerté, s'essuya le front d'un air pensif et reprit sa bêche. Notre trou occupait déjà toute l'étendue du cercle de quatre pieds de diamètre; nous entamâmes légèrement creusâmes encore de deux pieds. cette limite, et nous Rien n'apparut. Mon chercheur d'or, dont j'avais sé- rieusement pitié, sauta enfin hors du trou avec le plus affreux désappointement écrit sur le visage, et se décida, lentement et comme à regret, à reprendre son habit qu'il avait ôté avant de se mettre à l'ouvrage. Pour moi, je me gardai bien de faire aucune remarque. Jupiter, à un signal de son maître, commença à ras- sembler les outils. Cela fait, selé, et le chien étant dému- nous reprîmes notre chemin dans un profond silence. Nous avions peut-être fait une douzaine de pas, quand Legrand, poussant un terrible juron, sauta sur Jupiter et l'empoigna au collet. vrit les yeux et la Le nègre stupéfait ou- bouche dans toute leur ampleur, lâcha les bêches et tomba sur les genoux. — Scélérat! criait Legrand en faisant silller les LE SCARABKE D'OPx. 453 syllabes entre ses dents, infernal noir! gredin de noir! — parle, te dis-je ! — réponds-moi à sur— Quel quel est ton œil l'instant, et tout ne prévarique pas! est, gauche? — Ah miséricorde massa Will , ! ! n'est-ce pas là, pour sûr, mon œil gauche? rugissait Jupiter épouvanté plaçant sa main sur l'organe droit de la vision, et l'y maintenant avec s'il l'opiniâtreté comme du désespoir, eût craint que son maître ne voulût le lui arracher, — Je m'en doutais ! — je le bien savais ! hourra ! vociféra Legrand, en lâchant le nègre, et en exécutant une série de gambades et de cabrioles, au grand éton- nement de son domestique, qui, en se relevant, pro- menait, sans mot dire, ses regards de son maître à moi et de moi à son maître. — Allons, il nous faut retourner, dit celui-ci; la partie n'est pas perdue. Et il reprit son chemin vers le tulipier. — de Jupiter, dit-il l'arbre, viens quand nous fûmes arrivés au pied ici ! — Le crâne est-il cloué à la branche avec la face tournée à l'extérieur ou tournée contre la branche ? — La face est tournée à l'extérieur, massa, de sorte que les corbeaux ont pu manger les yeux sans aucune peine. — Bien. Alors, est-ce par cet œil-ci ou par celui-là que tu as fait couler le scarabée? Et Legrand touchait alternativement les deux yeux de Jupiter. 0. HlSTOIRi:S EXTRAORDIxNAIRES. 154 — Par cet massa, œil-ci, — par l'œil gauche, — juste comme vous me l'aviez dit. Et c'était encore son œil droit qu'indiquait le pauvre nègre. — Allons, allons! il nous faut recommencer. Alors, mon ami dans la folie duquel je voyais main- tenant, ou croyais voir certains indices de méthode, re- porta la cheville qui marquait l'endroit où le scarabée était tombé, à trois pouces vers l'ouest de sa première position. Étalant de nouveau son cordeau du point le plus rapproché du tronc jusqu'à la cheville, comme il avait déjà fait, et continuant à l'étendre en ligne droite à une distance de cinquante pieds, il marqua un nou- veau point éloigné de plusieurs yards de l'endroit oi^i nous avions précédemment creusé. Autour de ce nouveau centre, un cercle fut tracé, un peu plus large que le premier, et nous nous mîmes derechef à jouer de tigué ; la bêche. J'étais effroyablement fa- mais, sans me rendre compte de ce qui occa- sionnait un changement dans plus une aussi m'était imposé. Je je dirai plus, je ma pensée, je ne sentais grande aversion pour m'y le labeur qui intéressais inexplicablement; me sentais excité. Peut-être y avait-il dans toute l'extravagante conduite de Legrand un certain air délibéré, une certaine allure prophétique qui m'impressionnaient moi-même. Je bêchais ai^demment et de temps à autre je me surprenais cherchant, pour ainsi dire, des yeux, avec un sentiment qui ressem- blait à de l'attente, ce trésor imaginaire dont la vision LE SCARABEE D'OR. 155 avait affolé mon infortuné camarade. Dans un de ces moments oià ces rêvasseries s'étaient plus singulière- ment emparées de moi, travaillé comme nous avions et déjà une heure et demie à peu près, nous fûmes de nouveau interrompus par les violents hurlements du chien. Son inquiétude, dans le premier cas, n'était évidemment que le d'un résultat caprice ou d'une gaieté folle; mais, cette fois, elle prenait un ton plus Comme Jupiter s'efforçait violent et plus caractérisé. de nouveau de le museler, il fit une résistance furieuse, et, bondissant dans le trou, se mit à gratter fréné- il tiquement la terre avec ses griffes. En quelques se- condes, il avait découvert une masse d'ossements hu- mains, formant deux squelettes complets et mêlés de plusieurs boutons de métal, nous parut émiettée. de être la avec quelque chose qui vieille laine pourrie et Un ou deux coups de bêche firent sauter la lame d'un grand couteau espagnol; nous creusâmes encore, et trois ou quatre pièces de monnaie d'or et d'argent apparurent éparpillées. A cette vue, Jupiter put à peine contenir sa joie, mais la physionomie de son maître exprima un affreux désappointement. nuer nos 11 efforts, et à nous supplia toutefois de contipeine avait-il fini de parler que je trébuchai et tombai en avant; la pointe de ma botte s'était engagée dans un gros anneau de fer qui gisait à moitié enseveli sous un amas de terre fraîche. Nous nous remîmes au travail avec une ardeur nouvelle; jamais je n'ai passé dix minutes dans une aussi HISTOIRES EXTRAORDINAIRES. 156 vive exaltation. Durant cet intervalle, nous déterrâmes complètement un coffre de forme oblongue, qui, à en juger par sa parfaite conservation et son étonnante dureté, avait été évidemment soumis à quelque procédé de minéralisation, — peut-être au bichlorure de mercure. Ce coffre avait trois pieds et demi de long, trois de large et deux et demi de profondeur. Il était solide- ment maintenu par des lames de fer forgé, rivées et formant tout autour une espèce de treillage. De chaque côté du coffre, près du couvercle, étaient trois anneaux de fer, six en tout, au moyen desquels six personnes pouvaient s'en emparer. Tous nos efforts réunis ne réussirent qu'à le déranger légèrement de son lit. Nous vîmes tout de suite l'impossibilité d'emporter un si énorme poids. Par bonheur, le couvercle n'était retenu que par deux verrous que nous fîmes glisser, — trem- blants et pantelants d'anxiété. En un instant, un trésor d'une valeur incalculable s'épanouit, étincelant, devant nous. Les rayons fosse, et des lanternes tombaient dans la faisaient jaillir d'un amas confus d'or et de bijoux des éclairs et des splendeurs qui nous éclaboussaient positivement les yeux. Je n'essayerai pas de décrire les sentiments avec les- quels je contemplais ce trésor. La stupéfaction, comme on peut le supposer, dominait tous les autres. Legrand paraissait épuisé par son excitation même, et ne pro- nonça que quelques paroles. Quant à Jupiter, sa figure devint aussi mortellement pâle- que cela est possible à une figure de nègre. Il semblait slupéQé, foudroyé. LE SCARABEC D'OR. 157 Bientôt il tomba sur ses genoux dans la fosse, et plon- geant ses bras nus dans laissa longtemps, jusqu'au coude, l'or comme jouissait des s'il d'un bain. Enfin, il s'écria avec comme se parlant à lui-même que : joli scara- pauvre petit scarabée d'or que j'injuriais, le calomniais! N'as-tu pas honte de je les y un profond soupir, — Et tout cela vient du scarabée d'or? Le bée d'or! il voluptés toi. vilain n,ègre? — hein! qu'as-tu à répondre? 11 fallut cependant que je réveillasse, le maître et le valet, et que pour ainsi dire, je leur fisse qu'il y avait urgence à emporter le trésor. tard, et il nous fallait comprendre 11 se faisait déployer quelque activité, si nous voulions que tout fût en sûreté chez nous avant le jour. Nous ne savions quel perdions beaucoup de nous avions allégeâmes les en enlevant les deux tiers de son pûmes l'arracher de encore, en avions nous idées en désordre. Finalement nous le coffre contenu, et nous parti prendre, et temps en délibérations, tant tirés furent enfin, mais non sans peine son trou. Les objets que nous déposés parmi confiés à la garde du chien, à qui les ronces, et Jupiter enjoignit strictement de ne bouger sous aucun prétexte, et de ne pas même ouvrir la bouche jusqu'à notre retour. Alors, nous nous mîmes précipitamment en route avec le coffre; nous atteignîmes la hutte sans accident, mais après une fatigue effroyable et à une heure du matin. Epuisés comme nous l'étions, nous ne pouvions immé- diatement nous remettre à la besogne, c'eût été dépas- HISTOIRES EXTRAORDINAIRES. lo8 ser les forces de la nature. Nous nous reposâmes jus- deux heures, puis nous soupâmes; enfin nous qu'à nous remîmes en route pour les montagnes, munis de gros sacs que nous trouvâmes par bonheur dans trois Nous arrivâmes un peu avant quatre heures la hutte. à notre fosse, nous que possible la le nous partageâmes aussi également reste du butin, et, sans nous peine de combler marche vers notre seconde fois le case, trou, donner nous nous remîmes en où nous déposâmes pour nos précieux fardeaux, juste la comme les premières bandes de l'aube apparaissaient à l'est, audessus de la cime des arbres. Nous étions absolument brisés; mais la profonde excitation actuelle nous refusa le repos. Après un som- meil inquiet de trois ou quatre heures, nous nous vâmes, le- comme si nous nous étions concertés, pour procéder à i' examen de notre trésor. Le coffre avait été rempli jusqu'aux bords, et nous passâmes toute la journée la nuit suivante avait à et la plus inventorier son contenu. On n'y mis aucune espèce d'ordre ni d'arrangement; tout y avait été empilé pêle-mêle. fait grande partie de Quand nous eûmes soigneusement un classement général, nous nous trouvâmes en possession d'une fortune qui dépassait tout ce que nous avions supposé. plus de 450,000 dollars, 11 y avait en espèces — en estimant la valeur des pièces aussi rigoureusement que possible d'après les tables de l'époque. d'argent. Dans tout Tout était en cela, or de pas' une vieille parcelle date et d'une LE SCARABEE DOR. grande variété 1Ô9 monnaies française, espagnole et alle- : mande, quelques guinées anglaises, et quelques jetons dont nous n'avions jamais vu aucun modèle. plusieurs pièces de monnaie, Il y avait très-grandes et très- lourdes, mais si usées, qu'il nous fut impossible de déchiffrer les inscriptions. Aucune monnaie américaine. Quant à l'estimation des bijoux, ce fut une affaire un peu plus difficile. Nous trouvâmes des diamants, dont quelques-uns très-beaux et d'une grosseur singulière, — en tout, cent dix, rubis d'un éclat remarquable; dont pas un n'était petit; dix-huit raudes, toutes très-belles opale. Toutes ces ; trois cent dix éme- vingt et un saphirs et une pierres avaient été arrachées de leurs montures et jetées pêle-mêle dans le coffre. Quant' aux montures elles-mêmes, dont nous fîmes une catégorie distincte de Tautre or, été broyées à elles coups de marteau paraissaient avoir comme pour rendre toute reconnaissance impossible. Outre tout cela, il y avait une énorme quantité d'ornements en or massif; — près de deux cents bagues ou boucles d'oreilles mas- sives ;debelles chaînes, au nombre de trente, si j'ai bonne mémoire; quatre-vingt-trois très-lourds ; crucilix très-grands cinq encensoirs d'or d'un grand prix gigantesque bol à punch en or, ; et un orné de feuilles de vigne et de figures de bacchantes largement ciselées; deux poignées d'épée merveilleusement travaillées, et une foule d'autres articles plus petits et dont j'ai perdu le souvenir. Le poids de toutes ces valeurs dépassait 350 livres ; et dans cette estimation j'ai omis cent HISTOIRES ËXTRAORDINAIUES. leO quatre-vingt-dix-sept montres d'or superbes, dont trois valaient chacune cinq cents dollars. Plusieurs étaient comme pièces d'hor- très-vieilles, et sans aucune valeur logerie, lesmouvementsayant plus ou moins souffert de l'action corrosive de la terre ; mais toutes étaient magni- fiquement ornées de pierreries, et les boîtes étaient d'un grand prix. Nous évaluâmes cette nuit le contenu total du coffre à un million et demi de dollars; et, lorsque plus tard nous disposâmes des bijoux et des pierreries, — après en avoir gardé quelques-uns pour notre usage personnel, — nous trouvâmes que nous avions singulièrement sous-évalué le trésor. Lorsque nous eûmes enfin terminé notre inventaire et que notre terrible exaltation fut en grande partie apaisée, Legrand, qui voyait que je mourais d'impa- tience de posséder la solution de cette prodigieuse énigme, entra dans un détail complet de toutes les circonstances qui s'y rapportaient. — Vous vous rappelez,. dit-il, le soir oi^i je vous fis passer la grossière esquisse que j'avais faite du scarabée. Vous vous souvenez aussi que je fus passable- ment choqué de votre insistance à me soutenir que mon dessin ressemblait à une tête de mort. La première fois que vous lâchâtes cette assertion, je crus que vous plaisantiez; ensuite je lières sur le me rappelai les taches particu- dos de l'insecte, et je reconnus en moi- même que votre remarque avait en somme quelque fondement. Toutefois, votre ironie à l'endroit de mes facultés graphiques m'irritaient, car on me regarde SCARABÉE D'OR. Lt: 101 comme un artiste fort passable; aussi, quand vous me j'étais au moment tendîtes le morceau de parchemin, de le froisser avec humeur et de le jeter dans le feu. — Vous voulez parler du morceau de papier, dis-je. — Non; cela avait toute l'apparence du papier, et, moi-même, j'avais d'abord supposé que c'en était; mais, quand je voulus dessiner dessus, je découvris tout de suite que c'était un morceau de parchemin très-mince. Il était fort sale, vous vous le rappelez. Au moment même où j'allais le chiffonner, mes yeux tombèrent sur le dessin que vous aviez regardé, et vous pouvez concevoir quel fut mon étonnement quand j'aperçus l'image positive d'une tête de mort à l'endroit même où j'avais cru dessiner un scarabée. Pendant un moment, je me sentis trop étourdi pour penser avec rectitude. Je savais que mon croquis différait de ce nouveau dessin par tous ses détails, bien qu'il y eût une certaine analogie dans le contour général. Je pris alors une chandelle, et, m'asseyant à l'autre bout de la chambre, je procédai à une analyse plus attentive du parchemin. En le retournant, je vis ma propre esquisse sur le revers, juste comme je l'avais faite. Ma première simplement de la surprise ; il impression fut y avait une analogie réellement remarquable dans le contour, et c'était une coïncidence singulière que ce fait de l'image d'un crâne, inconnue à moi, occupant l'autre côté du parchemin immédiatement au-dessous de mon dessin du scarabée, HISTOIRES EXTRAORDINAIRES. 162 — crâne qui ressemblait si exactement à mon et d'un dessin, non-seulement par le contour, mais aussi par dimension. Je dis que la singularité de cette coïn- la cidence me stupéfia positivement pour un instant. C'est l'effet ordinaire de ces sortes de coïncidences. L'esprit s'efforce d'établir un rapport, une liaison de cause à — et, se trouvant impuissant à y réussir, subit une espèce de paralysie momentanée. Mais, quand je effet, revins de cette stupeur, je sentis luire en grés une conviction me frappa qui moi par de- bien autrement encore que cette coïncidence. Je commençai à me rappeler distinctement, positivement, qu'il n'y avait aucun dessin sur le parchemin quand j'y fis mon croquis du scarabée. J'en acquis la parfaite certitude; car je me souvins de l'avoir tourné et retourné en cherchant l'endroit le plus propre. "Si le crâne avait été visible, l'aurais infailliblement je remarqué. 11 y avait réelle- ment là un mystère que je me sentais incapable de débrouiller; mais, dès ce voir moment même, il me sembla prématurément poindre une faible lueur dans les régions les plus profondes et les plus secrètes de mon entendement, une espèce de ver luisant intellectuel, une conception embryonnaire de la vérité, dont aventure de l'autre nuit nous a fourni une si notre splen- dide démonstration. Je me levai décidément, et, ser- soigneusement le parchemin, rant réflexion être » ultérieure jusqu'au je renvoyai toute moment où je pourrais seul. Quand vous fûtes parti et quand Jupiter fut bien LE SCARABEE, D"OR. 163 endormi, je me livrai à une investigation un peu plus méthodique de la chose. Et d'abord je voulus comprendre de quelle manière ce parchemin était tombé dans mes mains. L'endroit où nous découvrîmes le scarabée était sur la côte du continent, à un mille environ à l'est de l'île, mais à une petite distance au-dessus du niveau de la marée haute. Quand je m'en emparai, il me mordit cruellement, et je le lâchai. Jupiter, avec sa pru- dence accoutumée, avant de prendre l'insecte, qui s'était envolé de son côté, chercha autour de lui une feuille ou quelque chose d'analogue, emparer. Ce fut en ce avec quoi il pût s'en moment que ses yeux et les miens tombèrent sur le morceau de parchemin, que je pris alors pour du papier. Il était à moitié enfoncé dans le sable, avec un coin en l'air. nous le trouvâmes, j'observai Près les de l'endroit où restes d'une coque de grande embarcation, autant du moins que j'en pus juger. Ces débris de naufrage étaient ment depuis bien longtemps, car on y retrouver la là probable- peine pouvait- à physionomie d'une charpente de bateau. )) Jupiter ramassa donc le parchemin, enveloppa l'in- secte et me le donna. Peu de temps après, nous reprîmes le chemin de la hutte, et nous rencontrâmes le lieute- nant G Je lui montrai l'insecte, et il me pria de lui permettre de l'emporter au fort. J'y consentis, et il le fourra dans la poche de son gilet sans qui lui servait à la main le parchemin d'enveloppe, et que je tenais toujours pendant qu'il examinait le scarabée. Peut- HISTOIRES EXTRAORDINAIRES. 164 être eut-il peur que je ne changeasse d'avis, et jugea- prudent de s'assurer l-il d'abord de sa prise vous ; savez qu'il est fou d'histoire naturelle et de tout ce qui s'y rattache. j'ai >; Il est évident qu'alors, sans y penser, remis le parchemin dans ma poche. Vous vous rappelez que, lorsque je m'assis à la table pour faire un croquis du scarabée, je ne trouvai pas de papier à l'endroit où on gardai dans le chais dans lettre, le met ordinairement. Je re- il n'y en avait point. Je cher- mes poches, espérant trouver une vieille tiroir, quand mes doigts rencontrèrent le parchemin. minutieusement toute Je vous détaille la série constances qui l'ont jeté dans mes mains ; de cir- car toutes ces circonstances ont singulièrement frappé mon es- prit. » Sans aucun doute, vous me considérerez comme un rêveur, — mais j'avais déjà établi une espèce de con- nexion. J'avais uni deux anneaux d'une grande chaîne. Un bateau échoué à la côte, et non loin de ce bateau un parchemin, — non pas un papier, — portant l'image d'un crâne. Vous allez naturellement me demander où est le rapport? Je répondrai que le crâne ou la tête de mort est l'emblème bien connu des ont pirates. Ils toujours, dans tous leurs engagements, hissé le pavillon à tête de mort. . » Je vous ai dit que c'était un morceau de parchemin et non pas de papier. rable, Le parchemin est une chose du- presque impérissable. On confie rarement au parchemin des documents d'une minime importance, LE SCARABÉE D'OR. 165 puisqu'il répond beaucoup moins bien que le papier aux ordinaires besoins de l'écriture et du dessin. Cette réflexion m'induisit à penser qu'il devait y avoir dans la tête de mort quelque rapport, quelque sens singu- lier. Je ne faillis pas non plus à remarquer la forme du parchemin. Bien que l'un des coins eût été détruit par quelque accident, on voyait bien que la forme primitive était oblongue. C'était donc une de ces bandes qu'on choisit pour écrire, pour consigner un document impor- tant, une note qu'on veut conserver longtemps et soi- gneusement. — Mais, interrompis-je, vous dites que n'était le le crâne pas sur le parchemin quand vous y dessinâtes scarabée. Comment donc pouvez-vous établir un bateau et rapport entre crâne, — puisque ce dernier, d'après votre propre aveu, a dû être dessiné — le Dieu sait le comment ou par qui! — postérieurement à votre dessin du scarabée? — Ah! c'est là-dessus que roule tout le mystère; bien que j'aie eu comparativement peu de peine à ré- soudre ce point de l'énigme. Ma marche était sûre, et ne pouvait me conduire qu'à un seul sonnais ainsi, par exemple : résultat. Je rai- quand je dessinai mon sca- rabée, il n'y avait pas trace de crâne sur le parchemin; quand j'eus fini mon dessin, je vous le fis passer, et je ne vous perdis pas de vue que vous ne me l'eussiez rendu. Conséquemment ce n'était pas vous qui aviez dessiné le crâne, et il n'y avait là aucune autre per- sonne pour le faire. Il n'avait donc pas été créé par HISTOIRES EXTRAORDINAIRES. 160 l'action humaine; et cependant, yeux! )) Arrivé à ce point de mes réflexions, je m'appliquai à me rappeler et je me rappelai en effet, et avec une parfaite exactitude, tous les incidents survenus dans l'in- tervalle en question. La température oh! l'heureux, le rare accident! — bait dans la cheminée. J'étais par l'exercice, et je m'assis était — froide, et un bon feu flam- suffisamment réchauffé près de la table. Vous, cependant, vous aviez tourné votre chaise tout près de moment où je vous mis le parchemin dans la main, et comme vous alliez l'examiner, Wolf, mon terre-neuve, entra et vous sauta sur les la cheminée. Juste au épaules. Vous le caressiez avec la main gauche, et vt)us cherchiez à l'écarter, en laissant tomber nonchalam- ment votre main droite, celle qui tenait le parchemin, entre vos genoux et tout près du feu. Je crus un mo- ment que la flamme allait l'atteindre, et j'allais vous dire de prendre garde ; mais avant que j'eusse parlé vous l'aviez retiré, et vous vous étiez Quand j'eus bien je mis à l'examiner. considéré toutes ces circonstances, ne doutai pas un instant que la chaleur n'eût été l'agent qui avait fait apparaître sur le parchemin le crâne dont je voyais l'image. Vous savez bien qu'il y a — il y en a eu de tout temps — des préparations chi- miques, au moyen desquelles on peut écrire sur du papier ou sur du vélin des caractères qui ne deviennent visibles que lorsqu'ils sont soumis à l'action du feu. On emploie quelquefois le safre, digéré dans l'eau régale et délayé LE SCARABÉE D'OR. dans quatre fois son poids d'eau il ; 1G7 en résulte une teinte verte. Le régule de cobalt, dissous dans l'esprit de nitre, donne une couleur rouge. Ces couleurs disparaisssent plus ou moins longtemps après que la sub- stance sur laquelle on a écrit s'est refroidie^ mais reparaissent à volonté par une application nouvelle de la chaleur. » J'examinai alors la tête soin. de mort avec le plus grand Les contours extérieurs, c'est-à-dire les plus rap- prochés du bord du vélin, étaient beaucoup plus distincts que rique avait les autres. été Évidemment imparfaite ou du calo- l'action inégale. J'allumai immédiatement du feu, et je soumis chaque partie du parchemin à une chaleur brûlante. D'abord, cela n'eut d'autre effet que de renforcer les lignes un peu pâles du crâne; mais, en continuant l'expérience, je vis apparaître, dans un coin de la bande, au coin diagona- lement opposé à celui où était tracée la tête de mort, une figure que je supposai d'abord être celle d'une chèvre. Mais un examen plus attentif me convainquit qu'on avait voulu représenter un chevreau. — Ah! ah! pas le droit de me dis-je, je n'ai certes moquer de vous; — un million c'est chose trop sérieuse et demi de dollars! pour qu'on en plaisante; — mais vous n'allez pas ajouter un troisième anneau à votre chaîne ; vous ne trouverez aucun rapport spécial entre vos pirates et une chèvre savez, n'ont rien à faire regarde les fermiers. ; — les pirates, avec les chèvres. vous le — Gela HISTOIRES EXTRA ORDIN AIRES. 1G8 — Mais que l'image n'était pas je viens de vous dire celle d'une chèvre. — Bon va pour un chevreau ! ; c'est presque la môme chose. — Presque, mais pas tout à fait, dit Legrand. — Vous avez entendu parler peut-être d'un certain capitaine Kidd. Je considérai cet anima] comme une tout de suite la espèce de figure de signature logogri- phique ou hiéroglyphique {Jdd, chevreau). Je dis signature, parce que la place qu'elle occupait sur le vélin suggérait naturellement cette idée. Quant à la tète de mort placée au coin diagonalement opposé, elle avait l'air d'un sceau, d'une estampille. Mais je fus cruelle- — du corps ment déconcerté par l'absence du reste, du texte de mon même de mon document rêvé, — contexte. — Je présume que vous espériez trouver une lettre entre le timbre et la signature. — Quelque chose comme cela. sentais comme irrésistiblement timent d'une immense bonne Le fait est que je me pénétré du fortune pressen- imminente. Pourquoi? je ne saurais trop le dire. Après tout, peutêtre était-ce plutôt un désir qu'une croyance positive; — mais croiriez-vous que le dire absurde de Jupiter, que le scarabée était en or massif, a eu une influence remarquable sur mon imagination? Et puis cette série d'accidents et de coïncidences était vraiment ordinaire! si extra- Avez-vous remarqué tout ce qu'il y a de fortuit là dedans ? Il a fallu que tous ces événements LE SCARABEE D'OR. 1G9 arrivassent le seul jour de toute l'année où il a fait, où il a pu faire assez froid pour nécessiter du feu; et, sans ce feu et sans l'intervention du chien au moment précis où il a paru, je n'aurais jamais eu connaissance de la tète de mort et n'aurais jamais possédé ce trésor. -— — sur des charbons. — Eh bien, vous avez donc connaissance je suis Allez, allez, ô.\\ne foule d'histoires qui courent, tives de mille rumeurs vagues rela- aux trésors enfouis quelque part sur la côte de l'Atlantique, par Kidd et ses associés? En somme, tousces bruits devaient avoir quelque fondement. Et si ces bruits duraient depuis si longtemps et avec tant de persistance, cela ne pouvait, selon moi, tenir qu'à un fait, c'est que le trésor enfoui était resté enfoui. Si Kidd avait caché son butin pendant un certain temps et l'avait ensuite repris, ces rumeurs ne seraient pas sans doute venues jusqu'à nous sous leur forme actuelle et invariable. Remarquez que les histoires en question roulent toujours sur des chercheurs et jamais sur des trouveurs de trésors. Si le pirate restée là. avait Il repris son argent, l'affaire en serait me semblait que quelque accident, par exemple la perte de la note qui indiquait l'endroit précis, avait supposais du le priver des moyens de le recouvrer. Je que cet accident était arrivé à la connais- sance de ses compagnons, qui autrement n'auraient jamais su qu'un trésor avait été enfoui, et qui, par leurs recherches infructueuses, sans guide et sans notes positives, avaient donné naissance à cette rumeur uni- verselle et à ces légendes aujourd'hui si communes. HISTOIRES EXTRAORDINAIRES. 170 Avez-vous jamais entendu parler d'un trésor important qu'on aurait déterré sur la côte? — Jamais. — Or, menses il est notoire que Kidd avait accumulé d'im- richesses. Je considérais donc sûre que la terre les gardait encore ; et comme chose vous ne vous étonnerez pas trop quand je vous dirai que je sentais — une espérance qui montait — certitude; que parchemin, en moi une espérance, presque à la c'est si sin- le gulièrement trouvé, contiendrait l'indication disparue du lieu où. avait été fait le dépôt. — Mais comment avez-vous procédé? — J'exposai de nouveau vélin au le feu, après avoir augmenté la chaleur; mais rien ne parut. Je pensai que la couche de crasse pouvait bien être pour quelque chose dans cet insuccès ; aussi je nettoyai soigneuse- ment le parchemin en versant de l'eau chaude dessus, puis je le plaçai dans une casserole de fer-blanc, le crâne en dessous, et je posai la casserole sur un ré- chaud de charbons allumés. Au bout de quelques minutes, la casserole ét^nt parfaitement retirai la bande de vélin, et je chauffée, je m'aperçus, avec une joie inexprimable, qu'elle était mouchetée en plusieurs endroits de signes qui ressemblaient à des chiffres rangés en lignes. Je replaçai la chose dans la casserole, je l'y laissai encore une minute, et, quand je l'en retirai, elle était juste Ici, comme vous allez la voir. Legrand, ayant de nouveau chauffé le vélin, le soumit à mon examen. Les caractères suivants appa- LE SCARABÉE D'OR. 171 raissaient en rouge, grossièrement tracés entre la tète de mort et le chevreau. 53:[:]:+305])6*;4826)4:{:.)4:[:);806*;48+8^60))85;l + (;:+*8 +83(88)5*+;46(;88*96*?;8)*+(;48o];o*+2:*+(;49o6*2(5*— 4)818*;4069285);)6+8}4:{:+;1(+9;48081;8:8:[:'l;48+85;4)48o +528806*81 (+9;48;(88;4(:{:?34;48)4+;161,:l 88;+?; — Mais, dis-je, en lui rendant la bande de vélin, — je n'y vois pas plus clair. Si tous les trésors deGolconde devaient être pour moi le prix de la solution de cette énigme, je serais parfaitement sûr de ne pas les gagner. — Et cependant, dit Legrand, la solution tainement pas aussi difficile premier coup Ces caractères, d'oeil. n'est cer- qu'on se l'imaginerait au pourrait le deviner facilement, comme chacun forment c'est-à-dire qu ils présentent un sens ; un chiffre, mais, d'après ce que nous savons de Kidd, je ne devais pas le supposer capable de fabriquer un échantillon de cryptographie bien abstruse. Je jugeai donc tout d'abord que celui-ci était d'une espèce simple, telligence grossière du — marin tel il cependant qu'à l'indût paraître absolu-, ment insoluble sans la clef. — Et vous — Très-aisément l'avez résolu, ; fois plus compliqués. inclination d'esprit j'en ai résolu d'autres dix mille Les circonstances et une certaine m'ont amené à prendre intérêt à ces sortes d'énigmes, et l'ingéniosité vraiment? il est vraiment humaine puisse créer une douteux que énigme de ce HISTOIRES EXTRAORDINAIUES. \TÎ genre dont l'ingéniosité humaine ne vienne à bout par que j'eus une application suffisante. réussi à établir une série de caractères lisibles, je dai- Aussi, une fois gnai à peine songer à la difficulté d'en dégager la signification. )) — en somme, dans tous — première question à vider, Dans le cas actuel, cas d'écritui'e secrète, les et, la c'est la langue du chiffre: car les principes de solution, particulièrement quand il s'agit des chiffres les plus dépendent du génie de chaque idiome, simples, peuvent en être modifiés. En général, il et n'y a pas d'autre moyen que d'essayer successivement, en se di- rigeant suivant les probabilités, toutes les langues qui vous sont connues, jusqu'à ce que vous ayez trouvé la bonne. Mais, dans le chiffre qui nous occupe, toute dif- égard ficulté à cet était résolue par la signature. Le rébus sur le mot Kldd n'est possible que dans la langue anglaise. Sans cette circonstance, j'aurais commencé mes essais par l'espagnol et le français, comme étant langues dans lesquelles un pirate des mers espa- les gnoles avait dû le plus secret de cette nature. naturellement enfermer un Mais, dans le cas actuel, je présumai que le cryptogramme était anglais. )) Vous remarquez qu'il n'y a pas d'espaces entre les mots. S'il y avait eu des espaces, la tâche eût été sin- gulièrement plus facile. Dans ce cas, j'aurais commencé par faire une collation et une analyse des mots les plus courts, et, si j'avais trouvé, comme cela est tou- jours probable, un mot d'une seule lettre, a ou 1 (un, LE SCARABEE D'OU. je) par exemple, j'aurais considéré 173 la solution comme mon assurée. Mais, puisqu'il n'y avait pas d'espaces, premier devoir était de relever les lettres prédomi- nantes, ainsi que celles qui se rencontraient rarement. Je que voici les le plus comptai toutes, et je dressai la table : cardclère 8 HISTOIRES EXTRAORDINAIRES. 174 conjecture. L'usage général qu'on peut faire de cette table est évident; mais, pour ce chiffre particulier, nous ne nous en servirons que très-médiocrement. Puisque notre caractère dominant est 8, nous par le commencerons prendre pour Ve de l'alphabet naturel. Pour vé- rifier cette supposition, voyons si le 8 se rencontre sou- vent double; car Ve se redouble très-fréquemment en anglais, comme par exemple dans les mots speed, scen, been, agrée, etc. Or, dans le : meet, fleet, cas présent, nous voyons qu'il n'est pas redoublé moins de cinq fois, bien que le cryptogramme soit très-court. » Donc 8 représentera e. Maintenant, de tous les mots de la langue, the est le plus usité; conséquemment, il .lous faut voir si nous ne trouverons pas répétée plu- sieurs fois la même combinaison de trois caractères, ce 8 étant le dernier des trois. Si nous trouvons des répétitions de ce genre, elles représenteront très-proba- blement le mot the. Vérification faite, nous n'en trouvons pas moins de 7 pouvons donc ; et les caractères sont ;Zt8. supposer que représente ; représente h, et que 8 représente e, — la Nous que k valeur du t, dernier se trouvant ainsi confirmée de nouveau. Il y a maintenant un grand pas de fait. » Nous n'avons déterminé qu'un mot, mais ce seul mot nous permet d'étabhr un point beaucoup plus important, c'est-à-dire les commencements et les terminai- sons d'autres mots. Voyons, par exemple, l'avant-dernier cas où se présente la combinaison ;/i8, presque à la fin du chiffre. Nous savons que le ; qui vient immédia- LE SCARABÉE D'OR. 175 ment après est le commencement d'un mot, et, des six caractères qui suivent ce ^/le, nous n'en connaissons pas moins de cinq. Remplaçons donc ces caractères par les lettres qu'ils représentent, l'inconnu en laissant un espace pour : ' eeth. t )) Nous devons tout d'abord écarter le Ih comme ne pouvant pas faire partie du mot qui commence par le premier t, puisque nous voyons, en essayant successi- vement toutes les lettres de l'alphabet pour combler la lacune, qu'il est impossible de former un mot dont ce th puisse faire partie. Réduisons donc nos caractères à et ''] ee, t "*'\'. reprenant de nouveau tout l'alphabet, s'il le faut, nous concluons au mot tree (arbre), comme à la seule Nous gagnons une nouvelle version possible. lettre, r, représentée par (, plus deux mots juxtaposés, ainsi the tree (l'arbre). Un peu plus loin, nous retrouvons la combinaison ;/j8, et nous nous en servons comme de terminaison à ce qui précède immédiatement. Gela nous donne l'ar- rangement suivant : the tree ;4(:{:?34 the, ou, en substituant les lettres naturelles aux caractères que nous connaissons, the tree thr ;{:?3/i the. . HISTOIRES EXTRAORDINAIRES. 176 Maintenant, aux caractères inconnus nous substi- si tuons des blancs ou des points, nous aurons : the Iree Ihr... h the, et le mot through (par, à travers) se dégage pour ainsi dire de lui-même. Mais cette découverte nous donne trois lettres de plus, o,u et g, représentées par ^;]? et 3. » Maintenant, cherchons attentivement dans le cryp- togramme des combinaisons de caractères connus, et nous trouverons, non loin du commencement, l'arran- gement suivant : 83(88, ou egree, qui est évidemment la terminaison du mot degree (degré), et qui nous livre encore une lettre d, représentée par )) + Quatre lettres plus loin que ce mot degree, nous trouvons la combinaison ;46(;88, dont nous traduisons les caractères connus et représentons l'inconnu par un point; cela nous donne th . : riee, arrangement qui nous suggère immédiatement le mot tliirteen (treize), et nous fournit deux lettres nouvelles, i et n, représentées par 6 et *. )) Reportons-nous maintenant au commencement du cryptogramme, nous trouverons la combinaison S3+++. L\L SCARABÉE DOn. 177 Traduisant comme nous avons déjà fait, nous obtenons . ce qui nous montre que et les good, que la première lettre est un deux premiers mots sont a good rt, (un bon, une bonne). » Il serait temps maintenant, pour éviter toute confu- sion, de disposer toutes nos découvertes sous forme de table. Cela nous fera un 5 commencement de clef : HISTOIRES EXTIlAORDINAItUlS. 178 qu'à vous donner la traduction complète du document, comme si nous avions déchiffré successivement tous les caractères. La voici : A good glass in thebishop's hostel in the devil's seal fortyone degrees and thirleen minutes northeast and by nortii main brandi seventh liinb east side shoot from the left eye of the death's-head a bee line from the tree through the shot fifty feet out. (Un bon verre dans l'hostei de l'évoque dans la chaise du diable quarante et un degrés et treize minutes nord-est quart de nord prin:!ipale tige septième branche côté est lâchez de l'œil gauche de la tête de mort une ligne d'abeille de l'arbre à travers la balle cinquante pieds au large.) — Mais, dis-je, Ténigme me paraît d'une qualité Comment peut- tout aussi désagréable qu'auparavant. on tirer chaise un sens quelconque de tout ce jargon de du diable, de tête de mort et d'hostel de l'èvê- que? — Je conviens, l'air encore rétiliqua Legrand, que l'affaire a passablement sérieux, quand on y jette un simple coup d'oeil. Mon premier soin fut d'essayer de retrouver dans la phrase les divisions naturelles qui étaient dans l'esprit de celui qui l'écrivit. — De ponctuer, voulez-vous dire? — Quelque chose comme — Mais comment diable avez-vous — que la cela. fait? Je réfléchis l'écrivain s'était fait une loi d'assembler ses mots sans aucune division, espérant LE SCARABKK D'OR. rendre ainsi la solution plus qui n'est pas excessivement difficile. 179 un Or, homme sera presque toujours fin enclin, dans une pareille tentative, à dépasser la me- sure. Quand, dans le cours de sa composition, il arrive à une interruption de sens qui demanderait naturel- lement une pause ou un point, il est fatalement porté à serrer les caractères plus que d'habitude. Examinez ce manuscrit, et vous découvrirez facilement cinq endroits de ce genre où il y a pour. ainsi dire encombre- ment de caractères. En me dirigeant d'après cet indice j'établis la division suivante : A good glass in the bishop's hostel in the devil's seat — forty-one degrees and thirteen minutes — northeast and by north — main brandi seventh limb east side — shoot from the eye of the deatli's-head — a bee-line from the tree left through the shot fifty feet out. (Un bon verre dans l'hostel de l'évoque dans la chaise du — quarante et un degrés et treize minutes — nord — principale tige septième branche côté est quart de nord diable est — lâchez de l'œil gauche de la tête de mort — une ligne d'abeille de l'arbre à travers la balle cinquante pieds au large.) — Malgré votre division, dis-je, je reste toujours dans les ténèbres. — J'y restai moi-même pendant quelques jours, répliqua Legrand. Pendant ce temps, je fis force re- cherches dans le voisinage de l'île de Sullivan sur un bâtiment qui devait s'appeler rHôlel de VÈvêque, car HISTOIRES EXTRAOUDINAIP.ES. 180 je 110 m'inquiétai pas de la vieille orthographe du mot hoslel. N'ayant trouvé aucun renseignement à ce sujet, j'étais sur le point d'étendre la sphère de mes re- cherches et de procéder d'une manièiK^ plus systématique, quand, un matin, je m'avisai tout à coup que ce Blshop's hoslel pouvait bien avoir rapport vieille famille du à une nom de Bessop, qui, de temps immé- morial, était en possession d'un ancien manoir à quatre milles environ au nord de l'île. J'allai donc à la plan- tation, et je recommençai mes questions parmi les plus vieux nègres de l'endroit. Enfin, une des femmes les pkis âgées me dit qu'elle avait entendu parler d'un endroit comme Bessop' s castle (château de Bessop), et qu'elle croyait bien pouvoir m'y conduire, mais que ce n'était ni un château, ni une auberge, mais un grand rocher. » Je lui offris de la bien payer pour sa peine, et, quelque hésitation, elle consentit à après m'accompagner jusqu'à l'endroit précis. Nous le découvrîmes sans trop de difficulté, je la congédiai, et commençai à examiner la localité. Le château consistait en un assemblage irré- gulier de pics et de rochers, dont l'un était aussi re- marquable par sa hauteur que par son isolement et sa configuration quasi-artificielle. Je grimpai au sommet, et, là, je me sentis fort embarrassé de ce que j'avais désormais à faire. Pendant que j'y rêvais, mes yeux tombèrent sur une étroite saillie dans la face orientale du rocher, à un yard environ au-dessous de la pointe où j'étais placé. Cette saillie se projetait de dix-huit pouces à peu près. LE SCARABÉE D'OR. et n'avait guère plus creusée 181 d'un pied de large; dans le pic juste au-dessus lui une niche donnait une grossière ressemblance avec les chaises à dos concave dont se servaient nos ancêtres. Je ne doutai pas que ce ne fût la chaise du diable dont il était fait mention dans le manuscrit, et il me sembla que je tenais désormais tout le secret de l'énigme. Le bon verre, je le savais, ne pouvait pas signifier )) autre chose qu'une longue-vue; car nos marins ploient rarement le mot glass dans compris tout de suite qu'il un autre em- sens. Je fallait ici se servir d'une longue-vue, en se plaçant à un point de vue défini et n'admctlant aucune variation. Or, les phrases et un degrés nord, et treize minutes, : et nord-est — je n'hésitai pas un instant à quarante quart de le croire, — de- pour pointer la longue-vue. vaient* donner la direction je me me procurai une longue-vue, Fortement remué par toutes ces découvertes, précipitai chez moi, et je retournai » Je je au rocher. me laissai glisser sur la corniche, et je m'aperçus qu'on ne pouvait s'y tenir assis que dans une certaine position. Ce fait confirma ma conjecture. Je pensai alors à me servir de la longue-vue. Naturellement, les quarante et un degrés et treize r)iinutes ne pouvaient avoir trait qu'à l'élévation au-dessus de l'horizon sensible, puisque la direction horizontale était clairement indiquée par les mots nord-est quart de nord. J'établis cette direction au puis, pointant, moyen d'une boussole de poche; aussi juste que possible par approxi- HISTOIRES EXTRAORDINAIRES. 182 mation, ma longue-vue à un angle de quarante et un degrés d'élévation, je la fis mouvoir avec précaution de haut en bas et de bas en haut, jusqu'à ce que mon attention fût arrêtée par une espèce de trou circulaire ou de lucarne dans le feuillage d'un grand arbre qui dominait tous ses voisins dans l'étendue visible. Au centre de ce trou, j'aperçus un point blanc, mais je ne pus pas tout d'abord distinguer ce que c'était. Après avoir ajusté le foyer de ma longue-vue, je regardai de nouveau, et je m'assurai enfin que c'était un crâne hu- main. » Après cette découverte qui je considérai l'énigme principale tige, comme me combla de confiance, résolue septième branche, avoir trait qu'à la position ; côté car la phrase est, du crâne sur : ne pouvait l'arbre, et celle-ci : lâchez de l'œil gauche de la tête de mort, n'ad- mettait aussi qu'une interprétation, puisqu'il s'agissait de la recherche d'un trésor enfoui. fallait laisser crâne, et tomber une balle de qu'une ligne d'abeille, Je compris qu'il l'œil gauche du ou, en d'autres termes, une ligne droite, partant du point le plus rap- proché du tronc, et s'étendant, à travers la balle, c'està-dire à travers le point où querait l'endroit précis, qu'il était pour le — tomberait la balle, indi- et sous cet endroit je jugeai moins possible qu'un dépôt précieux fût encore enfoui. — Tout cela, dis-je, est excessivement clair, et tout à la fois ingénieux, simple et explicite. Et, quand vous eûtes quitté l'hôtel de rÉvêque, que fîtes-vous? LE SCARABEE D'OR. 183 — Mais, ayant soigneusement noté mon arbre, sa forme et sa position, je retournai chez moi. A peine eus-je quitté la chaise du Diable, que le disparut, et, de quelque côté que je trou circulaire me tournasse, il me fut désormais impossible de l'apercevoir. Ce qui me paraît le chef-d'œuvre de l'ingéniosité dans toute cette affaire, c'est ce fait (car me suis convaincu que et j'ai c'est répété l'expérience un fait), que l'ouver- ture circulaire en question n'est visible que d'un seul point, et cet unique point de vue, c'est l'étroite cor- niche sur le flanc du rocher. » Dans cette expédition à l'hôtel de VÈvèque j'avais été suivi par Jupiter, qui observait sans doute depuis quelques semaines mon air préoccupé, et mettait un soin particulier à ne pas me laisser seul. Mais, le jour suivant, je lui me levai de très-grand matin, je réussis à échapper, et je courus dans les montagnes à la re- cherche de mon arbre. J'eus beaucoup de peine à le trouver. Quand je revins chez moi mestique se disposait à à la nuit, mon do- me donner la bastonnade. Quant au reste de l'aventure, vous êtes, je présume, aussi bien renseigné que moi. — Je suppose, fouilles, dis-je, vous aviez que, lors de nos premières manqué l'endroit par suite de bêtise de Jupiter, qui laissa Toeil droit tomber le la scarabée par du crâne au lieu de le laisser filer par l'œil gauche. — Précisément. Cette méprise faisait une diffé- rence de deux pouces et demi environ relativement à HISTOIRES EXTRAORDINAIRES. 184 la balle, de c'est-à-dire à la position de la l'arbre; marqué si par la trésor le balle, avait cheville près sous été erreur cette l'endroit eût été sans importance; mais la balle et le point le plus rappro- ché de l'arbre étaient deux points ne servant qu'à établir fort une ligne de direction; naturellement, Terreur, minime au commencement, augmentait en pro- portion de la longueur de la ligne, fûmes arrivés à une distance de et, .quand nous cinquante pieds, elle nous avait totalement dévoyés. Sans dont j'élais possédé, qu'il y l'idée fixe avait positivement là, quelque part, un trésor enfoui, nous aurions peut-être bien perdu toutes nos peines. — Mais votre emphase, vos attitudes solennelles, scarabée! — quelles bizarreries! Je en balançant le vous croyais positivement fou. absolument voulu laisser Et pourquoi avez-vous tomber du crâne votre insecte, au lieu d'une balle? — Ma foi ! pour être franc, je vous avouerai que je me sentais quelque peu vexé par vos soupçons relativement à l'état de mon esprit, et je résolus de vous punir tranquillement, à ma manière, par un petit brin de mystification froide. Voilà pourquoi je balançais le scarabée, et voilà pourquoi je voulus le faire tomber du haut de l'arbre. Une observation que vous fîtes sur son poids singulier me suggéra cette dernière idée. — Oui, je comprends; et maintenant il n'y a plus qu'un point qui m'embarrasse. Que dirons-nous des squelettes trouvés dans le trou? — LE SCARABÉE D'OR. — Ah ! c'est 185 une question à laquelle je ne saurais pas mieux répondre que vous. Je ne vois qu'une nière plausible de l'expliquer, implique une atrocité croire. Il est clair telle, — et ma- mon hypothèse que cela est horrible que Kidd, — si à c'est bien Kidd qui a enfoui le trésor, ce dont je ne doute pas, pour mon compte, — il est clair dans son travail. Mais, que Kidd a dû la besogne se faire finie, il aider a pu juger convenable de faire disparaître tous ceux qui possédaient son secret. Deux bons coups de pioche ont peut- être suffi, pendant que ses aides étaient encore occupés dans la fosse; il Qui nous le dira? en a peut-être fallu une douzaine. LE CANARD AU BALLON — Étonnantes nouvelles par exprès, via Norfolk L'Atlantique traversée en trois jours!! Triomphe signalé de la machine î — volante de M. Monck Mason!!! —Arrivée à l'île de Sullivan, près Charleston, S. G., de MM. Mason, Robert Holland, Henson, Harrison Ainsworth, et de quatre autres personnes, par le ballon dirigeable Victoria, après une traversée de soixante-cinq heures d'un continent à l'autre !!!— Détails circonstanciés du voyage! ! ! ! ! Le jeu d'esprit ci-dessous, avec l'en-tête qui précède en magnifiques capitales, soigneusement émaillé de points d'admiration, fut publié primitive- ment, comme un fait positif, dans le New-Yoï'k Sun feuille périodique, et y remplit complètement le but de fournir un aliment indigeste aux insatiables badauds durant les quelques heures d'intervalle entre deux courriers de Charleston. La cohue qui se fit pour se disputer le seul journal qui eût les nouvelles fut quelque chose qui dépasse même le prodige; et, en somme, si, comme quelques-uns l'affirment, le Victoria n'a pas absolument accompl la traversée en question, il serait difficile de trouver une raison quelconque qui l'eût empêché de l'accomplir. , Le grand problème est à la fin résolu! L'air, aussi bien que la terre et l'Océan, a été conquis par la science, et deviendra pour l'humanité une grande voie com- mune et commode. L'Atlantique vient d'être traversée — sans trop de — avec une machine dont on absolument maître, — dans l'espace inconcevable- en ballon ! et cela, sans difficultés, grand danger apparent, est et HISTOIRES EXTRAORDINAIRES. 188 ment court de soixante-cinq heures d'un continent à l'autre ! Grâce à l'activité d'un correspondant de Char- leston, nous sommes en mesure de donner les premiers au public un récit détaillé de cet extraordinaire voyage, qui a été accompli, — du samedi 6 du courant, à quatre heures du matin, au mardi 9 du courant, à deux heures de l'après-midi, — par sir Everard Bringhurst, M. Os- borne, un neveu de lord Bentinck, MM. Monck Mason et Robert Holland, les célèbres aéronautes, M. Harrison Ainsworth, auteur de Jach Sheppard, son, inventeur du malheureux projet de machine volante, — la M. Hendernière deux marins de Woolwich, et en tout huit personnes. Les peuvent être considérés — détails fournis ci-dessous comme parfaitement authen- thiques et exacts sous tous sont, à etc., rapports, les puisqu'ils une légère exception près, copiés mot à mot d'après les journaux réunis et Harrison Ainsworth, à la de MM. Monck Mason politesse desquels notre agent doit également bon nombre d'explications verbales relativement au ballon lui-même, à sa construction, et à d'autres matières d'un altération haut intérêt. La seule dans le manuscrit communiqué a été faite dans le but de donner au récit hâtif de notre agent, M. Forsyth, une forme suivie et intelligible. LE BALLON. — ceux de M. Hen— avaient beaucoup Deux insuccès notoires et récents s©n et de sir George Gayley LE CANARD AU BALLON. 189 amorti Tintérêt du public relativement à la navigation aérienne. Le plan de M. Henson (qui fut d'abord considéré comme très-praticable, même par les hommes de science), était fondé sur principe d'un plan in- le hauteur par une force intrinsèque cliné, lancé d'une créée et continuée par la rotation de palettes semblables, en forme et en nombre, aux ailes d'un moulin à vent. Mais, dans toutes les expériences qui furent faites avec des modèles à VAdelaide-Gallery, se trouva que l'opé- il ration de ces ailes, non-seulement ne faisait pas avan- cer la machine, mais empêchait positivement son vol, La seule force propulsive qu'elle ait jamais montrée fut le simple mouvement acquis par la descente du plan incliné; et ce mouvement portait la quand les palettes fonctionnaient, — leur inutilité servait en ; et, machine plus loin étaient au repos fait que quand qui démontrait elles suffisamment en l'absence du propulseur, qui lui même temps d'appui, toute la machine de- vait nécessairement descendre vers le sol. Cette consi- dération induisit sir George Cayley à ajuster un propul- seur à une machine qui aurait en elle-même la de se soutenir, néanmoins, n'était ou nouvelle Georges, qu'en ce qui regardait pratique. 11 force — en un mot, à un ballon. L'idée, exhiba un le originale, chez sir mode d'application modèle de son invention à l'Institution polytechnique. La force motrice, ou prin- cipe propulseur, était, ici encore, attribuée à des sur- faces non continues ou ailes étaient au nombre de quatre ; tournantes. Ces ailes mais il se trouva qu'elles 11. HISTOIRES EXTRAORDINAIRES. 100 étaient totalement impuissantes à mouvoir le ballon ou à aider sa force ascensionnelle. Tout le projet, dès lors, n'était plus qu'un four- complet. Ce fut dans cette conjoncture que M. Monck Mason (dont le voyage de Douvres à Weilburg sur le ballon le Nassau excita un si grand intérêt en 1837) eut l'idée d'appliquer le principe delà vis d'Archimède au projet de la navigation aérienne, attribuant judicieusement l'insuccès des plans de M. Henson et de sir George Cayley à la non-continuité des surfaces dans l'appareil des roues. Willis's Il fit sa première expérience publique à Rooms, puis plus tard porta son modèle à VAdelaide-Gallery. Comme le ballon de sir George Cayley, le sien était un ellipsoïde. Sa longueur était de treize pieds pouces, sa hauteur de six pieds huit pouces. Il six conte- nait environ trois cent vingt pieds cubes de gaz, qui, si c'était de l'hydrogène pur, pouvaient supporter vingt et une livres aussitôt après qu'il était enflé, avant que le gaz n'eût eu le temps de se détériorer ou de fuir. Le poids de toute la machine et de l'appareil était de dix-sept livres, ~ donnant ainsi une économie de quatre livres environ. Au centre du ballon, en dessous, était une charpente de bois fort léger, longue d'envi- ron neuf pieds, et attachée au ballon par un réseau de l'espèce ordinaire. A cette charpente était suspendue une corbeille ou nacelle d'osier. La vis consiste en un axe formé d'un tube de cuivre creux, long de six pouces, à travers lequel, sur une spirale LE GAiNARD AU BALLON. lUl inclinée à un angle de quinze degrés, passe une série de rayons de fil d'acier, longs de deux pieds et se projetant d'un pied de chaque côté. Ces rayons sont réunis à leurs extrémités externes par deux lames de fil métallique aplati, — le tout formant ainsi la char- pente de lavis, qui est complétée par un tissu de soie huilée, coupée en pointes et tendue de manière à présenter une surface passablement lisse. Aux deux bouts de son axe, cette vis est surmontée par des montants cylin- driques de cuivre descendant du cerceau. Aux bouts inférieurs de ces tubes sont des trous dans lesquels tournent les pivots de l'axe. le Du bout de Taxe qui est plus près de la nacelle part une flèche d'acier qui une machine à levier fixée à relie la vis à Par l'opération de ce ressort, la nacelle. la vis est forcée et tour- née avec une grande rapidité, communiquant à l'en- semble un mouvement de progression. Au moyen du gouvernail, la machine ment s'orienter dans toutes était pouvait aisé- les directions. Le levier d'une grande puissance, comparativement à sa dimension, pouvant soulever un poids de quarante- cinq livres sur un cylindre de quatre pouces de dia- mètre après le premier tour, qu'il fonctionnait. Le gouvernail 11 et davantage à mesure pesait en tout huit livres six onces. était une légère charpente de roseau re- couverte de soie, façonnée à peu près comme une raquette, de trois pieds de long à peu près, et d'un pied dans sa plus grande largeur. Son poids était de deux onces environ. 11 pouvait se tourner aplat et se diriger HISTOIRES EXTRAORDINAIIIES. 192 en haut et en bas, aussi bien qu'à droite et à gauche, et donner à l'aéronaute la faculté de transporter la ré- sistance de l'air, qu'il devait, dans une position inclinée, créer sur son passage, du côté sur lequel il désirait agir, déterminant ainsi pour le ballon la direction opposée. Ce modèle (que, faute de temps, nous avons nécessairement décrit d'une manière imparfaite) fut mis en mouvement dans V Aclelaide-Gallery où il donna une , vélocité de cinq milles à l'heure; et, chose étrange à dire, il n'excita qu'un mince intérêt en comparaison de la précédente machine compliquée de M. Henson, — tant le monde est décidé à mépriser toute chose qui se présente avec un air de simplicité! Pour accomplir le grand desideratum de la navigation aérienne, on supposait généralement l'application singulièrement compliquée de quelque principe extraordinairement profond de dynamique. Toutefois, M. Mason était tellement satisfait du ré- cent succès de son invention, qu'il résolut de construire immédiatement, s'il était possible, un ballon d'une capacité suffisante pour voyage de quelque était avec vérifier étendue ; le problème par un — son projet primitif de traverser la Manche comme il avait déjà le sollicita ballon le fait Nassau. Pour favoriser ses vues, et obtint le patronage il de sir Everard Bring- hurst et de M. Osborne, deux gentlemen bien connus par leurs lumières scientifiques et spécialement pour l'intérêt qu'ils l'aéiostation. ont manifesté pour les progrès de Le projet, selon le désir de M. Osborne, LE CANARD AU BALLON. fut soigneusement sonnes auxquelles caché au public; il fut confié engagées dans la construction de — 193 les seules per- furent les personnes la machine, qui fut de MM. Mason, HoUand, établie sous la surveillance de sir Everard Bringhurst et de M. Osborne, dans l'habitation de ce dernier, près de Penstruthal, dans le pays de Galles. M. Henson, accompagné de son ami M. Ainsworth, fut admis à examiner le ballon samedi dernier, — après les derniers arrangements pris par ces messieurs pour être admis à la participation de l'entreprise. Nous ne savons pas pour quelle firent aussi raison partie de l'expédition, délai d'un ou deux jours les deux marins — mais dans un nous mettrons le lecteur en possession des plus minutieux détails concernant cet extraordinaire voyage. Le ballon est fait de soie recouverte d'un vernis de caoutchouc. 11 est conçu dans de grandes proportions et contient plus de ZiO,000 pieds cubes de gaz; mais, comme le gaz de houille a été employé préférablement à l'hydrogène, dont la trop grande force d'expansion a des inconvénients, la puissance de l'appareil, quand il estparfaitement gonflé et aussitôt après son gonflement, n'enlève pas plus de 2,500 livres environ. Non-seule- ment le gaz de houille est moins coûteux, mais on peut se le procurer et le gouverner plus aisément. L'introduction de ce gaz dans les procédés usuels de l'aérostation est due à M. Charles Green. Avant sa découverte, le procédé du gonflement était non-seule- HISTOIRES EXTRAORDINAIRES. 194 ment excessivement dispendieux, mais peu sur. On a souvent perdu deux et même trois jours en futiles efforts pour se procurer la quantité sufTisante d'hydro- gène pour un ballon d'où il avait toujours une tendance à fuir, grâce à son excessive subtilité et à son affinité pour l'atmosphère ambiante. Un ballon assez bien fait pour retenir sa contenance de gaz de houille intacte, en qualité et en quantité, pendant six mois, ne pourrait pas conserver six semaines la même quantité d'hydro- gène dans une égale intégrité. La force de support étant estimée à 2,500 livres, et les poids réunis de cinq individus environ, il restait un seulement à 1,200 de 1,300, dont 1,200 surplus étaient prises par le lest,, dont marqué sur chacun, le poids cordages, les était baromètres, réparti en différents sacs, les télescopes, — par les les barils contenant des provisions pour une quinzaine, les barils d'eau, les portemanteaux, les sacs de nuit et divers autres objets indispensables, y compris une cafetière à faire bouillir le café à la chaux, pour se dispenser totalement de feu, si cela était jugé prudent. Tous ces articles à l'exception du lest et de quelques bagatelles, étaient appendus au cerceau. La nacelle est plus lé- gère et plus petite à proportion que celle qui la représente dans le modèle. Elle est faite d'un osier fort ger, et singulièrement forte l'air si fragile. Elle lé- pour une machine qui a a environ quatre pieds de profon- deur. Le gouvernail diffère aussi de celui du modèle en ce qu'il est beaucoup plus large, et que la vis est LE CANARD AU BALLON. 195 considérablement plus petite; Le ballon est en outre muni d'un grappin et d'un guide-rope, ce dernier étant de la plus indispensable utilité. Quelques mots d'explication seront nécessaires ici pour ceux de nos lecteurs qui ne sont pas versés dans les détails de l'aérostation. Aussitôt que le ballon quitte la terre, il est sujet à l'influence de mille circonstances qui tendent à créer une différence dans wSon poids, augmentant ou diminuant sa force ascensionnelle. Par exemple, il y a parfois sur la soie une masse de rosée qui peut aller à quelques centaines de livres; il faut alors jeter du lest, sinon l'aérostat descendra. Ce lest jeté, et un bon soleil vaporisant la rosée et augmentant la force d'ex- pansion du gaz dans la soie, le tout montera de nouveau très-rapidement. Pour modérer notre ascension, le seul moyen est (ou plutôt était jusqu'au guide-rope inventé par M. Charles Green) de faire échapper du la faculté gaz par une soupape; mais la perte du gaz impliquait une déperdition proportionnelle de sion ; la force d'ascen- bien que, dans un laps de temps compara- si tivement très -bref, le ballon le mieux vait nécessairement épuiser toutes s'abattre sur le sol. C'était là le construit de- ses ressources et grand obstacle aux voyages un peu longs. Le guide-rope remédie à la plus simple la difficulté de la manière du monde. C'est simplement une très- longue corde qu'on laisse traîner hors de la nacelle, le ballon de changer de sensible. Si, par exemple, la soie et dont l'effet est d'empêcher niveau à un degré HISTOIRES EXTRAORDINAIRES. 196 est chargée d'humidité, et si conséquemment la ma- chine commence à descendre, il n'y a pas nécessité de jeter du lest pour compenser l'augmentation du poids, car on y remédie on on la neutralise, dans une proportion exacte, en déposant à terre autant de longueur de corde qu'il est nécessaire. Si, au contraire, quelques amènent une légèreté excessive circonstances et une légèreté sera immédiate- ascension précipitée, cette ment neutralisée par le poids additionnel de la corde qu'on ramène de terre. Ainsi le ballon ne peut monter ou descendre que dans des proportions très-petites, et ses ressources en gaz et en lest restent à peu près intactes. Quand on passe au-dessus d'une étendue d'eau, il devient nécessaire d'employer de petits barils de cuivre ou de bois remplis d'un lest liquide plus léger que l'eau. Ils flottent et remplissent l'office d'une corde sur la terre. Un autre office très-important du guide-rope est de marquer la direction du ballon. La corde drague, pour ainsi dire, soit sur terre, soit sur mer, quand le ballon est libre; ce dernier conséquemment, toutes les fois qu'il marche, est en avance ; ainsi, une appréciation faite, au compas, des positions des deux objets, indiquera toujours direction. la De la même façon, l'angle formé par la corde avec Taxe vertical de la machine indique la vitesse. — Quand il n'y a pas d'angle, en d'autres termes, quand la corde descend perpendiculairement, c'est que la machine est stationnaire vert, ; mais plus l'angle est ou- c'est-à-dire plus le ballon esj en avance sur le LE CANARD AU BALLON. bout de grande la corde, plus 197 est la vitesse; — et ré- ciproquement. Comme le projet des voyageurs, dans le principe, était de traverser le canal de la Manche, et de descen- dre aussi près de Paris qu'il serait possible, ils avaient pris la précaution de se munir de passe-ports visés pour toutes les parties du continent, spécifiant la nature de l'expédition, comme dans le cas du voyage sur le Nassau, et assurant aux courageux aventuriers une dis- pense des formalités usuelles de bureaux; mais des événements inattendus rendirent les passe-ports superflus. L'opération du gonflement commença fort tran- quillement samedi matin, 6 du courant, au point du jour, dans la grande cour de Weal-Vor-House, résidence de M. Osborne, à un mille environ de Penstruthal, dans la Galles du Nord ; et, à onze heures sept minutes, tout étant prêt pour le départ, le ballon fut lâché et s'éleva doucement, mais direction presque sud. constamment, dans une On ne fit point usage, pendant la première demi-heure, de la vis ni du gouvernail. Nous nous servons maintenant du journal, tel qu'il a été transcrit par M. Forsyth d'après les manuscrits réunis de MM. Monck Mason et Ainsworth. Le corps du journal, tel que nous le donnons, est de la main de M. Mason, et il y a été ajouté un post-scriptum ou appendice de M. Ainsworth, qui a en préparation et donnera très-prochainement au public un compte rendu plus minutieux du voyage, et, sans aucun doute, d'un intérêt saisissant. 198 HISTOIRES EXTRAORDINAIRES. LE JOURNAL. Samedi, 6 avril, — Tous les préparatifs qui pouvaient nous embarrasser ont été finis cette nuit commencé le gonflement ; nous avons ce matin au point du jour mais, par suite d'un brouillard épais qui ; chargeait d'eau les plis de la soie et la rendait peu maniable, nous ne nous sommes pas élevés avant onze heures à peu près. Alors, nous fîmes tout larguer, dans un grand enthousiasme, et nous nous élevâmes doucement, mais sans interruption, par une jolie brise du nord, qui nous porta dans la direction du canal de la Manche. Nous trouvâmes la force ascensionnelle plus forte que nous ne l'avions espéré, assez haut pour et, comme dominer toutes nous montions les falaises et nous trouver soumis à Faction plus prochaine des rayons du soleil, notre ascension devenait de plus en plus rapide. Cependant, je désirais commencement de fallait ne pas perdre de gaz dès monter pour le moment présent. Nous retirâmes bien vite à nous notre guide-rope; mais, l'avoir à le notre tentative, et je résolus qu'il même après absolument enlevé de terre, nous continuâmes monter très-rapidement. Le ballon marchait avec une assurance singulière et avait un aspect magnifique. Dix minutes environ après notre départ, le baro- mètre indiquait une hauteur de 15,000 pieds. Le temps était remarquablement beau, et l'aspect de la campagne placée sous nos pieds, — un des plus LE CANARD AU BALLON. romantiques à tous les points de vue, particulièrement sublime. 199 — était alors Les gorges nombreuses et profondes présentaient l'apparence de lacs, en raison des épaisses vapeurs dont elles étaient remplies, et les hauteurs et les rochers situés au sud-est, empilés dans un inextricable chaos, ressemblaient absolument aux cités géantes de la fable orientale. Nous approchions rapidement des montagnes vers le sud; mais notre élévation était plus que suffisante pour nous permettre de les dépasser en toute sûreté. En quelques minutes, nous planâmes au-dessus magnifiquement, et M. Ainsworth, ainsi que les marins, furent frappés de leur apparence pou élevée, vues ainsi de la nacelle; une grande élévation en ballon ayant pour résultat de réduire les inégalités de la surface située au-dessous à un niveau presque uni. A onze heures et demie, nous dirigeant toujours vers le sud, ou à peu près, nous aperçûmes pour la première fois le canal de Bristol et, ; quinze minutes après, la ligne des brisants de la côte apparut brusquement au-dessous de nous, et nous marchâmes rondement au-dessus de la mer. Nous résolûmes alors de lâcher assez de gaz pour laisser notre guidc-ropc traîner dans l'eau avec les bouées attenantes. Cela fut fait à la minute, et nous commençâmes à des- cendre graduellement. Au bout de vingt minutes environ, notre première bouée toucha, et, au plongeon de la seconde, nous restâmes à une élévation fixe. Nous étions tous très-inquiets de vérifier l'efficacité du gouvernail et de la vis, et nous les mîmes immédia- HISTOIRES EXTRAORDINAIRES. 200 tement en réquisition dans le but de déterminer davantage notre route vers Test et de mettre k cap sur Paris. Au moyen du gouvernail, nous effectuâmes à Tinstant le changement nécessaire de direction, et notre route se trouva presque à angle droit avec nous mîmes en mouvement le ressort le de vent; puis la vis, et nous fûmes ravis de voir qu'elle nous portait docilement dans le sens voulu. Là-dessus, nous poussâmes neuf fois un fort vivat, et nous jetâmes à la mer une bou- teille qui contenait une bande de parchemin avec le bref compte rendu du principe de l'invention. Toutefois, nous en avions à peine fini avec nos manifestations de triomphe, qu'il survint un accident imprévu qui n'était pas peu propre à nous décourager. La verge d'acier qui reliait le levier au propulseur fut soudainement jetée hors de sa place par le bout qui confinait à la nacelle (ce fut l'effet de l'inclinaison de la nacelle par suite de quelque mouvement de des marins que nous avions pris avec nous), et, l'un en un instant, se trouva suspendue et dansante hors de notre portée, loin du pivot de l'axe de la vis. Pendant que nous nous efforcions de la rattraper, et que toute notre attention y était absorbée, nous fûmes enveloppés dans un violent courant d'air de l'est qui nous porta avec une force rapide et croissante du côté de l'Atlantique. Nous nous trouvâmes chassés en mer par une vitesse qui n'était certainement pas moins de cinquante ou de soixante milles à l'heure, si bien que nous atteignîmes LE CANARD AU BALLON. le cap 201 Clear, à quarante milles vers notre nord, avant d'avoir pu assurer la verge d'acier d'avoir eu et temps de penser à virer de bord. Ce fut alors M. Ainsworlh qui, dans fit le que une proposition extraordinaire, mais mon opinion, n'était nullement déraison- nable ni chimérique, dans laquelle tement encouragé par M. Holland, nous pourrions profiter de portait, et tenter, au immédia- fut il — à savoir, que la forte brise qui nous em- de rabattre sur Paris, d'at- lieu teindre la côte du Nord-Amérique. Après une légère donnai de bon gré réflexion, je mon assentiment à cette violente proposition, qui, chose étrange à dire, ne trouva d'objections que dans les deux marins. comme nous Toutefois, étions la majorité, nous maîtrisâmes leurs appréhensions, et nous maintînmes résolument notre l'ouest; mais, route. Nous gouvernâmes droit à comme le traînage des bouées faisait un obstacle matériel à notre marche, et que nous étions suffisamment maîtres du ballon, soit pour monter, soit pour descendre, nous jetâmes tout d'abord cinquante livres de manivelle, lest, et nous ramenâmes, au moyen d'une toute la corde hors de la mer. Nous con- statâmes immédiatement l'effet de cette manœuvre par un prodigieux accroissement de vitesse; et, comme la brise fraîchissait, nous filâmes avec une vélocité presque inconcevable ; le guide-ropc s'allongeait nacelle comme un sillage de navire. Il derrière la est superflu de dire qu'il nous suffit d'un très-court espace de temps pour perdre la côte de vue. Nous passâmes au-dessus HISTOIUHS EXTRAORDINAIRES. 202 d'innombrables navires de toute espèce, dont quel- ques-uns louvoyaient avec peine, mais dont la plupart restaient en panne. Nous causâmes à leur bord le plus enthousiasme grand — enthousiasme fortement , savouré par nous-mêmes, et particuHèrement par nos deux hommes, qui, maintenant, sous l'influence de quelques petits verres de genièvre, semblaient résolus au vent toutes craintes et tous scrupules. Plu- à jeter sieurs navires tirèrent le canon de signal; et tous nous saluèrent par de grands vivat que nous entendions avec une netteté surprenante, et par l'agitation des chapeaux et des mouchoirs. Nous marchâmes ainsi tout le jour, sans incident matériel, et, comme les premières se formaient autour de nous, nous fîmes une ombres approximative de estimation la distance parcourue. Elle ne pouvait pas être de moins de cinq cents milles, probablement davantage. Pendant tout ce temps, le propulseur fonctionna, et, sans aucun doute, aida positivement notre marche. Quand le soleil se coucha, la brise fraîchit et se transforma en une vraie tempête. Au-dessous de nous, l'Océan était parfaitement visible en raison de sa phosphorescence. Le vent souffla de l'est toute la nuit, et nous donna les plus brillants présages de succès. Nous ne souffrîmes pas peu du froid, et pénible ; l'humidité de l'atmosphère nous était fort mais la place libre dans la nacelle était assez vaste pour nous permettre de nous coucher, et, au moyen de nos manteaux et de quelques couvertures^ nous nous tirâmes passablement d'alTaire. LE CANARD AU BALLON. Post'Scriptum {par M. Ainsworth). 203 — Ces neuf der- nières heures ont été incontestablement les plus en- flammées de ma vie. Je ne peux rien concevoir de plus enthousiasmant que l'étrange péril et la nouveauté d'une pareille aventure. Dieu veuille nous donner le demande pas le succès pour succès! Je ne simple le salut de mon insignifiante personne, mais pour l'amour de la science humaine et pour l'immensité du triomphe. Et cependant l'exploit est si évidemment faisable, que mon seul étonnement est que les hommes aient reculé jusqu'à présent devant la tentative. Qu'une simple brise comme celle qui nous favorise maintenant, pareille rafale pousse — qu'une un ballon pendant quatre ou cinq jours (ces brises durent quelquefois plus long- temps), et le voyageur sera facilement porté, dans ce laps de temps, d'une rive à l'autre. Avec une pareille brise, la vaste Atlantique n'est plus qu'un lac. Je suis plus frappé, au moment où j'écris, du silence suprême qui règne sur la mer, malgré son agitation, que d'aucun autre phénomène. Les eaux ne jettent pas de voix vers les cieux. L'immense Océan flamboyant audessous de nous se tord et se tourmente sans pousser une plainte. Les houles montagneuses donnent l'idée d'innombrables démons, gigantesques et muets, qui se tordraient dans une impuissante agonie. Dans une nuit telle qu'est pour moi celle-ci, un homme vit, tout un siècle de vie ordinaire, — et je — il vit ne donnerais pas ce délice ravissant pour ce siècle d'existence vulgaire. 204 HISTOIRES EXTRAORDINAIRES. Dimanche, 7 [Manuscrit de M. Mason). — Ce matin, vers dix heures, la tempête n'était plus qu'une brise de huit ou neuf nœuds (pour un navire en mer), et elle nous fait parcourir peut-être trente milles à l'heure, peut-être davantage. Néanmoins, elle a tourné ferme vers le nord; et, maintenant, au coucher du soleil, nous nous dirigeons droit à l'ouest, grâce surtout à la vis et au gouvernail, qui fonctionnent Je regarde Tentreprise la admirablement. comme entièrement réussie, et navigation aérienne dans toutes les directions (si ce n'est peut-être avec le vent absolument debout), comme un problème résolu. Nous n'aurions pas pu faire tête à la rude brise d'hier; mais, en montant, nous aurions pu sortir d» champ de son action, si nous en avions eu besoin. Je suis convaincu qu'avec notre propulseur, nous pourrions marcher contre une jolie brise carabinée. Aujourd'hui, à midi, nous nous sommes élevés à une hauteur de 25,000 pieds, en jetant du lest. Nous avons agi ainsi pour chercher un courant plus direct, mais nous n'en avons pas trouvé de plus favorable que celui dans lequel nous sommes à présent. Nous avons surabondamment de gaz pour traverser ce petit lac, dût le voyage durer trois semaines. Je n'ai pas la plus légère crainte relativement à l'issue de notre entreprise. Les difficultés ont été étrangement exagérées et incomprises. Je puis choisir mon courant, et, eussé-je con|;re moi tous les courants, je puis faire passablement ma route avec mon propulseur. Nous n'avons pas eu d'incidents notables. La nuit s'annonce bien. LE CANARD AU BALLON. Post-Scriptum {par M. Ainsworth). chose à noter, excepté le fait (fort — 205 J'ai moi), qu'à une élévation égale à celle du Cotopaxi, je n'ai éprouvé ni froid trop intense, ni migraine, ficulté peu de surprenant pour ni dif- de respiration; M. Mason, M. Holland, sir Eve- rard, n'ont pas plus souffert que moi, je crois. M. Os- borne s'est plaint d'une constricdon de la poitrine, — mais cela a disparu assez vite. Nous avons filé avec une grande vites,se toute la journée, et nous devons être plus de ipoitié chemin de l'Atlantique. à Nous avons passé au-dessus de vingt ou trente navires de toute sorte, et tous semblaient délicieusement étonnés. Tra- verser l'Océan en ballon n'est pas une affaire si difficile après tout! Omne ignotum pro magnifico. — A une hauteur de 25,000 pieds, Nota. le ciel apparaît presque noir, et les étoiles se voient distinc- tement; pendant que la mer, au lieu de paraître convexe, comme on pourrait le supposer, semble absolu- ment et entièrement concave *. \. M. Ainsworth n'a pas essayé de se rendre compte de ce phénomène, dont l'explication est cependant bien simple. Une ligne abaissée perpendiculairement sur la surface de la terre fou de la mer) d'une hauteur de 25,000 pieds, formerait la perpendiculaire d'un triangle rectangle, dont la base s'étendrait de l'angle droit à l'horizon, et l'hypoténuse de l'horizon au ballon . Mais les 25,000 pieds de hauteur sont peu de chose ou presque rien relativement à l'étendue de la perspective. En d'autres termes, la base et l'hypoténuse du triangle supposé seraient si longues, comparées avec la perpendiculaire, qu'elles pourraient être regardées comme presauc parallèles. De cette façon, l'horizon de l'aéi'onaute devait lui ap- paraître de niveau avec la nacelle. Mais, comme le point situé im***** 12 HISTOIRES EXTRAORDINAIRES. 206 Lundi, 8 {Manuscrit de M. Mason). — Ce matin, nous avons encore eu quelque embarras avec la lige du propulseur, qui devra être entièrement modifiée, de crainte de sérieux accidents; — je parle de la tige d'acier non pas des palettes; ces dernières ne et laissaient rien à désirer. Le vent a soufflé tout le jour du nordest, roide et sans interruption, tant la fortune semble résolue à nous favoriser. Juste avant jour, le fûmes tous un peu alarmés par quelques bruits nous sin- guliers et quelques secousses dans le ballon, accom- pagnés de la soudaine interruption du jeu de la machine. Ces phénomènes étaient occasionnés par l'expansion du gaz, résultant d'une augmentation de chaleur dans l'atmosphère, et la débâcle naturelle des particules de glace dont le filet s'était incrusté pendant la nuit. Nous avons jeté quelques bouteilles aux navires que nous avons aperçus. L'une d'elles a été recueillie par un grand navire, vraisemblablement un des paquebots qui font le service de New-York. Nous avons essayé de déchif- frer son nom, mais nous ne sommes pas sûrs d'y avoir réussi. Le télescope de M. Osborne nous a laissé quelque chose comme VAlalante. Il lire maintenant est minuit, et nous marchons toujours à peu près vers médiatement au-dessous de lui, paraît et est en effet à une grande distance, il lui semble naturellement à une grande distance audessous de l'horizon. De là l'impression de concavité, et cette impression durera jusqu'à ce que l'élévation se trouve dans une telle proportion avec l'étendue de l'horizon, que le parallélisme appaalors la réelle rent de la base et de l'hypoténuse disparaisse, convexité de la terre deviendra sensible. — — E. A. P. LE CANARD AU BALLON. l'ouest d'une allure rapide. 207 La mer est singulièrement phosphorescente. — Post-Scriptam (par M. Ainsworth). est Il mainte- nant deux heures du matin, et il fait presque calme, — mais autant du moins que j'en peux juger; c'est un point qu'il est fort difficile d'apprécier, depuis que nous nous mouvons si complètement avec et dans l'air. Je dormi depuis que n'ai point j'ai Wheal-Vor, quitté mais je ne peux plus y tenir, et je vais faire un somme. Nous ne pouvons pas être loin de côte la d'Amé- rique. Mardi 9 {Manuscrit de M. Ainsworth). de r après-midi. — Une heure — Nous sommes en vue de la côte basse de la Caroline du Sud! Le grand problème est résolu. Nous avons traversé versée en l'Atlantique, ballon, facilement, loué! Qui osera dire — nous l'avons rondement maintenant qu'il ! tra- Dieu soit y a quelque chose d'impossible? Ici finit le ont été journal. Quelques détails sur la descente communiqués toutefois par M. Ainsworth à M. Forsyth. Il faisait presque un calme plat quand le? voyageurs arrivèrent en vue de la côte, qui fut immé- diatement reconnue par les deux marins et par M. Osborne. Ce gentleman ayant des connaissances au fort Moultrie, on résolut immédiatement de descendre dans le voisinage. Le ballon fut porté vers basse, le sable ferme, uni, la plage ; la marée était admirablement approprié flISTOIIiRS 208 à une EXTRAORDINAIRKS. descente, et le grappin mordit du premier coup et tintbon. Leshabitants de l'île et du fort se pressaient naturellement pour voir le ballon mais ; ce n'était qu'avec difficulté qu'on ajoutait foi au voyage accompli, — la traversée de V Atlantique ! L'ancre mordait à deux heures de l'après-midi; ainsi le voyage entier avait duré soixante-quinze heures; ou plutôt un peu moins si on compte simplement le trajet d'un rivage à l'autre. Il n'était arrivé aucun accident sérieux. On n'avait eu à craindre aucun danger réel. Le ballon fut dégonflé et serré sans peine Moultrie , ; et ces messieurs étaient encore au fort quand les manuscrits d'oili ce récit est partaient par le courrier de Charleston. rien de positif sur leurs intentions tire On ne sait ultérieures ; mais nous pouvons promettre en toute sûreté à nos lecteurs quelques informations supplémentaires, soit pour lundi, soit pour le jour suivant au plus tard. Voilà certainement l'entreprise la plus prodigieuse, la plus intéressante, la plus importante qui ait jamais été accomplie ou même tentée par un homme. Quels magnifiques résultats on en peut perflu maintenant de le tirer, n'est-il déterminer? pas su- AVENTURE SANS PAREILLE D'UN CERTAIN HANS PFAALL Avec un cœur plein de fantaisies délirantes Dont je suis le capitaine, Avec une lance de feu et un cheval d'air, A travers l'immensité je voyage. Chanson de Tom O'Uedlam. D'après les nouvelles les plus récentes de Rotterdam, il paraît que cette ville est dans un singulier état d'ef- fervescence philosophique. En réalité, il s'y est produit des phénomènes d'un genre si complètement inattendu, si entièrement nouveau, si absolument en contradiction avec toutes les opinions reçues, que je ne doute pas qu'avant peu toute l'Europe ne soit sens dessus dessous, toute la physique en fermentation, et que la rai- son et l'astronomie ne se prennent aux cheveux. Il pas paraît que le... du mois positivement date), la de... (je ne me rappelle une foule immense était rassemblée, dans un but qui n'est pas spécifié, sur la grande place de la Bourse de la Rotterdam. La journée singulièrement chaude pour la saison, — il était confortable ville de y avait à peine un souffle d'air, et 12. 210 HISTOIRES EXTRAORDINAIRES. la foule n'était pas trop fâchée de se trouver de temps à autre aspergée d'une ondée amicale de quelques mi- nutes, qui s'épanchait des vastes masses de blancs abondamment éparpillés à travers la nuages voûte bleue du firmament. Toutefois, vers midi, il se manifesta dans l'assemblée une légère mais remarquable agitation, suivie du brou- haha de dix mille langues; une minute après, dix mille visages se tournèrent vers le ciel, dix mille pipes simultanément descendirent bouches, et un cri, du coin de dix mille qui ne peut être comparé qu'au rugissement du Niagara, retentit longuement, haute- ment, furieusement, à travers toute la cité et tous les environs de Rotterdam. L'origine de ce vacarme devint bientôt suffisam- ment manifeste. On vit déboucher et entrer dans une des lacunes de l'étendue azurée, du fond d'une de ces vastes masses de nuages aux contours vigoureusement définis, un être étrange, hétérogène, d'une apparence solide, si singulièrement configuré, si fantastiquement organisé, que la foule de ces gros bourgeois qui le re- gardaient d'en bas, bouche béante, ne pouvait absolu- ment y rien comprendre ni se lasser de l'admirer. Qu'est-ce que cela pouvait être? Au nom de tous les diables de Rotterdam, qu'est-ce que cela pouvait pré- personne ne pouvait sager? Personne ne le savait, deviner; personne, — pas même le bourgmestre xMyn- heer Superbus Von Underduk, — ne possédait la le plus légère donnée pour éclaircir ce mystère; en sorte que, AVENTURE D'UN CERTAIN HANS PFAALL. 211 n'ayant rien de mieux à faire, tous les Rotterdamois, à un homme près, remirent sérieusement leurs pipes dans le coin de leurs bouches, et, gardant toujours un œil braqué sur le phénomène, se mirent à pousser leur fumée, firent une pause, se dandinèrent de droite à gauche, et grognèrent significativement, — puis se dandinèrent de gauche à droite, grognèrent, firent une pause, et finalement — se remirent à pousser leur fumée. Cependant, on voyait descendre, toujours plus bas vers la béate ville de Rotterdam, l'objet d'une grande curiosité et la cause d'une si grosse fumée. En quelques minutes, la chose arriva assez près pour qu'on pût la distinguer exactement. c'était Cela semblait être, indubitablement une espèce — oui ! de ballon, mais jusqu'alors, à coup sûr, Rotterdam n'avait pas vu de pareil ballon. Car qui — je vous le demande — a jamais entendu parler d'un ballon entièrement fabriqué avec des journaux crasseux? Personne en Hollande, certai- nement; et cependant, là, sous le nez même du peuple ou plutôt à quelque distance au-dessus de son nez, apparaissait la chose en faite — j'ai avec cette question, la chose elle-même, de bonnes autorités pour Taffirmer — même matière à laquelle personne n'avait jamais pensé pour un pareil dessein. C'était une énorme insulte au bon sens des bourgeois de Rotterdam. Quant à la forme du phénomène, elle était encore plus répréhensible, — ce n'était guère qu'un gigan- tesque bonnet de fou tourné sens dessus dessous. Et HISTOIRES EXTRAORDINAIRES. 212 cetle d'être amoindrie, similitude fut loin quand, en l'inspectant de plus près, la foule vit un énorme gland pendu à la pointe, et autour du bord supérieur ou dç la base du cône un rang de petits instruments qui res- semblaient à des clochettes de brebis, et tintinnabulaient incessamment sur l'air de Betty Martin. Mais voilà qui était encore plus violent par des rubans bleus au bout : — suspendu de la fantastique ma- chine, se balançait, en manière de nacelle, un immense chapeau de castor gris américain, à bords superlative- ment larges, à calotte hémisphérique, avec un ruban noir et une boucle d'argent. Chose assez remarquable toutefois, maint citoyen de Rotterdam aurait juré qu'il connaissait déjà ce chapeau, et, en vérité, toute l'as- semblée le regardait presque avec des yeux familiers ; pendant que dame Grettel Pfaall poussait en le voyant une exclamation de joie et de surprise, et déclarait que homme c'était positivement le chapeau de son cher lui-même. Or, une circonstance d'autant plus c'était importante à noter, que Pfaall, avec ses trois compa- gnons, avait disparu de Rotterdam, depuis cinq ans environ, d'une manière soudaine et inexplicable, et, jus- qu'au moment où commence ce récit, tous les efforts pour obtenir des renseignemefats sur eux avaient échoué. 11 est vrai qu'on avait découvert récemment, dans une partie retirée de la ville, à l'est, quelques ossements humains, mêlés à un amas de décombres d'un aspect bizarre; et quelques profanes avaient été jusqu'à supposer qu'un hideux meurtre avait dû être AVENTURE D'UN CERTAIN IIANS PFAALL. 213 commis en cet endroit, et que Hans Pfaall et ses camarades en avaient été très-probablement les victimes. Mais revenons à notre récit. Le ballon (car c'en était un, décidément) était maintenant descendu à cent pieds du sol, et montrait dis- tinctement à la foule le personnage qui l'habitait. Un singulier individu, en vérité. ne pouvait guère avoir Il plus de deux pieds de haut. Mais sa taille, toute petite qu'elle était, ne l'aurait pas empêché de perdre l'équilibre, et de passer par-dessus le bord de sa toute pe- tite nacelle, sans l'intervention d'un rebord circulaire qui lui montait jusqu'à la poitrine, et aux cordes du ballon. Le corps du petit se rattachait homme était volumineux au delà de toute proportion, et donnait à l'ensemble de son individu une apparence de rotondité singulièrement absurde. ment, on De ses pieds, naturelle- n'en pouvait rien voir. Ses monstrueusement grosses, ses cheveux, mains étaient gris et ras- semblés par derrière en une queue; son nez, prodi- gieusement long, crochu et empourpré; ses yeux bien fendus brillants joues, et perçants, — quoique ridés par la son menton vieillesse, et ses — larges, boursouflés, doubles; mais, sur les deux côtés de sa tête, il était impossible d'apercevoir le semblant d'une oreille. Ce drôle de petit monsieur était habillé d'un paletotsac de satin bleu de ciel et de culottes collantes assorties, serrées aux genoux par une boucle d'argent. Son gilet était d'une étoffe jaune et brillante; un bonnet de HISTOIRES EXTRAORDINAIRES. 214 taffetas blanc était tête et, ; gentiment posé sur le côté de sa pour compléter cet accoutrement, un foulard écarlate entourait son cou, et, contourné en un nœud superlatif, laissait traîner sur sa poitrine ses bouts prétentieusement longs. Étant descendu, comme je l'ai dit, à cent pieds environ du sol, le vieux petit monsieur fut soudainement saisi d'une agitation nerveuse, et parut peu soucieux de s'approcher davantage de la terre ferme. 11 jeta donc une quantité de sable d'un sac de toile qu'il souleva à grand' peine, et resta stationnaire pendant un instant. Il s'appliqua alors à extraire de la poche de son paletot, d'une manière feuille agitée de maroquin. et précipitée, 11 le pesa un grand porte- soupçonneusement dans sa main, l'examina avec un air d'extrême surprise, il comme évidemment étonné de son poids. Enfin l'ouvrit, en tira une énorme lettre scellée de cire de môme rouge et soigneusement entortillée de fil couleur, et la laissa tomber juste aux pieds du bourg- mestre Superbus von Underduk. Son Excellence se baissa pour la ramasser. Mais l'aéronaute, toujours fort inquiet, et n'ayant apparem- ment pas d'autres affaires qui le retinssent à Rotter- dam, commençait déjà à faire préparatils de départ; et, comme il précipitamment fallait ses décharger une portion de son lest pour pouvoir s'élever de- nouveau, une demi-douzaine de sacs qu'il jeta l'un après l'autre, sans se donner la peine de les vider, tombèrent coup sur coup sur le dos de l'infortuné bourgmestre. AVENTURE D'UN CERTAIN H ANS PFAALÎ,. '215 une demi-douzaine de fois à la et le culbutèrent juste face de tout Rotterdam. Il ne faut pas supposer toutefois que le grand Under- duk ait laissé passer impunément cette impertinence de la part du vieux petit bonhomme. On dit, au contraire, qu'à chacune de ses six culbutes il ne poussa pas moins de six bouffées, distinctes et furieuses, de sa chère pipe qu'il retenait pendant tout ce temps et de toutes ses forces, et qu'il se propose de tenir ainsi Dieu si le permet Cependant, et, ballon s'élevait le — — jusqu'au jour de sa mort. planant au-dessus de comme une alouette, par disparaître la cité, finit tranquillement derrière un nuage semblable à celui d'où il avait si singulièrement émergé, et fut ainsi perdu pour les yeux éblouis des bons citoyens de Rotterdam. Toute l'attention se porta alors sur la lettre, dont la transmission avec les accidents qui la suivirent avaient failli être si fatale à la personne et à la dignité de Son Excellence von Underduk. Toutefois, ce fonctionnaire n'avait pas oublié durant ses de mettre qui, en sûreté l'objet d'après la mains suscription, légitimes, mouvements giratoires important, puisqu'elle était — la lettre, tombée était dans adressée à — des lui d'abord, et au professeur Rudabub, en leurs qualités respectives de président et de vice-président du Collège astronomique de Rotterdam. Elle fut donc ouverte surle-champ par ces dignitaires, communication suivante, foi, très-sérieuse : et ils y trouvèrent très-extraordinaire, et, la ma HISTOIRES EXTRAORDINAIRES. 210 A Leurs Excellences von Underduh et Ruclahuh, président et vice-président du Collège national astrono- mique de la ville de Rotterdam. Vos Excellences se souviendront d'un peut-être humble artisan, du nom de Hans Pfaall, raccommodeur de soufflets de son métier, qui disparut de Rotterdam, il y a environ cinq ans, avec trois individus, et d'une manière qui a dû être regardée comme inexplicable. C'est moi, Hans Vos Excellences nication. Il Pfaall lui-même — n'en déplaise à — qui suis l'auteur de cette commu- est de notoriété parmi la mes plupart de concitoyens que j'ai occupé, quatre ans durant, la petite maison de briques placée à l'entrée de la ruelle demeurais encore au moment de ma disparition. Mes aïeux y ont toujours rédite Sauerhraut, et que j'y sidé, de temps immémorial, et ils y ont invariablement exercé comme moi-même la très-respectable et très- lucrative profession de raccommodeurs de soufflets; car, pour dire la vérité, jusqu'à ces dernières années, où toutes les têtes de la population ont été mises en feu par la politique, jamais plus fructueuse industrie n'avait été exercée par un honnête citoyen de Rotter- dam, et personne n'en était plus digne que moi. Le ferme, on ne crédit était bon, la pratique donnait manquait bonne volonté. ni d'argent ni de Mais, comme je l'ai dit, nous ressentîmes bientôt les effets de la liberté, des grands discours, du radicalisme de toutes les drogues de cette espèce. Les gens et qui AVENTURE D'UN CERTAIN HANS PFAALL. jusque-là avaient été les meilleures pratiques 217 du monde n'avaient plus un moment pour penser à nous. Ils en avaient à peine assez pour apprendre l'histoire des révolutions et pour surveiller dans sa marche l'intelligence et l'idée du siècle. journal. avaient besoin de S'ils souffler leur feu, ils se faisaient un soufflet avec un A mesure que le gouvernement devenait plus faible, j'acquérais la conviction que le cuir et le fer devenaient de plus en plus indestructibles; et bientôt il n'y eut pas dans tout Rotterdam un seul soufflet qui eût besoin d'être repiqué, ou qui réclamât l'assistance du marteau. C'était un état de choses impossible. Je fus bientôt aussi gueux qu'un rat, et, comme j'avais une femme et des enfants à nourrir, mes charges devinrent à la longue intolérables, et je passai toutes mes heures à réfléchir sur le mode le plus convenable pour me dé- barrasser de la vie. Cependant, mes chiens de créanciers me laissaient peu de loisir pour la méditation. Ma maison était littéralement assiégée du matin au soir. 11 y avait particu- lièrement trois gaillards qui me tourmentaient au delà du possible, montant continuellement la garde devant ma porte, et me menaçant toujours de la loi. Je me promis de tirer de ces trois êtres une vengeance amère, si jamais j'étais assez heureux pour les tenir dans mes griffes; et je crois que cette espérance ravissante fut la seule chose qui m'empêcha de mettre immédiatement à exécution mon plan de suicide, qui était de me faire gauter la cervelle d'un coup ***** d'espingole. Toutefois, je 13 HISTOIRES EXTRAORDINAIRES. 218 jugeai qu'il valait mieux dissimuler ma rage, et les bourrer de promesses et de belles paroles, jusqu'à ce que, par un caprice heureux de la destinée, l'occasion de la vengeance vînt s'offrir à moi. Un jour que j'étais parvenu à leur échapper, et que je me sentais encore plus abattu que d'habitude, je continuai à errer pendant longtemps encore et sans but à travers les rues les plus obscures, jusqu'à ce qu'enfin je buttai contre le coin d'une échoppe de bouquiniste. Trouvant sous des pratiques, je ma main un fauteuil à l'usage m'y jetai de mauvaise humeur, et, sans savoir pourquoi, j'ouvris le premier volume qui me tomba sous la main. Il se trouva que c'était une petite brochure traitant de l'astronomie spéculative, et écrite, soit par le professeur Encke, de Berlin, soit par un Français dont le nom ressemblait beaucoup au sien. J'avais une légère teinture de cette science, et je fus bientôt tellement absorbé par la lecture de ce livre, que je le lus deux fois d'un bout à l'autre avant de revenir au sentiment de ce qui se passait autour de moi. Cependant, il commençait à faire nuit, et je repris le chemin de mon traité logis. Mais la lecture de ce petit (coïncidant avec une découverte pneumatique qui m'avait été récemment communiquée par un cousin de Nantes, comme un secret d'une haute importance) avait fait sur mon esprit une impression indélébile; et, tout en flânant à travers les rues crépusculeuses, je repassais minutieusement dans ma mémoire les rai- AVENTURE D'UN CERTAIN HANS PFÀALL. 210 sonnements étranges, et quelquefois inintelligibles, de récrivain. affecté Il mon y avait quelques imagination passages qui avaient manière d'une extraor- dinaire. Plus j'y rêvais, plus intense devenait l'intérêt qu'ils avaient excité en moi. Mon éducation, généralement fort limitée, mon ignorance spéciale des sujets relatifs à la philosophie naturelle, loin de m'ôter toute confiance dans mon aptitude à comprendre ce que j'avais lu, ou de m'induire à mettre en suspicion les notions confuses et vagues qui avaient surgi naturellement de ma lecture, devenaient simplement un aiguillon plus puissant pour mon imagination; et j'étais assez vain, ou peut-être assez raisonnable, pour me demander si ces idées indigestes qui surgissent dans les esprits mal réglés ne contiennent pas souvent en elles elles en ont la parfaite apparence toute la réalité, et toutes les — comme — toute la force, autres propriétés inhé- rentes à l'instinct et à l'intuition. II était tard quand j'arrivai à la maison, et je me mis immédiatement au lit. Mais mon esprit était trop préoccupé pour que je pusse dormir, et je passai la nuit entière en méditations. Je me levai de grand matin, et je courus vivement à l'échoppe du bouquiniste, oii j'employai tout le peu d'argent qui me restait à Tacquisition de quelques volumes de mécanique et d'as- tronomie pratiques. Je les transportai chez moi comme un trésor, et je consacrai à les lire tous mes instants de loisir. Je fis ainsi assez de progrès dans mes nou- HISTOIRES EXTRAORDINAIRES. 220 velles études pour mettre à exécution certain projet qui m'avait été inspiré par le diable ou par mon bon génie. Pendant tout ce temps, je fis tous mes efforts pour me concilier les trois créanciers qui m'avaient causé tant de tourments. Finalement, j'y réussis, tant en vendant une assez grande partie de mon mobilier pour satisfaire à moitié leurs réclamations, qu'en leur faisant la promesse de solder la différence après la réalisation d'un petit projet qui me trottait dans la tête, et pour l'accomplissement duquel je réclamais leurs services. Grâce à ces moyens (car c'étaient des gens fort ignorants), je n'eus pas grand'peine à les faire entrer dans mes vues. Les choses ainsi arrangées, je m'appliquai, avec ma femme, avec les plus grandes précautions l'aide de et dans le plus parfait secret, à disposer du bien qui me restait, et à réaliser par de petits emprunts, et sous différents prétextes, une assez bonne quantité d'argent comptant, sans m'inquiéter le moins du monde, je l'avoue à ma honte, des moyens de remboursement. Grâce à cet accroissement de ressources, je me procurai, en diverses fois, plusieurs pièces de très-belle — — de une provision de vernis de caoutchouc, — un vaste sur commande, — profond panier quel- batiste, de douze yards chacune, la ficelle, et d'osier, fait et ques autres articles nécessaires à la construction et à l'équipement d'un ballon d'une dimension extraordinaire. Je chargeai ma femme de le confectionner le AVENTURE D'UN CERTAIN HANS PFAALL. 221 plus rapidement possible, et je lui donnai toutes les instructions nécessaires pour la manière de procé- der. même temps, je fabriquais avec de la ficelle un En d'une dimension suffisante, j'y adaptais un cerceau filet et des cordes, et je faisais l'emplette des nombreux instruments et des matières nécessaires pour faire des expériences dans les plus hautes régionsde l'atmosphère. Une nuit, je transportai prudemment dans un endroit retiré de Rotterdam, à l'est, cinq barriques cerclées de qui pouvaient contenir chacune environ cinquante fer, gallons, et une sixième d'une dimension plus vaste; tubes en fer-blanc, de trois pouces de diamètre et six de quatre pieds de long, façonnés ad hoc; une bonne quantité d'une certaine substance métallique ou demimétal, que je ne nommerai pas, et une douzaine de dames-jeannes remplies d'un acide très-commun. Le gaz qui devait résulter de cette combinaison est gaz qui n'a jamais été, jusqu'à présent, un fabriqué que par moi, ou du moins qui n'a jamais été appliqué à un pareil objet. Tout ce que je puis dire ici, c'est qu'il est une des parties constituantes de l'azote, qui a été si longtemps regardé comme irréductible, et que sa densité est moindre que celle de l'hydrogène d'environ trente-sept fois et quatre dixièmes. mais non sans odeur; il brûle, Il est sans saveur, quand il est pur, avec une flamme verdàtre; il attaque instantanément la vie animale. Je ne ferais aucune difficulté d'en livrer tout le secret, mais il appartient de droit, comme je l'ai HISTOIRES EXTRAORDINAIRES. 222 déjà fait entendre, à un citoyen de Nantes, en France, par qui il m'a été communiqué sous condition. même individu m'a confié, sans être le moins Le du monde au fait de mes intentions, un procédé pour fabriquer les ballons avec un certain tissu animal, qui rend la fuite du gaz chose presque impossible; mais je trouvai ce moyen beaucoup trop dispendieux, et, d'ailleurs, il se pouvait que la batiste, revêtue d'une couche de caoutchouc, fût tout aussi bonne. Je ne mentionne cette circonstance que parce que je crois probable que l'individu en question tentera, un de ces jours, une ascension avec le nouveau gaz et la matière dont j'ai parlé, et que je ne veux pas le priver de l'honneur d'une invention très-originale. A chacune des Tun des petit, petits places qui devait être occupée par tonneaux, je creusai secrètement un trou; les trous formant de cette façon un cercle de vingt-cinq pieds de diamètre. Au centre du cercle, qui était la place désignée pour la plus grande bar- rique, je creusai un trou plus profond. Dans chacun des cinq petits trous, je déposai une boîte de fer-blanc, contenant cinquante livres de poudre à canon, et dans le plus grand un baril qui en tenait cent cinquante. Je reliai convenablement le baril et les cinq boîtes par des traînées couvertes, et, ayant fourré dans l'une des boîtes le bout d'une mèche longue de quatre pieds environ, je comblai le trou et plaçai la barrique par-dessus, laissant dépasser l'autre bout de la pouce à peu près au delà de la mèche d'un barrique, et d'une AVENTURE D'UN CERTAIN HANS PFAALL. 223 manière presque invisible. Je comblai successivement les autres trous, et disposai chaque barrique à la place qui lui était destinée. Outre les articles que j'ai énumérés, je transportai mon dépôt général et j'y cachai un des appareils perfectionnés de Grimm pour la condensation de l'air à atmosphérique. Toutefois, je découvris que cette ma- chine avait besoin de singulières modifications pour devenir propre à l'emploi auquel je la destinais. Mais, grâce à un travail entêté et à une incessante persé- vérance, j'arrivai à des résultats excellents dans tous mes préparatifs. Mon ballon fut bientôt parachevé. 11 pouvait contenir plus de quarante mille pieds cubes de gaz; il pouvait facilement m'enlever, selon mes calculs, moi et tout mon attirail, et même, en le gouvernant convenablement, cent soixante-quinze livres de lest pardessus le marché. 11 avait reçu trois couches de vernis, et je vis que la batiste remplissait parfaitement l'ofïice de la soie; elle était également solide et coûtait beaucoup moins cher. Tout étant prêt, j'exigeai de ma femme qu'elle me jurât le secret sur toutes mes actions depuis le jour de ma première visite à l'échoppe du bouquiniste, et je lui promis de mon côté de revenir aussitôt que les circonstances me le permettraient. Je lui donnai le peu d'argent qui me restait, et je lui fis mes adieux. En réalité, Elle je était n'avais pas d inquiétude sur son compte. ce que les gens appellent une maîtresse femme, et pouvait très-bien faire ses affaires sans mon HISTOIRES EXTRAORDINAIRES. 224 assistance. crois Je même, pour un simple complément de poids, tout dire, qu'elle — — un remplissage, — m'avait toujours regardé comme un triste fainéant, une espèce d'homme bon pour bâtir des châteaux en l'air, et rien de plus, — et qu'elle n'était pas fâchée d'être débarrassée de moi. Il faisait nuit sombre quand je lui fis mes adieux, et, prenant avec moi, en manière d'aides de camp, les trois créanciers qui m'avaient causé tant de souci, nous portâmes le ballon avec sa nacelle et tous ses accessoires, par une route détournée, à l'endroit où j'avais déposé les autres articles. les y trouvâmes parfaitement intacts, et je Nous me mis im- médiatement à la besogne. Nous étions au 1^^ avril. La nuit, comme je Tai dit, était sombre ; et on ne pouvait pas apercevoir une étoile une bruine ; qui tombait par intervalles, épaisse, nous incommodait fort. Mais ma grande inquiétude, c'était le ballon, qui, en dépit du vernis qui le proté- geait, commençait à s'alourdir par l'humidité; la poudre aussi pouvait s'avarier. Je lis donc travailler rudement mes trois gredins, je leur fis piler de la glace autour de la barrique centrale et agiter l'acide dans les autres. Cependant, il ne cessaient de m'importuner de questions pour savoir ce que attirail, et terrible je voulais faire avec tout cet exprimaient un vif mécontentement de besogne à laquelle je les condamnais. comprenaient pas résulter de — disaient-ils bon à leur faire uniquement pour les — ce Ils la ne qu'il pouvait ainsi se mouiller la peau rendre complices d'une aussi AVENTURE D'UN CERTAIN HANS PFAALL. 225 abominable incantation. Je commençais'à çtre un peu inquiet, et j'avançais l'ouvrage de toute ma force; car, en vérité, ces idiots s'étaient figuré, j'imagine, que j'avais fait un pacte avec le diable, et que dans tout ce que je faisais J'avais maintenant il n'y avait rien de bien rassurant. donc une très-grande crainte de les voir me planter là. Toutefois, je m'efforçai de les apaiser en leur promettant de payer jusqu'au dernier sou, les aussitôt que j'aurais mené à bonne fm la besogne en préparation. Naturellement ils interprétèrent ces beaux discours comme ils voulurent, s'imaginant sans doute que de toute manière j'allais me rendre maître d'une immense quantité d'argent comptant; et, pourvu que je leur payasse ma dette, et un petit brin en plus, en considéiation de leurs services, j'ose affirmer qu'ils s'inquiétaient fort peu de ce qui pouvait advenir de mon âme ou de ma carcasse. Au bout de quatre heures et demie environ, le ballon me parut suffisamment gonflé. J'y suspendis donc la nacelle, et j'y plaçai tous mes bagages, — un téles- cope, un baromètre avec quelques modifications importantes, un thermomètre, un électromètre, un compas, une boussole, une montre à secondes, une cloche, un porte-voix, etc., etc., ainsi qu'un globe de j'avais fait le vide, et verre on hermétiquement bouché, sans oublier f appareil condensateur, de la chaux vive, un bâton de cire à cacheter, une abondante provision pem- d'eau, et des vivres en quantité, tels que le mkan, qui énorme nutritive contient une matière 13. HISTOIRES EXTRAORDINAIRES. 226 comparativement à son petit volume. J'installai aussi dans ma nacelle un couple de pigeons et une chatte. Nous étions presque au point du jour, et je pensai mon départ. comme par accident, qu'il était grandement temps d'effectuer Je laissai donc tomber par terre, un cigare allumé, et, en me baissant pour le ramasser, j'eus soin de mettre sournoisement le feu à la mèche, dont le bout, comme je l'ai dit, dépassait un peu le bord inférieur d'un des petits tonneaux. J'exécutai cette manœuvre sans être vu le moins du monde par mes trois bourreaux; je sautai dans la nacelle, je coupai immédiatement Tunique corde qui me retenait à la terre, et je m'aperçus avec j'étais enlevé bonheur que avec une inconcevable rapidité; le ballon emportait très-facilement ses cent soixante-quinze livres de lest de plomb ; il aurait pu en porter le double. Quand je quittai la terre, le baromètre marquait trente pouces, et le thermomètre centigrade 19 degrés. Cependant, j'étais à peine monté à une hauteur de cinquante yards, quand arriva derrière moi, avec un rugissement et un grondement épouvantables, une si épaisse trombe de feu et de gravier, de bois et de métal enflammés, mêlés à des membres humains déchi- rés, que je sentis mon cœur jetai tout au défaillir, et que je me fond de ma nacelle, tremblant de terreur. Alors, je compris que j'avais horriblement chargé la mine, et que j'avais encore à subir les principales con- séquences de la secousse. En effet, en moins d'une seconde, je sentis tout mon sang refluer vers mes tempes, AVENTURE D'UN CERTAIN HANS PFAALL. et '121 immédiatement, inopinément, une commotion que je n'oublierai jamais éclata à travers les ténèbres, et sembla déchirer en deux le firmament lui-même. Plus tard, quand j'eus le temps de la réflexion, je ne man- quai pas d'attribuer l'extrême violence de l'explosion, re- lativement à moi, à sa véritable cause, — c'est-à-dire à ma position, directement au-dessus de la mine et dans de son action la ligne plus puissante. iMais, en ce la moment, je ne songeais qu'à sauver ma vie. D'abord, le ballon s'affaissa, puis il se dilata furieusement, puis il se mit à pirouetter avec une vélocité vertigineuse, un homme me jeta par-dessus le bord de la nacelle, et me et finalement, vacillant et roulant comme ivre, il laissa accroché à une épouvantable hauteur, la tête en bas, par un bout de corde fort mince, haut de trois pieds de long environ, qui pendait par hasard à travers une crevasse, près du fond du panier d'osier, et dans lequel, au milieu de ma chute, mon pied gauche s'engagea providentiellement. 11 est impossible, absolu- ment impossible, de se faire une idée juste de reur de ma J'ouvrais situation. bouche pour respirer, accès de fièvre la — un frisson ressemblant à un secouait tous muscles de mon être, l'hor- convulsivement — les nerfs et tous les je sentais mes yeux jaillir de leurs orbites, une horrible nausée m'envahit, — enfin je m'évanouis et perdis toute conscience. Combien de temps restai-je dans cet état, il m'est impossible de le dire. 11 s'écoula toutefois un assez long temps, car, lorsque je recouvrai en partie l'usage HISTOIRES EXTllAORDINAIRES. •228 de mes sens, je vis le jour qui se levait; se trouvait à — le ballon une prodigieuse hauteur au-dessus de l'immensité de l'Océan, et dans les limites de ce vaste horizon, aussi loin que pouvait s'étendre ma vue, je n'apercevais pas trace de terre. Cependant, mes sensations, quand je revins à moi, n'étaient pas aussi étran- gement douloureuses que j'aurais dû m'y attendre. En réalité, il y avait plation placide beaucoup de folie dans la contem- avec laquelle j'examinai d'abord situation. Je portai ma mes deux mains devant mes yeux, l'une après l'autre,. et me demandai avec étonnement quel accident pouvait avoir gonflé mes veines et noirci si horriblement mes ongles. Puis j'examinai soigneu- sement ma tête, je la secouai à plusieurs la tâtai je reprises, et avec une attention minutieuse, jusqu'à ce que me fusse heureusement assuré qu'elle n'était pas, ainsi que j'en avais eu l'horrible idée, plus grosse que homme qui sait où sont ses poches, je tâtai les deux poches de ma culotte, et, m'apercevant que j'avais perdu mon calepin et mon étui à cure-dent, je m'efforçai de me rendre mon ballon. Puis, compte de leur j'en ressentis avec l'habitude d'un disparition, et, ne pouvant y réussir, un inexprimable chagrin. Il me sembla alors que j'éprouvais une vive douleur à la cheville de mon pied gauche, et une obscure conscience de ma situation commença à poindre dans mon esprit. Mais — chose étrange — ! je n'éprouvai ni éton- nement ni horreur. Si je ressentis une émotion quelconque, ce fut une espèce de satisfaction ou d'épa- AVENTURE D'UN CERTAIN HANS PFAALL. '229 me faudrait noLiissement en pensant à l'adresse qu'il déployer pour me tirer de cette singulière alternative et je ne fis pas de mon ; salut définitif l'objet d'un doute d'une seconde. Pendant quelques minutes, je restai plongé dans la plus profonde méditation. Je me rappelle distinctement que j'ai souvent serré les lèvres, que j'ai appliqué mon index sur le côté de mon nez, et que j'ai pratiqué les gesticulations et grimaces ha- bituelles aux gens qui, installés tout à leur aise dans leur fauteuil, méditent sur des matières embrouillées ou importantes. Quand je crus avoir suflisamment rassemblé idées, je portai avec la plus mes grande précaution, la plus parfaite délibération, mes mains derrière mon dos, et je détachai la grosse boucle de fer qui terminait ceinture de mon pantalon. Cette boucle avait dents qui, étant un peu rouillées, tournaient la trois difficile- ment sur leur axe. Cependant, avec beaucoup de patience, je les amenai à angle droit avec le corps de la boucle et m'aperçus avec joie qu'elles restaient fermes dans cette position. Tenant entre mes dents cette espèce d'instrument, je m'appliquai à dénouer le nœud de ma cravate. Je fus obligé de avant me reposer plus d'une fois d'avoir accompli cette longue, j'y réussis. manœuvre; mais, A l'un des bouts de la à la cravate, j'assujettis la boucle, et, pour plus de sécurité, je nouai étroitement l'autre bout autour de mon poing. Sou- levant alors mon corps par un déploiement prodigieux de force musculaire, je réussis du premier coup à jeter HISTOIRES EXTRAORDINAIRES. 230 la boucle par-dessus la nacelle et à l'accrocher, comme je l'avais espéré, dans le rebord circulaire de l'osier. Mon corps faisait alors avec la paroi de la nacelle un angle de quarante-cinq degrés environ ; mais il ne faut pas entendre que je fusse à quarante-cinq degrés audessous de la perpendiculaire; bien loin de là, j'étais toujours placé dans un plan presque parallèle au niveau de l'horizon; car la nouvelle position que j'avais conquise avait eu pour effet de chasser d'autant le fond de la nacelle, et conséquemment ma position était des plus périlleuses. Mais qu'on suppose que, dans le principe, lorsque je tombai de la nacelle, je fusse tombé la face tournée vers le ballon au lieu de l'avoir tournée du côté opposé, comme elle était maintenant, — ou, en second lieu, que la corde par laquelle j'étais accroché eût pendu par hasard du rebord supérieur, au lieu de passer par une crevasse du fond, — on concevra facilement que, deux hypothèses, il m'eût été impossible d'accomplir un pareil miracle, — et les présentes ré- dans ces vélations eussent été entièrement perdues pour la postérité. J'avais donc toutes les raisons de bénir le hasard; mais, en somme, j'étais tellement stupéfié, que je me sentais incapable de rien sus- faire, et que je restai pendu, pendant un quart d'heure peut-être, dans cette extraordinaire situation, sans tenter de nouveau le plus léger effort, une béatitude perdu dans un singulier calme et dans idiote. Mais cette disposition de être s'évanouit bien vite et fit mon place à un sentiment AVENTURE D'UN CERTAIN HANS PFAALL. 231 d'horreur, d'effroi, d'absolue désespérance et de destruction. En dans vaisseaux de la tête et de les réalité, avait jusque-là créé l'acLion le sang si longtemps accumulé la gorge, et qui en moi un délire salutaire dont suppléait à Ténergie, commençait maintenant à refluer et à reprendre son niveau; et la clairvoyance qui me revenait, augmentant la perception du danger, ne servait qu'à me priver du sang-froid et du courage nécessaires pour l'affronter. Mais, par bonheur pour moi, cette faiblesse ne fut pas de longue durée. L'énergie du désespoir me revint à propos, et, avec des cris et des efforts frénétiques, je m'élançai convulsi- vement et à plusieurs reprises par une secousse générale, jusqu'à ce qu'enfin, désiré' avec des tortillai m' accrochant au bord si qu'un étau, je griffes plus serrées mon corps par-dessus et tombai la tête la pre- mière et tout pantelant dans le fond de la nacelle. Ce ne fut qu'après un certain laps de temps que je fus assez maître de moi pour m'occuper de mon ballon. Mais alors je l'examinai avec attention et découvris, à ma grande joie, qu'il n'avait subi aucune avarie. Tous mes instruments étaient sains et saufs, et, très-heu- reusement je n'avais perdu ni lest ni provisions. A la vérité, je les avais si bien assujettis à leur place qu'un pareil accident était chose tout à fait improbable. Je re- gardai à ma montre, elle marquait six heures. Je continuais à monter rapidement, et le baromètre me donnait alors une hauteur de trois milles trois quarts. Juste au-dessous de moi apparaissait dans l'Océan un petit HISTOIRES EXTRAORDINAIRES. 232 objet noir, d'une forme légèrement allongée, à peu près de la dimension d'un domino, et ressemblant for- tement, à tous égards, à l'un de ces petits joujoux. Je dirigeai mon télescope sur lui, et je vis distinctement que c'était un vaisseau anglais de quatre-vingt-quatorze canons, tanguant lourdement dans la mer, au plus le cap à l'ouest-sud-ouest. A l'excep- tion de ce navire, je ne vis rien que l'Océan et le ciel, près du vent, et et le soleil qui était levé depuis longtemps. Il est grandement temps que j'explique à Vos Excel- mon voyage. Vos Excellences se souma situation déplorable à Rotterdam lences l'objet de viennent que m'avait à la longue poussé à la résolution du suicide. Ce n'était pas cependant que j'eusse un dégoût positif de la vie elle-même, mais j'étais harassé, à n'en pouvoir plus, par les misères accidentelles de ma position. Dans cette disposition d'esprit, désirant vivre encore, et cependant fatigué de la vie, le traité que je lus à l'échoppe du bouquiniste, appuyé par l'opportune dé- couverte de mon cousin de iNantes, ouvrit une res- source à mon imagination. Je pris enfin un parti décisif. Je résolus de partir, mais de vivre, — de quitter — monde, mais de continuer mon existence ; le bref, et pour couper court aux énigmes, je résolus, sans m'inquiéter du reste, de me frayer, si je pouvais, un pas- sage jitsgu'à la lune. Maintenant, pour qu'on ne me croie pas plus fou que je ne le suis, je vais exposer en détail, et le mieux que je pourrai, les considérations qui m'induisirent à AVENTURE D'UN CERTAIN HANS PFAALL. 233 croire qu'une entreprise de cette nature, quoique difficile sans doute et pleine de dangers, n'était pas ab- solument, pour un esprit audacieux, située au delà des limites du possible. La première chose à considérer était la distance positive de la lune à la terre. ou approximative entre Or, la distance les moyenne centres de ces deux pla- nètes est de cinquante-neuf fois, plus une fraction, le rayon équatorialdela terre,ou environ 237,000 milles. moyenne ou approximative, mais il Je dis la distance est facile de concevoir que, la forme de l'orbite lunaire étant une ellipse d'une excentricité qui n'est pas de moins de 0,05k^k de son demi-grand axe, et le centre de la terre occupant le foyer de cette ellipse, si je pouvais réussir d'une manière lune à son périgée , quelconque à rencontrer la la distance ci-dessus évaluée se trou- verait sensiblement diminuée. Mais, pour laisser de côté cette hypothèse, il était positif qu'en tout cas j'avais à déduire des 237,000 milles le rayon de la terre, c'est- à-dire /t,000, et le rayon de la lune, c'est-à-dire 1,080, en tout 5,080, et qu'il ne me resterait ainsi à franchir qu'une distance approximative de 231,920 milles. Cet espace, pensais-je, n'était pas vraiment extraordinaire. On a fait nombre de fois sur cette terre des voyages d'une vitesse de 60 milles par heure, et, en réalité, il y a tout lieu de croire cité ; qu'on arrivera à une plus grande vélo- mais, même en me contentant de la vitesse dont je parlais, il ne me faudrait pas plus de cent soixante et un jours pour atteindre la surface de la lune. HISTOIRES EXTRAORDINAIRES. '234 11 y avait toutefois de nombreuses circonstances qui m'induisaient à croire que la vitesse approximative de mon voyage dépasserait de beaucoup cielle de soixante milles à l'heure; et, comme ces considérations produisirent sur moi une impression profonde, je les expli- querai plus amplement par la suite. Le second point à examiner était d'une bien autre importance. D'après les indications fournies par le ba- romètre, nous savons que, lorsqu'on s'élève, au-dessus de la surface delà terre, à une hauteur de 1,000 pieds, on laisse au-dessous de soi environ un trentième de la masse atmosphérique; qu'à 10,000 pieds, nous arrivons à peu près à un tiers; et qu'à 18,000 pieds, ce qui est presque la hauteur du Cotopaxi, nous avons dépassé la moitié de la masse fluide, ou, en tout cas, la moitié de la partie pondérable de l'air qui enveloppe notre globe. On a aussi calculé qu'à une hauteur qui n'excède pas la centième partie du diamètre terrestre, à-dire 80 milles, la vie — — c'est- la raréfaction devait être telle, animale ne pouvait en aucune façon s'y que main- tenir; et, de plus, que les moyens les plus subtils que nous ayons de constater présence de l'atmosphère la devenaient alors totalement insuffisants. Mais je ne manquai pas d'observer que ces derniers calculs étaient uniquement basés sur notre connaissance expérimentale des propriétés de l'air et des lois mécaniques qui régissent sa dilatation et sa compression dans ce qu'on peut appeler, comparativement parlant, la proximité immédiate de la terre. Et, en même temps, on regarde AVENTURE D'UN CERTAIN HANS PFAALL. 235 comme chose positive, qu'à une distance quelconque donnée, mais inaccessible, de sa surface, la vie animale est et doit être essentiellement incapable cation. de modifi- Maintenant, tout raisonnement de ce genre, et d'après de pareilles données, doit évidemment purement analogique. La plus grande l'homme être hauteur où jamais parvenu est de 25,000 pieds; je soit parle de l'expédition aéronautique de MM. Gay-Lussac et Biot. une hauteur assez C'est même médiocre, quand on la compare aux 80 milles en question; ne pouvais m'empêcher de penser que laissait une place la et je question au doute et une grande latitude aux conjectures. Mais, en fait, en supposant une ascension opérée à une hauteur donnée quelconque, la quantité d'air pondérable traversée dans toute période ultérieure de l'ascension n'est nullement en proportion avec la hau- teur additionnelle acquise, comme on peut le voir d'après ce qui a été énoncé précédemment, mais dans une raison constamment décroissante. Il est donc évident que, nous élevant aussi haut que possible, nous ne pouvons pas, littéralement parlant, arriver à une limite au delà de laquelle l'atmosphère cesse absolu- ment d'exister. Elle doit exister, concluais-je, qu'elle puisse, est vrai, exister à il quoi- un état de raré- faction infinie. D'un autre côté, je savais manquent pas pour prouver que les qu'il existe arguments ne une limite réelle et déterminée de l'atmosphère, au delà de la- HISTOIRES EXTRAORDINAIRES. 23G quelle il n'y a absolument plus d'air respirable. Mais une circonstance a été omise par ceux qui opinent pour non pas une réfutation pé- cette limite, qui semblait, remptoire de leur doctrine, mais un point digne d'une sérieuse investigation. les Comparons les intervalles entre retours successifs de la comète d'Encke à son pé- rihélie, en tenant compte de toutes les perturbations dues à l'attraction planétaire, et nous verrons que les périodes diminuent graduellement, c'est-à-dire que le grand axe de l'ellipse de la comète va toujours se raccourcissant dans une proportion lente, 'mais parfai- tement régulière. Or, c'est précisément le cas qui doit avoir lieu, si nous supposons que la comète subisse une résistance par le fait d'im milieu éthèrè excessivement rare qui pénètre les régions de son orbite. Car il est évident qu'un pareil milieu doit, en retardant la vitesse de la comète, accroître sa force centripète sa force centrifuge. soleil En d'autres termes, et affaiblir l'attraction du deviendrait de plus en plus puissante, et la co- mète s'en rapprocherait davantage à chaque révolution. Véritablement, il n'y a pas d'autre moyen de se rendre compte de la variation en question. Mais voici un autre fait : on observe que le diamètre réel de la partie nébuleuse de cette même comète se contracte rapidement à mesure qu'elle approche du soleil, et se dilate avec la même rapidité quand elle repart vers son aphélie. N'avais-je pas quelque raison de supposer avec M. Valz que cette apparente condensation de volume prenait son origine dans la com- AVENTURE D'UN CERTAIN HANS PFAALL. pression de ce milieu 237 éthéré dont je parlais tout à riieure, et dont la densité est en proportion de la proxi- mité du soleil? Le phénomène qui affecte la forme lenticulaire et qu'on appelle la lumière zodiacale était aussi un point digne sible sous les tropiques, et prendre d'attention. Cette lumière si vi- pour une lumière qu'il est impossible météorique de quelconque, s'élève obliquement de l'horizon et suit généralement la ligne de l'équateur du soleil. Elle me semblait évi- demment provenir d'une atmosphère rare qui s'étendrait depuis le soleil jusque par delà l'orbite de Vénus au moins, et même, selon moi, indéfiniment plus loin. Je ne pouvais pas supposer que ce milieu fût limité par la hgne du parcours de la comète, ou fût confiné dans le Voisinage immédiat du soleil. Il était si simple d'imaginer au contraire qu'il envahissait toutes les régions de notre système planétaire, condensé autour des planètes en ce que nous appelons atmosphère, et peutêtre modifié chez quelques-unes par des circonstances purement géologiques, c'est-à-dire modifié ou varié dans ses proportions ou dans sa nature essentielle par les matières volatilisées émanant de leurs globes respectifs. Ayant pris la question sous ce point de vue, je n'avais plus guère à hésiter. En supposant que dans mon pas- sage je trouvasse une atmosphère essentiellement semblable à celle qui enveloppe la surface de la terre, je réfléchis qu'au moyen du très-ingénieux appareil de M. Grimm je pourrais facilement la condenser en suffisante quantité pour les besoins de la respiration. 'm HISTOIRES EXTRAORDINAIRES. Voilà qui écartait le principal obstacle à un voyage à donc dépensé quelque argent et beau- la lune. J'avais coup de peine pour adapter l'appareil au but que je me proposais, et j'avais pleine confiance dans son appli- pourvu que je pusse accomplir le voyage dans cation, un espace de temps suffisamment court. Ceci me ra- mène à la question de la vitesse possible. Tout le monde sait que les ballons, -dans la première période de leur ascension, s'élèvent avec une vélocité modérée. Or, comparativement consiste uniquement dans la biant relativement au gaz du ballon vue, il ne paraît pas du tout probable blable que le ballon, à mesure d'ascension force la pesanteur de f air am- qu'il ; et, à première ni vraisem- gagne en éléva- tion et arrive successivement dans des couches atmo- sphériques d'une densité décroissante, puisse gagner en vitesse autre côté, D'un et accélérer sa vélocité je n'avais pas souvenir que, dans primitive. un compte rendu quelconque d'une expérience antérieure, l'on eût jamais constaté une diminution apparente dans la vitesse absolue de l'ascension, quoique tel eût pu être le cas, en raison de la fuite du gaz à travers un aérostat mal confectionné et généralement revêtu d'un vernis insuffisant, ou pour toute autre cause. Il me semblait donc que l'effet de cette déperdition pouvait seulement contre-balancer l'accélération acquise par le ballon à mesure qu'il s'éloignait tation. Or, je considérai que, versée je trouvasse le du centre de gravi- pourvu que dans ma tra- milieu que j'avais imaginé, et AVENTURE D'UN CERTAIN HANS PFAALL. pourvu qu'il fût 23:» de même essence que ce que nous appelons l'air atmosphérique, il importait relativement assez peu que je le trouvasse à tel ou tel degré de ra- relativement à ma force ascen- réfaction, c'est-à-dire sionnelle; car non-seulement le gaz du ballon serait soumis à la même raréfaction (et, dans cette occurrence, je n'avais qu'à lâcher une quantité proportionnelle de gaz, suffisante pour prévenir une explosion), mais, par la nature de ses parties intégrantes, il devait, en tout cas, être toujours spécifiquement plus composé quelconque de pur azote avait donc une chance, forte probabilité, — et et léger qu'un d'oxygène. Il y même, en somme, une pour qu'a aucune 'période de mon ascension'je n'arrivasse à un point oii les différentes pesanteurs réunies de mon immense ballon, du gaz in- concevablement rare qu'il renfermait, de la nacelle et de son contenu, pussent égaler la pesanteur de la masse d'atmosphère arnbiante déplacée; et Ton conçoit facile- ment que c'était là l'unique condition qui pût arrêter ma fuite ascensionnelle. Mais encore, si jamais j'atteignais ce point imaginaire, il me restait la faculté d'user de mon lest et d'autres poids montant à peu près à un total de 300 livres. En même temps, la force centripète devait toujours décroître en raison du carré des distances, et ainsi je devais, avec une vélocité prodigieusement accélérée, arri- ver à la longue dans ces lointaines régions où la force d'attraction de la lune serait substituée à celle de la terre. HISTOIRES EXTRAORDINAIRES. 2iU Il y avait une autre difficulté qui ne laissait pas de me causer quelque inquiétude. On a observé que dans les ascensions poussées à une hauteur considérable, outre la gêne de la respiration, on éprouvait dans la tête et dans tout le corps un immense malaise, souvent accompagné de saignements de nez et d'autres symptômes passablement alarmants, et qui devenait de plus en plus insupportable à mesure qu'on s'élevait ^ C'était là une considération passablement effrayante. N'était-il pas probable que ces symptômes augmenteraient jusqu'à ce qu'ils se terminassent par la mort elle-même? Après mûre réflexion, je conclus que non. 11 fallait en chercher l'origine dans la pression la surface disparition la progressive de atmosphérique, à laquelle est accoutumée de notre corps, et danâ vitable des vaisseaux sanguins la distension iné- superficiels, — et non dans une désorganisation positive du système animal, comme dans le cas de difficulté de respiration, où la densité atmosphérique est pour la rénovation chimiquement insuffisante régulière du sang dans un ven- tricule du cœur. Excepté dans le cas oii cette rénovation ferait défaut, je ne voyais vie ne se maintînt pas, pas de raison pour que même dans le vide ; la car l'ex- pansion et la compression de la poitrine, qu'on appelle 1. Depuis la première publication de Hans Pfaal, j'apprends que M. Green, le célèbre aéronaute du ballon le Nassau, et d'autres expérimentateurs contestent à cet égard les assertions de M. de Humboldt, et parlent au contraire d'una incommodité toujours dé- croissante, ce qui s'accorde précisément avec la théorie présentée ici. — E. A. P. AVENTURE D'ON CERTAIN HANS PFAALL. communément respiration, est une action purement musculaire; elle est la cause et non piration. l'effet En un mot, je concevais que, bituant à l'absence de pression sensations douloureuses 241 le de la res- corps s'ha- atmosphérique, ces devaient diminuer graduel- lement; et, pour les supporter tant qu'elles dureraient, j'avais toute confiance dans la solidité de fer de ma constitution. J'ai donc exposé quelques-unes des considérations — non pas toutes certainement — qui m'induisirent à former le projet d'un voyage à la lune. Je vais main- tenant, s'il plaît à Vos Excellences, vous exposer le résultat d'une tentative dont la conception paraît si au- dacieuse, et qui, dans tous les cas, n'a pas sa pareille dans les annales de l'humanité. Ayant atteint la hauteur dont il a été parlé ci-dessus, c'est-à-dire trois mille trois quarts, je jetai hors de la nacelle une quantité de plumes, et je vis que je montais toujours avec une rapidité suffisante; il n'y avait donc pas nécessité de jeter du lest. J'en fus très-aise, car je désirais garder avec moi autant de lest que j'en pourrais porter, par la raison bien simple que je n'avais aucune donnée positive sur la puissance d'attraction et sur la densité atmosphérique. Je ne souffrais jus- qu'à présent d'aucun malaise physique, je respirais avec une parfaite liberté et n'éprouvais aucune douleur dans la tête. La chatte était couchée fort solennellement sur mon habit que j'avais ôté, et regardait les pigeons avec un air de nonchaloir. Ces derniers, que j'avais at14 rtlSTOIRKS EXTRAORDINAIRES. 242 tachés par la patte, pour les empêcher de s'envoler, grains de étaient fort occupés à piquer quelques riz éparpillés pour eux au fond de la nacelle. A six heures vingt minutes, le baromètre donnait une élévation de 26,^00 pieds, ou cinq milles, à une fraction près. La perspective semblait sans bornes. Rien de plus facile d'ailleurs que de calculer à l'aide de la trigonométrie sphérique l'étendue de surface terrestre qu'embrassait mon regard. La surface convexe d'un segment de sphère est à la surface entière de la sphère comme le sinus verse du segment est au diamètre de la sphère. Or, dans mon cas, le sinus verse — c'està-dire l'épaisseur du — était à segment situé au-dessous de moi peu près égal à mon élévation, ou à l'éléva- tion du point de vue au-dessus de la surface. La pro- portion de cinq milles à huit milles exprimerait donc l'étendue de la surface que j'embrassais, c'est-à-dire que j'apercevais la seize centième partie de la surface totale du globe. La mer apparaissait polie comme un bien qu'à l'aide du télescope je découvrisse miroir, qu'elle était dans un état de violente agitation. Le navire n'était plus visible, il avait sans doute dérivé vers l'est. Je commençai dès lors à ressentir par intervalles une forte douleur à la tête, bien que je conti- nuasse à respirer à peu près librement. La chatte et les pigeons semblaient n'éprouver aucune incom- modité. A sept heures moins vingt, le ballon entra dans la région d'un grand et épais nuage qui me causa beau- AVENTURE D'UN CERTAIN HANS PFAALL. coup d'ennui ; mon appareil condensateur en 243 fut en- dommagé, et je fus trempé jusqu'aux os. C'est, à coup sûr, une singulière rencontre, car je n'aurais pas sup- posé qu'un nuage de cette nature pût se soutenir à une si grande élévation. Je pensai faire pour le mieux en jetant deux morceaux de lest de cinq livres chaque, me laissait encore cent-soixante-cinq livres de ce qui lest. Grâce à cette opération, je traversai bien vite l'obstacle, et je m'aperçus immédiatement que j'avais gagné prodigieusement en vitesse. Quelques secondes après que j'eus quitté le nuage, un éclair éblouissant le traversa d'un bout à l'autre et l'incendia dans toute son étendue, lui donnant l'aspect masse de d'une charbon en ignition. Q^i'on se rappelle que ceci se passait en plein jour. Aucune pensée ne pourrait rendre la sublimité d'un pareil phénomène se déployant dans les ténèbres de la nuit. L'enfer son image exacte. Tel que me fit dresser les lui-même aurait trouvé je le vis, ce spectacle cheveux. Cependant, au loin mon regard dans les abîmes béants mon je ; dardais je laissais imagination plonger et se promener sous d'é- tranges et immenses voûtes, dans des gouffres em- pourprés, dans les abîmes rouges et sinistres d'un feu effrayant et insondable. Je l'avais échappé belle. Si le ballon était resté une minute de plus dans le nuage, — c'est-à-dire si l'incommodité dont je souffrais ne m'avait pas déterminé à jeter du lest, — ma destruction pouvait en être et en eût très-probablement été la con- séquence. De pareils dangers, quoiqu'on y fasse peu HISTOIRES EXTRAORDINAIRES, 244 d'attention, sont les plus grands peut-être qu'on puisse courir en ballon. J'avais pendant ce temps atteint une hauteur assez grande pour n'avoir aucune inquiétude à ce sujet. Je m'élevais alors très-rapidement, et à sept heures le baromètre donnait une hauteur qui moindre de neuf milles éprouver une grande aussi et demi. difficulté Je n'était pas commençais à de respiration. Ma tête me faisait excessivement souffrir; et, ayant senti depuis quelque temps de l'humidité sur mes joues, je découvris à la fin que c'était du sang qui suintait continuellement du tympan de mes oreilles. Mes yeux me donnaient aussi beaucoup d'inquiétude. En passant ma main dessus, il me sembla qu'ils étaient poussés hors de leurs orbites, et à un degré assez considérable ; et tous les objets contenus dans la nacelle et le ballon lui-même se présentaient à ma vision sous une forme monstrueuse et faussée. Ces symptômes dépassaient ceux auxquels je m'attendais, et me causaient quelque alarme. Dans cette conjoncture, très-imprudemment et sans réflexion, je jetai hors de la nacelle trois morceaux de lest de cinq livres chaque. La vitesse dès lors accélérée de mon ascension m'emporta, trop raj>idement ej, sans gradation suffisante, dans une couche d'atmo- sphère singulièrement raréfiée, ce qui faillit amener un résultat fatal pour mon expédition et pour moi-même. Je fus soudainement pris par de cinq minutes, et, un spasme qui dura plus même quand il eut en partie cessé, il se trouva que je ne pouvais plus aspirer qu'à AVENTURE D'UN CERTAIN HANS PFAALL. (le 245 longs inlervalles et d'une manière conviilsive, sai- gnant copieusement pendant tout ce temps par le nez, par les oreilles, et même légèrement par les yeux. Les pigeons semblaient en proie à une excessive angoisse et à pendant que la chancelant çà et là se débattaient pour s'échapper, chatte miaulait lamentablement, travers la nacelle comme sous l'influence d'un poison. Je découvris alors trop tard l'immense imprudence que j'avais commise en jetant du lest, et mon trouble devint extrême. Je n'attendais pas moins que la mort, et la mort dans quelques minutes. La souffrance phy- sique que j'éprouvais contribuait aussi à me rendre presque incapable d'un effort quelconque pour sauver ma vie. 11 me restait à peine la faculté de réfléchir, et violence de mon mal de tête semblait augmenter la de minute en minute. Je m'aperçus alors que mes sens allaient bientôt m'abandonner tout à fait, et j'avais déjà empoigné une des cordes de la soupape, quand le souvenir du mauvais tour que j'avais joué aux trois créanciers, et la crainte des conséquences qui pouvaient m'accueillir à mon retour, m'effrayèrent et m'arrêtèrent pour le moment. Je me couchai au fond de la nacelle et m'efforçai de rassembler mes facultés. J'y réussis un peu, et je résolus de tenter l'expérience d'une saignée. Mais, comme je n'avais pas de lancette, je fus obligé de procéder à cette opération tant bien que mal, et finalement j'y réussis en m'ouvrant une veine au bras li. HISTOIRES EXTRAORDINAIRES. 240 mon canif. Le sang avait à gauche avec la lame de peine commencé à couler, que j'éprouvais un soulage- ment notable, et, lorsque j'en eus perdu à peu près la valeur d'une demi-cuvette de dimension ordinaire, les plus dangereux symptômes avaient pour la plupart entièrement disparu. Cependant, je ne jugeai pas prudent d'essayer de me remettre immédiatement sur mes pieds; mais, ayant bandé mon bras du mieux que je pus, je restai immobile pendant un quart d'heure environ. Au bout de ce temps, je me levai et me sentis plus libre, plus dégagé de toute espèce de malaise que je ne l'avais été depuis une heure un quart. Cependant fort la de respiration n'avait que difficulté peu diminué, et je pensai qu'il y aurait bientôt nécessité urgente à faire usage du condensateur. En même temps, je jetai les yeux sur ma chatte qui s'était commodément réinstallée sur mon habit, et, à ma grande suiprise, je découvris qu'elle avait jugé à propos, pendant mon indisposition, de mettre au jour une ventrée de cinq petits chats. Certes, je ne m'attendais pas le moins du monde à ce supplément de passagers, somme, mais, en l'aventure fournissait l'occasion plus qu'aucune autre, de me fit plaisir. vérifier Elle me une conjecture qui, m'avait décidé à tenter cette ascension. J'avais imaginé que Vliabllade de la pression atmo- sphérique à la surface de la terre était en grande partie la cause des douleiu'S qui attaquaient la vie ani- male à une certaine distance au-dessus de cette sur- AVENTURE D'UN CERTAIN HANS PFAALL. 247 face. Si les petits chats éprouvaient du malaise au même ma théorie comme fausse, mais je pouvais regarder le cas contraire comme une excellente confirmation de mon degré que leur mère, je devais considérer idée. A huit heures, j'avais atteint une élévation de' dixsept milles. Ainsi ascensionnelle il me parut évident que ma vitesse non-seulement augmentait, mais que cette augmentation eût été légèrement sensible, même dans le cas où je n'aurais pas jeté de lest, comme je l'avais fait. Les douleurs de tête et d'oreilles revenaient par intervalles avec violence, j'étais repris par et, de temps à autre, mes saignements de nez; mais, en somme, je. souffrais beaucoup moins que je ne m'y étais attendu. Cependant, de minute en minute, ma respiration devenait plus difficile, et chaque inhalation était suivie d'un mouvement spasmodique de la poitrine, des plus fatigants. Je déployai alors l'appareil condensateur, de manière à le faire fonctionner immédia- tement. L'aspect de la terre, à cette période de mon ascension, était vraiment magnifique. A sud, aussi loin que pénétrait l'ouest, au nord et au mon regard, s'étendait une nappe illimitée de mer en apparence immobile, qui, de seconde en seconde, prenait une teinte bleue plus profonde. A une vaste distance vers l'est, s'allon- geaient très-distinctement les îles Britanniques, les côtes occidentales de la France et de l'Espagne, ainsi qu'une petite portion de la partie nord du continent HISTOIRES EXTRAORDINAIRES. 248 africain. 11 était impossible de découvrir une trace des édifices particuliers, et les plus orgueilleuses cités de l'humanité avaient absolument disparu de la surface de la terre. Ce qui m'étonna particulièrement dans l'aspect des choses situées au-dessous de moi, ce fut la concavité apparente de la surface du globe. Je m'attendais, assez sottement, à voir sa convexité réelle se manifester plus distinctement à proportion que je m'élèverais; mais quelques secondes de réflexion me suffirent pour expli- quer cette contradiction. Une ligne abaissée perpendiculairement sur la terre du point où je me trouvais aurait formé la perpendiculaire d'un triangle rectangle dont la base se serait étendue de l'angle droit à l'horizon, et l'hypoténuse de l'horizon au point occupé par mon ballon. Mais l'élévation où j'étais placé n'était rien ou presque rien comparativement à l'étendue em- brassée par mon regard; en d'autres termes, la base et l'hypoténuse du triangle supposé étaient si longues, comparées à la perpendiculaire, qu'elles pouvaient être considérées comme deux lignes presque parallèles. De cette façon l'horizon de l'aéronaute lui apparaît tou- jours au niveau de sa nacelle. Mais, situé est, il comme le poinj: immédiatement au-dessous de lui, lui paraît et en effet, à une immense distance, naturellement lui paraît aussi à une immense distance au-dessous de Thorizon. De là l'impression de concavité; et cette impression durera jusqu'à ce que l'élévation se trouve relativement à l'étendue de la perspective dans une AVENTURE D'UN CERTAIN HANS PFAALL. 249 proportion telle, que le parallélisme apparent de la base et de rhypoténuse disparaisse. Cependant, comme les pigeons semblaient souffrir horriblement, je résolus de leur donner la liberté. Je déliai d'abord l'un d'eux, un superbe pigeon gris sau- moné, et le plaçai sur le bord de la nacelle. 11 semblait excessivement mal à son aise, regardait anxieusement autour de lui, battait des ailes, faisait entendre un roucoulement très-accentué, mais ne pouvait pas se décider à s'élancer hors de la nacelle. A la fin, je le pris et le jetai à six yards environ du ballon. Cepen- dant, bien loin de descendre, comme je m'y attendais, il fit des efforts véhéments pour rejoindre le ballon, poussant en même temps des cris très-aigus et trèsperçants. Enfin, il réussit à rattraper sa première position sur le bord du panier; mais à peine s'y était-il posé qu'il pencha sa tête sur sa gorge et tomba mort au fond de la nacelle. L'autre n'eut pas un sort aussi déplorable. Pour l'empêcher de suivre l'exemple de son camarade et d'effectuer un retour vers le ballon, je le précipitai vers la terre de toute plaisir qu'il continuait à vélocité, ma force, et vis avec descendre avec une grande faisant usage de ses ailes très-facilement et d'une manière parfaitement naturelle. En très-peu de temps, il fut hors de vue, et je ne doute pas qu'il ne soit arrivé à bon port. Quand à la minette, qui semblait en grande partie remise de sa crise, elle se faisait maintenant un joyeux régal de l'oiseau mort, et finit par s'endormir avec toutes les apparences du conten- HISTOIRES EXTRAORDINAIRES. t>50 tement. Les petits chats étaient parfaitement vivants et ne manifestaient pas le plus léger symptôme de malaise. A huit heures un quart, ne pouvant pas respirer plus longtemps sans une douleur intolérable, je com- mençai immédiatement à ajuster autour de la nacelle l'appareil attenant au condensateur. Cet appareil de- mande quelques explications, et Vos excellences voudront bien se rappeler que mon but, en premier lieu, était de m'enfermer entièrement, moi et ma nacelle, et de me barricader contre l'atmosphère singulièrement raréfiée au sein de laquelle j'existais, et enfin d'intro- duire à l'intérieur, à l'aide de mon condensateur, une quantité de celte même atmosphère suffisamment con- densée pour les besoins de la respiration. Dans ce but, j'avais préparé un vaste sac de caout- chouc très-flexible, très-solide, absolument imper- méable. La nacelle tout entière se trouvait en quelque sorte placée dans ce sac dont les dimensions avaient été calculées pour cet objet, c'est-à-dire qu'il passait sous le fond de la nacelle, s'étendait sur ses bords, et mon- tait extérieurement le long des cordes jusqu'au cerceau où le filet était attaché. Ayant ainsi déployé le sac et fait hermétiquement fallait la clôture de tous les côtés, il maintenant assujettir le haut ou l'ouverture du sac en faisant passer le tissu de caoutchouc au-dessus du cerceau, en d'autres termes, entre le filet et le cerceau. Mais, si je détachais le filet du cerceau pour opérer ce passage, comment la nacelle pourrait-elle se AVENTURE D'UN CERTAIN HANS PFAALL. 251 soutenir? Or, le filet n'était pas ajusté au cerceau d'une manière permanente, mais attaché par une série de brides mobiles ou de nœuds coulants. Je ne défis donc qu'un petit nombre de ces brides à la fois, laissant la nacelle suspendue par les autres. Ayant fait passer ce que je pus de la partie supérieure du sac, je rattachai les brides, — non pas au cerceau, car l'interposition de l'enveloppe de caoutchouc rendait cela impossible, — mais à une série de gros boutons fixés à Tenveloppe elle-même, à trois pieds environ au-dessous de l'ouverture du sac, les intervalles des boutons corres- pondants aux intervalles des brides. Gela fait, je détachai du cerceau quelques autres brides, j'introduisis une nouvelle portion de l'enveloppe, et les brides dénouées furent à leur tour assujetties à leurs boutons respectifs. Par ce procédé, je pouvais faire passer toute la partie supérieure du sac entre le filet et le cerceau. Il est évident que le cerceau devait dès lors tomber dans la nacelle, tout contenu le poids de la nacelle et de son n'étant plus supporté que par la force des boutons. A première vue, ce système pouvait ne pas offrir une garantie sufiîsante; mais il n'y avait aucune raison de s'en défier, car non-seulement les boutons étaient solides par eux-mêmes, mais, de plus, ils étaient si rapprochés, que chacun ne supportait en réalité qu'une très-légère partie du poids total. La nacelle et son contenu auraient pesé trois fois plus, que je n'en aurais pas été inquiet le moins du monde. Je relevai alors le cerceau le long de l'enveloppe de caoutchouc HISTOIRES EXTRAORDINAIRES. 252 et je l'étayai sur trois perches légères préparées pour cet objet. Cela avait pour but de tenir le sac convena- blement distendu par le haut, et de maintenir la partie inférieure du filet dans la position voulue. Tout ce qui me restait à faire maintenant était de nouer l'ouverture du sac, — ce que j'opérai facilement en rassemblant plis du caoutchouc, et en les tordant étroitement les ensemble au moyen d'une espèce de tourniquet à demeure. Sur les côtés de l'enveloppe ainsi déployée autour de la nacelle, j'avais fait adapter trois carreaux de verre ronds, très-épais, mais très-clairs, au travers desquels je pouvais voir facilement autour de moi dans toutes les directions formait le horizontales. Dans la partie du sac qui fond était une quatrième fenêtre analogue, correspondant à une petite ouverture pratiquée dans le fond de la nacelle elle-même. Celle-ci me permettait de regarder perpendiculairement au-dessous de moi. Mais il m'avait été impossible d'ajuster une invention du même genre au-dessus de ma tête, en raison de la manière particulière dont j'étais obligé de fermer l'ouverture et des plis nombreux qui en résultaient; j'avais donc renoncé à voir les objets situés dans mon zénith. Mais c'était là une chose de peu d'importance; car, lors même que j'aurais pu placer une fenêtre au-dessus de moi, le ballon aurait fait obstacle à ma vue et m'aurait empêché d'en faire usage. A un pied environ au-dessous d'une des fenêtres latérales était une ouverture circulaire de trois pouces de AVENTURE D'UN GL^RrAIN IIANS PFAALL. t>53 diamùtre, avec un rebord do cuivre façonné intérieu- rement pour s'adapter à la spirale d'une vis. Dans ce rebord sévissait le large tube du condensateur, le corps de la macbine était naturellement placé dans la chambre de caoutchouc. En faisant le vide dans le corps de la machine, on attirait dans ce tube une masse d'at- mosphère ambiante raréfiée, qui de là était déversée à l'état la condensé et mêlé à l'air subtil déjà contenu dans chambre. Cette opération, répétée plusieurs fois, remplissait à la longue la chambre d'une atmosphère suffisant aux besoins de la respiration. Mais, dans un espace aussi étroit que celui-ci, elle devait nécessai- rement, au bout d'un temps très-court, se vicier et devenir impropre à la vie par son contact répété avec les poumons. Elle était alors rejetée par une petite sou- pape placée au fond de la nacelle, l'air dense se pré- cipitant promptemcnt dans l'atmosphère raréfiée. éviter à un certain total Pour moment l'inconvénient d'un vide dans la chambre, cette purification ne devait jamais être effectuée en une seule fois, mais graduellement, la soupape n'étant ouverte que pour quelques secondes, puis refermée, jusqu'à ce qu'un ou deux coups de pompe du condensateur eussent fourni de quoi remplacer l'atmosphère expulsée. Par amour des expériences, j'avais placé la chatte et ses petits chats dans un petit panier, et les avais suspendus en dehors de la nacelle par un bouton placé près du fond, tout auprès de la soupape, à travers laquelle leur faire passer de la nourriture je pouvais quand besoin était. HISTOIRES EXTRAORDINAIRES. 25i J'accomplis cette manœuvre avant de fermer Fou- verture de la chambre, et non sans quelque difficulté, car il me fallut, pour atteindre le dessous de la nacelle, me servir d'une des perches dont j'ai parlé, à laquelle était fixé un crochet. Aussitôt pénétré dans la chambre, devinrent inutiles ; le que l'air condensé eut cerceau et les perches l'expansion de l'atmosphère incluse distendit puissamment le caoutchouc. Quand j'eus fini tous ces arrangements et rempli la chambre d'air condensé, dix. il était neuf heures moins Pendant tout le temps qu'avaient duré ces opén^i- tions, j'avais horriblement souffert de la difficulté de respiration, et je me repentais amèrement de la nég]i- gence ou plutôt de l'incroyable imprudence dont je nf étais rendu coupable en remettant au dernier mo- ment une affaire d'une si haute importance. xMais enfin, lorsque j'eus fini, je commençai cueillir, etpromptement, les bénéfices de à re- mon invention. Je respirai de nouveau avec une aisance et une liberté parfaites; et vraiment, pourquoi n'en eùt-il pas éïé ainsi? Je fus aussi très-agréablement surpris de me trouver en grande partie soulagé des vives douleurs qui m'avaient affligé jusqu'alors. Un léger mal de tête accompagné d'une sensation de plénitude ou de distension dans les poignets, les chevilles et la gorge, était à peu près tout ce dont j'avais à me plaindre main- tenant. Ainsi, il était positif qu'une grande partie du malaise provenant de la disparition de la pression at- mosphérique s'était absolument évanouie, et que près- AVENTURL: D'UN CI'IITAIIS ILVNS PFAALL. 255 que toutes les douleurs que j'avais endurées pendant les deux dernières heures devaient être attribuées uni- quement aux effets d'une respiration insuffisante. A neuf heures moins vingt, — c'est-à-dire peu de temps après avoir fermé l'ouverture de ma chambre, — mercure avait atteint son extrême limite et était le retombé dans la cuvette du baromètre, qui, comme je dit, l'ai d'une vaste dimension. me donnait Il une hauteur de 132,000 pieds ou de 25 milles, alors et était conséquemment mon regard en ce moment n'em- brassait pas totale moins de la 32 0« partie de la superficie de la terre. A neuf heures, j'avais de nouveau perdu de vue la terre dans l'est, mais pas avant de ballon dérivait rapidement vers m'être aperçu que le le nord-nord-ouest. L'Océan, au-dessous de moi, gardait toujours son apparence de était concavité ; mais ma vue souvent interceptée par des masses de nuées qui flottaient çà et là. A neuf heures et demie, je recommençai l'expérience des plumes, j'en jetai une poignée à travers la soupape. Elles ne voltigèrent pas, comme je m'y attendais, mais tom])èrent perpendiculairement, eu masse, comme un boulet, et avec une telle vélocité, que je les perdis de vue en quelques secondes. Je ne savais d'abord que penser de cet extraordinaire phénomène ; je ne pou- vais croire que ma vitesse ascensionnelle se fût si sou- dainement prodigieusement accélérée. Mais je que l'atmosphère était maintenant trop raréfiée pour soutenir môme des plumes — qu'elles réfléchis et si bientôt HISTOIRES EXTRAORDINAIRES. 256 tombaient réellement, ainsi avec une excessive rapidité, qu'il m'avait semblé, — et que j'avais été sim- plement surpris par les vitesses combinées de leur chute et de mon ascension. A dix heures, il se trouva que je n'avais plus grand chose à faire et que rien no réclamait mon attention immédiate. Mes affaires allaient donc comme sur des persuadé que roulettes, et j'étais avec une vitesse le ballon montait incessamment croissante, quoique je n'eusse plus aucun moyen d'apprécier cette progression de vitesse. Je n'éprouvais de peine ni de malaise d'aucune espèce; je jouissais môme d'un bien-êtrequeje n'avais pas encore connu depuis mon départ de Hol- terdam. Je m'occupais tantôt à vérifier l'état de tous mes instruments, tantôt à renouveler l'atmosphère de la chambre. Quant à ce dernier point, je résolus de m'en occuper à des intervalles réguliers de quarante minutes, plutôt pour garantir complètement ma santé que par une absolue nécessité. Cependant, je !îe pou- vais pas m'empêcher de faire des rêves et des conjectures. Ma pensée s'ébattait dans les étranges et chirégions de la- lune. Mon imagination, se mériques sentant une bonne fois délivrée de toute entrave, errait à son gré parmi les merveilles multiformes d'une pla- nète ténébreuse et changeante. forêts et Tantôt c'étaient des chenues et vénérables, des précipices rocailleux des cascades retentissantes s' écroulant dans des gouffres sans fond. Tantôt j'arrivais tout à coup dans de calmes solitudes inondées d'un soleil de midi, où AVENTURE D'UN CERTAIN HANS PFAALL. ne s'introduisait jamais aucun vent du ciel, et 257 où s'étalaient à perte de vue de vastes prairies de pavots et de longues fleurs élancées semblables à des lis, toutes silencieuses et immobiles pour l'éternité. Puis je voyageais longtemps, longtemps, et je pénétrais dans une contrée qui n'était tout entière qu'un lac ténébreux et vague, avec une frontière de nuages. Mais ces images n'étaient pas les seules qui prissent possession de mon cerveau. Parfois des horreurs d'une nature plus noire, plus effrayante, s'introduisaient dans mon esprit, et ébranlaient les dernières profondeurs de mon âme par la simple hypothèse de leur possibilité. Cependant, je ne pouvais permettre à ma pensée de s'appesantir trop longtemps sur ces dernières contemplations; je pensais judicieusement que les dangers réels et palpables de mon voyage suffisaient largement pour absorber toute mon attention. A cinq heures de l'après-midi, comme j'étais occupé à renouveler l'atmosphère de la chambre, je pris cette occasion pour observer la chatte et ses petits à travers la soupnpe. La chatte semblait de nouveau souffrir beaucoup, et je ne doutai pas qu'il ne fallût attribuer particulièrement son malaise à la difficulté de respirer; mais mon expérience relativement aux petits avait eu un résultat des plus étranges. Naturellement je m'at- tendais à les voir manifester une sensation de peine, quoique à un degré moindre que leur mère, et cela eut été suffisant pour conQrmer . mon opinion touchant l'habitude de la pression atmosphérique. Mais je n'es- HISTOIRES EXTRA OP.DINAIP.ES. 258 pérais pas les trouver, après un examen scrupuleux, jouissant d'une parfaite santé et ne laissant pas voir le me rendre ma théorie, et en plus léger signe de malaise. Je ne pouvais compte de cela qu'en élargissant supposant que l'atmosphère ambiante hautement raréfiée pouvait bien, que contrairement à l'opinion j'avais d'abord adoptée comme positive, n'être pas chi- miquement insuffisante pour les fonctions vitales, et qu'une personne née dans un pareil milieu pourrait peut-être ne s'apercevoir respiration, tandis que, denses avoisinant la d'aucune incommodité de ramenée vers les couches plus terre, elle souffrirait vraisem- blablement des douleurs analogues à celles que j'avais endurées tout à l'heure. C'a été pour moi, depuis lors, l'occasion d'un profond regret, qu'un accident mal- heureux m'ait privé de ma petite famille de chats et m'ait enlevé le moyen d'approfondir cette question par une expérience continue. En passant m.a main à travers la soupape avec une tasse pleine d'eau pour la vieille minette, la manche de ma chemise s'accrocha à la boucle qui supportait le panier, et du coup la détacha du bouton. Quand même tout le panier se fût abso- lument évaporé dans l'air, il n'aurait pas été escamoté à ma vue d'une manière plus abrupte et plus instan- tanée. Positivement, il ne s'écoula pas la dixième partie d'une seconde entre le moment où le panier se décro- cha et celui où il disparut complètement avec tout ce qu'il contenait. Mes souhaits les plus heureux l'accom- pagnèrent vers la terre, mais, naturellement, je n'es- AVENTURE D'UN CERTAIN HANS PFAALL. pérai guère que la chatte ou ses petits 2o9 survécussent pour raconter leur odyssée. A six heures, je m'aperçus qu'une grande partie de la surface visible de la terre, vers Test, était plongée dans une ombre épaisse, qui s'avançait incessamment avec une grande rapidité; enfin, à sept heures moins cinq, toute la surface visible fut enveloppée dans les ténèbres de la nuit. Ce ne^fut toutefois que quelques instants plus tard que les rayons du soleil couchant cessèrent d'illuminer le ballon; et, cette circonstance, à laquelle je m'attendais parfaitement, ne manqua pas de me causer un immense plaisir, il était évident qu'au matin je contemplerais le corps lumineux à son lever plusieurs heures au moins avant les citoyens de Rotterdam, bien qu'ils fussent situés loin que moi dans l'est, et qu'ainsi, beaucoup plus de jour en jour, à mesure que je serais placé plus haut dans l'atmosphère, je jouirais de la lumière solaire pendant une période de plus en plus longue. Je résolus alors do rédiger un journal de mon voyage en comptant les jours de vingt- quatre heures consécutives, sans avoir égard aux intervalles de ténèbres. A dix heures, sentant venir le sommeil, je résolus de me coucher pour le reste de la nuit; mais ici se présenta une difficulté qui, quoique de nature à sauter aux yeux, avait échappé à mon attention jusqu'au dernier moment. Si je me mettais à dormir, avais l'intention, comment renouveler chambre pendant cet intervalle? Respirer comme j'en l'air de la cette atmo- HISTOIRES EXTRAORDINAIRES. 260 Sphère plus d'une heure, au maximum, était chose ab- solument impossible; et, en supposant ce terme poussé une heure un quart, jusqu'à les plus déplorables conséquences pouvaient en résulter. Cette cruelle alter- me causa native ne pas peu d'inquiétude; et Ton croira à peine qu'après les dangers que j'avais essuyés je pris la chose tellement au sérieux, que je déses- pérai d'accomplir mon dessein, et que finalement je me résignai à la nécessité d'une descente. Mais cette hésitation ne fut que momentanée. Je réfléchis que l'homme est le plus parfait esclave de l'ha- bitude, et que mille cas de la routine de son existence sont considérés comme essentiellement importants, qui ne sont tels que parce qu'il en a fait des nécessités de routine. 11 était positif que je ne pouvais pas ne pas dormir; mais je pouvais facilement m'accoutumor à me réveiller sans inconvénient d'heure en heure durant tout le temps consacré à mon repos. 11 ne me fallait de cinq minutes au plus pour renouveler pas plus complètement l'atmosphère; et la seule difficulté réelle était d'inventer nécessaire. un procédé pour m' éveiller au moment Mais c'était là un problème dont la solution, je le confesse, ne me causait pas peu d'em- barras. J'avais qui, certainement entendu parler de l'étudiant pour s'empêcher de tomber de sommeil sur ses livres, tenait la dans une main une boule de cuivre, dont chute retentissante dans un bassin de même métal placé par terre, à côté de sa. chaise, servait à le ré- AVENTURE D'UN CERTAIN II veilJer en sursaut, si quelquefois il ANS PFAALL. 201 se laissait aller à l'engourdissement. Mon cas, toutefois, était fort différent du sien et ne livrait pas de place à une pareille idée; car je ne désirais pas rester éveillé, mais réveiller à des intervalles réguliers. me Enfin, j'imaginai l'expédient suivant qui, quelque simple qu'il paraisse, fut salué par moi, au moment de ma découverte, comme une invention absolument comparable à celle du télescope, des machines à vapeur, et même de l'imprimerie. est nécessaire de remarquer d'abord que le ballon, Il à la hauteur où j'étais parvenu, continuait à monter en ligne droite avec une régularité parfaite, et que la nacelle le suivait eonséquemment sans éprouver la plus légère oscillation. Cette circonstance me favorisa gran- dement dans l'exécution du plan que j'avais adopté. Ma provision d'eau avait été embarquée dans des barils qui contenaient chacun cinq gallons et étaient soli- dement arrimés dans l'intérieur de la nacelle. Je dé- tachai l'un de ces barils, et, prenant deux cordes, je les attachai étroitement au rebord d'osier, de manière qu'elles traversaient la nacelle, parallèlement, et à une distance d'un pied l'une de l'autre; ainsi une sorte de tablette, baril et l'assujettis dans elles formaient sur laquelle je plaçai le une position horizontale. A huit pouces environ au-dessous de ces cordes et à quatre pieds du fond de la nacelle, je fixai une autre tablette, mais faite d'une planche mince, la seule de cette nature qui fût à ma disposition. Sur cette dernière, 15. HISTOIRES EXTRAORDINAIRES. '2G2 et juste au-dessous d'un des bords du baril, je déposai une petite cruche de terre. perçai alors Je un trou dans le fond du baril, au- dessus de la cruche, et j'y fichai une cheville de bois en cône, ou en forme de bougie. J'enfonçai et taillée je retirai cette cheville, qu'elle s'adaptât, assez plus ou moins, jusqu'à ce après plusieurs tâtonnements, juste pour que l'eau filtrant par le trou et tombant dans la cruche la remplît jusqu'au bord dans un intervalle de soixante cile minutes. Quant à ceci, il me fut fa- de m'en assurer en peu de temps; je n'eus qu'à observer jusqu'à quel point la cruche se remplissait dans un temps donné. Tout cela dûment arrangé, le reste se devine. Mon lit était disposé sur le fond de la nacelle de manière que ma tête, quand j'étais couché, se trouvait immédiatement au-dessous de la gueule de la cruche. Il était évident qu'au bout d'une heure la cruche rem- plie devait déborder, et le trop-plein s'écouler par la gueule qui était un peu au-dessous du niveau du bord. 11 était également certain que l'eau tombant ainsi d'une hauteur de plus de quatre pieds ne pouvait pas ne pas tomber sur ma face, être un réveil instantané, et que le résultat devait quand même j'aurais dormi du plus profond sommeil. 11 était au moins onze heures quand j'eus fini toule me mis immédiatement au lit, dans l'enicaclté de mon invention. cette installation, et je plein de confiance Et je ne fus pas désappointé dans mes espérances. De AVENTURE D'UN CERTAIN HANS PFAALL. soixante en soixante minutes, je éveillé par mon fidèle chronomètre 263 fus ponctuellement ; je vidais le contenu delà cruche par le trou de bonde du baril, je faisais fonctionner le condensateur, et je me remettais au lit. Ces interruptions régulières dans causèrent mon sommeil me même moins de fatigue que je ne m'y étais attendu; et, quand enfin je me levai pour tout de bon, il était sept heures, et le soleil avait atteint déjà quelques degrés au-dessus de la ligne de mon horizon. 3 avril. — Je trouvai que mon ballon était arrivé à une immense hauteur, et que la convexité de la terre manière frappante. Au-des- se manifestait enfin d'une sous de moi, dans l'Océan, se montrait un semis de points noirs qui devaient être indubitablement des îles. Au-dessus de ma et les étoiles tête, le ciel était d'un noir de jais, visibles et scintillantes; en réalité, elles m'avaient toujours apparu ainsi depuis le premier jour de mon ascension. Bien loin vers le nord, j'apercevais au bord de l'horizon une ligne ou une bande mince, blanche et excessivement brillante, et je supposai immédiatement que ce devait être la limite sud de la mer de glaces polaires. Ma excitée, car j'avais curiosité fut l'espoir grandement de m'avancer beaucoup })lus vers le nord, et peut-être, à un certain moment, de me trouver directement au-dessus du pôle lui- même. Je déplorai alors que l'énorme hauteur où j'étais placé m'empêchât d'en faire un examen aussi positif que je l'aurais désiré. Toutefois, il y avait encore quelques bonnes observations à faire. HISTOIRES EXTR AOnDINAlRES. 2(54 Il ne m'aniva d'ailleurs rien d'extraordinaire durant cette journée. Mon 'appareil fonctionnait toujours très- régulièrement, et le ballon montait toujours sans au- cune vacillation apparente. Le froid était intense m'obligeait de m'envelopper letot. Quand les ténèbres et soigneusement d'un pa- couvrirent la terre, je me mis au lit, quoique je dusse être pour plusieurs heures encore enveloppé de horloge hydraulique la lumière du plein jour. Mon accomplit ponctuellement son devoir, et je dormis profondément jusqu'au matin suivant, sauf les interruptions périodiques. k avril. joyeuse — Je me suis levé en bonne santé et en humeur, et j'ai été fort étonné du singulier changement survenu dans l'aspect de la mer. Elle avait perdu, en grande partie, la. teinte de bleu profond qu'elle avait revêtue jusqu'à présent; elle blanc grisâtre et d'un éclat qui éblouissait était d'u.n l'œil. La convexité de l'Océan était devenue si évidente, que la masse entière de ses eaux lointaines semblait s'é- crouler précipitamment dans l'abîme de l'horizon, et je me surpris prêtant l'oreille et cherchant les échos de la puissante cataracte. Les îles n'étaient plus visibles, soit qu'elles eussent passé derrière l'horizon vers le sud-est, soit que mon élévation croissante les eût chassées au delà de la portée de ma vue; c'est ce qu'il m'est impossible de dire. Toutefois, j'inclinais vers cette dernière opinion. La bande de glace, au nord, devenait de plus en plus apparente. Le froid avait beaucoup perdu de son intensité. 11 ne AVENTURE D'UN CERTAIN HÂNS PFAALL. m'arriva rien d'important, et je passai tout lire, 205 le jour à car je n'avais pas oublié de faire une provision de livres. 5 avril. soleil — J'ai contemplé le singulier phénomène du levant pendant c|ue presque toute la surface visible de la terre restait enveloppée dans les ténèbres. Toutefois, la lumière commença à se répandre sur toutes choses, et je revis la ligne de glaces au nord. Elle était maintenant très-distincte, et paraissait d'un ton plus foncé que les eaux de l'Océan. Évidemment, je m'en rapprochais, et avec une grande rapidité. Je m'imaginai que je distinguais encore une bande de terre vers l'est, et une autre vers fut impossible de m'en assurer. l'ouest, mais il me Température modért^e. Rien d'important ne m'arriva ce jour-là. Je me mis au lit de fort bonne heure. 6 avril. — J'ai été fort surpris de trouver la bande de glace aune distance assez modérée, et un immmense champ de glaces s'étendant à l'horizon vers le nord. 11 était évident que, si le ballon gardait sa tuelle, il direction ac- devait arriver bientôt au-dessus de l'Océan boréal, et maintenant j'avais voir le pôle. Durant tout le une jour, forte espérance de continuai à me je rapprocher des glaces. Vers la nuit, les limites de mon horizon s'agrandirent très-soudainement et très-sensiblement, ce que je devais sans aucun doute à la forme de notre planète qui est celle d'un sphéroïde écrasé, et parce que j'arrivais au-dessus des régions aplaties qui avoisinent le cercle 200 HISTOIRES EXTRAORDINAIRES. arctique. A la longue, quand les ténèbres m'envahi- rent, je me mis au lit dans une grande anxiété, trem- blant de passer au-dessus de l'objet d'une si grande curiosité sans pouvoir l'observer à loisir. — 7 avril. Je me levai de bonne heure, et, à ma grande joie, je contemplai ce que je n'hésitai pas à considérer comme le pôle lui-même. cun doute, Il était là, sans au- directement sous mes pieds; mais, hé- et las! j'étais maintenant placé à une si grande hauteur, que je ne pouvais rien distinguer avec netteté. En réalité, à en juger d'après la progression des chiffres in- diquant mes diverses hauteurs à différents moments, depuis le 2 avril à heures six du matin jusqu'à neuf heures moins vingt de la même matinée (moment où le mercure retomba dans la cuvette du baromètre), il y avait vraisemblablement lieu de supposer que le ballon devait maintenant matin — avoir — 7 avril, quatre heures du atteint une hauteur qui était au moins de 1,25k milles au-dessus du niveau de la mer. Cette élévation peut paraître elle était énorme; mais l'estime sur laquelle basée donnait très-probablement un résultat bien inférieur à la réalité. En tout cas, j'avais indubita- blement sous les yeux la totalité du plus grand diamètre terrestre; tout l'hémisphère nord s'étendait au-dessous de moi comme une carte en projection orthograpliique; même de l'équateur formait la ligne mon horizon. Vos Excellences, toutefois, et le grand cercle frontière de concevront facilement que les régions inexplorées jusqu'à présent et confinées dans les limites du cercle AVENTURE D'UN CERTAIN HANS PFAALL. 207 arctique, quoique situées directement au-dessous de moi, et conséquemment aperçues sans aucune appa- rence de raccourci, étaient trop rapetissées et placées à une trop grande distance du point d'observation, pour admettre un examen quelque peu minutieux. Néanmoins, ce que j'en voyais était d'une nature singulière et intéressante. Au nord de cette immense bordure dont j'ai parlé, et que l'on peut définir, sauf une légère restriction, la limite de l'exploration hu- maine dans ces régions, continue de s'étendre sans interruption ou presque sans interruption une nappe de glace. Dès son commencement, la surface de cette mer de glace s'affaisse sensiblement; plus loin, elle est dé- primée jusqu'à paraître plane, et finalement elle devient sigulièrement concave, et se termine au pôle lui-même en une cavité centrale circulaire dont les bords sont nettement définis, et dont le diamètre apparent soustendait alors, relativement à mon ballon, un angle de soixante-cinq secondes environ ; quant à la couleur, elle était obscure, variant d'intensité, toujours plus sombre qu'aucun de point l'hémisphère profondissant quelquefois jusqu'au delà, il était difficile visible, et s'ap- noir parfait. Au de distinguer quelque chose. A midi, la circonférence de ce trou central avait sensi- blement décru, l'avais et, à sept heures de l'après-midi, je entièrement perdu de vue; le ballon passait vers le bord ouest des glaces et filait rapidement dans la direction 8 avril. de l'équateur. — J'ai remarqué une sensible diminution IIISTOIUES EXTRAORDINAIRES. 208 dans le diamètre apparent de terre, la sans^parler d'une altération positive dans sa couleur et son aspect général. Toute la surface visible parlicipait alors, à différents degrés, de la teinte jaune pâle, et dans cer- taines parties elle avait revêtu un éclat presque dou- Ma vue était singulièrement gênée par la densité de l'atmosphère et les amas de nuages loureux pour l'œil. qui avoisinaient cette surface; c'est à peine si entre ces masses je pouvais de temps à autre apercevoir la pla- nète. Depuis les dernières quarante-huit heures, ma vue avait été plus ou moins empêchée par ces obstacles; mais mon élévation actuelle, qui était excessive, rap- prochait et confondait ces masses flottantes de vapeur, et l'inconvénient devenait de plus en plus sensible à mesure que je montais. Néanmoins, je percevais facilement que le ballon planait maintenant au-dessus du groupe des grands lacs du Nord-Amérique droit vers le sud, ce qui devait et courait m'amener bientôt vers les tropiques. Cette circonstance ne manqua pas de me causer la plus sensible satisfaction, et je la saluai comme un heu- reux présage de mon succès final. En réalité, la direction que j'avais prise jusqu'aJors m'avait rempli d'in- quiétude; car il était évident que, longtemps encore, je n'aurais si je l'avais suivie jamais pu arriver à la lune, dont l'orbite n'est inclinée sur l'écliptique que d'un petit angle de 5 degrés 8 minutes /i8 secondes. Quelque étrange que cela puisse paraître, ce ne fut qu'à cette période tardive que je commençai à com- AVENTURE D'UN CERTAIN HANS PFAALL. 209 prendre la grande faute que j'avais commise en n'effectuant pas situé mon départ de quelque point terrestre dans le plan de l'ellipse lunaire. 9 avril. — Aujourd'hui, le diamètre de la terre est grandement diminué, et la surface prend d'heure en heure une teinte jaune plus p?t)noncée. Le ballon a toujours filé droit vers le sud, et est arrivé à neuf heures de l'après-midi au-dessus de la côte nord du golfe du Mexique. 10 avril. — J'ai été soudainement tiré de mon som- meil vers cinq heures du matin par un grand bruit, un craquement terrible, dont je n'ai pu en aucune façon me rendre compte. 11 a été de courte durée ;^ mais, tant qu'il a duré, il ne ressemblait à aucun bruit terrestre dont j'eusse gardé la sensation. dire 11 est inutile de que je fus excessivement alarmé, car j'attribuai d'abord ce bruit à une déchirure du ballon. Cependant, j'examinai tout mon appareil avec une grande attention, et je n'y l;i pus découvrir aucune avarie. J'ai passé plus grande partie du jour à méditer sur un acci- dent aussi extraordinaire, mais je n'ai absolument rien trouvé de satisfaisant. Je me suis mis au lit fort mé- content et dans un état d'agitation et d'anxiété excessives. 11 avril. le — J'ai trouvé une diminution sensible dans diamètre apparent de coîtsidérable, observable la terre et un accroissement pour la première fois, dans celui de la lune, qui n'était qu'à quelques jours de son plein. Ce fut alors pour moi un très-long et très-pé- HISTOIRES EXTRAORDINAIRES, 2J0 nible labeur de condenser dans la chambre tité d'air une quan- atmosphérique suffisante pour l'entretien de la vie. 12 avril. — Un singulier changement a eu lieu dans > la direction du ballon, qui, bien que je m'y attendisse parfaitement, m'a causé le plus sensible plaisir. Il était parvenu dans sa direction première au vingtième parallèle de latitude sud, et il a tourné brusquement vers Test, à angle aigu, et a suivi cette route tout le jour, en se tenant à peu près, sinon absolument, dans le plan exact de l'ellipse lunaire. Ce qui était digne de remar- que, c'est que ce changement de direction occasion- nait une oscillation très-sensible de la nacelle, — oscil- lation qui a duré plusieurs heures à un degré plus ou moins vif. 13 avril. — J'ai été de nouveau très-alarmé par la répétition de ce grand bruit de craquement qui m'avait terrifié le 10. J'ai il longtemps médité sur ce sujet, mais m'a été impossible d'arriver à une conclusion satis- faisante. Grand décroissement rent de la terre. Il dans le diamètre appa- ne sous-tendait plus, relativement au ballon, qu'un angle d'un peu plus de 25 degrés. Quant à la lune, il m'était impossible de la voir, elle était presque dans mon zénith. Je marchais toujours dans le plan de l'ellipse, mais je faisais peu de progrès vers l'est. av7^lL — Diminution excessivement rapide dans le diamètre de la terre. Aujourd'hui, j'ai été fortement 1/t impressionné de l'idée que le ballon courait mainte- AVENTURE D'UN CERTAIN HANS PFAALL. '271 liant sur la ligne des apsides en remontant vers le pé- — en d'autres termes, qu'il suivait directement rigée, la route qui devait le conduire à la lune dans cette par- tie de son orbite qui est la plus rapprochée de la terre. La lune était juste au-dessus de ma tète, et conséquem- ment cachée à ma vue. Toujours travail indispensable ce grand et long pour la condensation de l'atmo- sphère. 15 avril. — Je ne pouvais même plus distinguer net- tement sur la planète les contours des continents et des mers. Vers midi, je fus frappé pour la troisième fois de ce bruit effrayant qui m'avait déjà si fort étonné. Cette fois-ci, cependant, prit de l'intensité. il dura quelques moments et A la longue, stupéfié, frappé de ter- reur, j'attendais anxieusement je ne sais quelle épou- vantable destruction, lorsque la nacelle oscilla une violence excessive, je n'eus pas le et avec une masse de matière que temps de distinguer passa à côté du ballon, gigantesque et enflammée, retentissante et ru- gissante comme la voix de mille tonnerres. Quand mes terreurs et mon étonnement furent un peu diminués, je supposai naturellement que ce devait être quelque énorme fragment volcanique vomi par ce monde dont j'approchais si rapidement, et, selon toute probabilité, un morceau de ces substances singulières qu'on ra- masse quelquefois sur la terre, et qu'on nomme aérolithes, faute d'une appellation plus précise. 16 avril. — Aujourd'hui, en regardant au-dessous do moi, aussi bien que je pouvais, par chacune des HISTOIRES EXTRAORDINAIRES. 272 deux fenêtres latérales alternativement, j'aperçus, à ma grande satisfaction, une très-petite portion du disque lunaire qui s'avançait, pour ainsi dire de tous les côtés, au delà de la vaste circonférence de mon ballon. Mon agitation devint extrême, car maintenant je ne doutais guère que je n'atteignisse bientôt le mon pé- but de rilleux voyage. En vérité, le labeur qu'exigeait alors le condensateur accru jusqu'à devenir obsédant, et ne laissait s'était presque pas de répit à mes efforts. De sommeil, il n'en était, pour ainsi dire, plus question. Je devenais réelle- ment malade , et tout mon être tremblait d'épuise- ment. La nature humaine ne pouvait pas supporter plus longtemps une pareille france. Durant l'intervalle intensité dans la souf- des ténèbres, bien court maintenant, une pierre météorique passa de nouveau dans mon voisinage, et la fréquence de ces phéno- mènes commença à me donner de fortes inquiétudes. 17 avril. — Cette matinée a voyage. On se rappellera que, dait relativement à fait le époque dans mon 13, la terre sous-tcn- moi un angle de 25. degrés. Le l/j, cet angle avait fortement diminué; le 15, j'observai une diminution encore plus rapide; et, le 16, avant de me coucher, j'avais estimé que l'angle n'était plus que de 7 degrés et 15 minutes. Qu'on se figure donc quelle dut être ma stupéfaction, quand, en m'éveillant co matin, 17, et sortant d'un sommeil court et troublé, je m'aperçus que la surface planétaire placée au-des- sous de moi avait si inopinément et si effroyablement AVENTURE D'EN CERTAIN HANS PFAALL. 273 auijmcnlè de volimiG que son diamètre apparent soiislendait un angle qui ne mesurait pas moins de 39 degrés! J'étais foudroyé! Aucune parole ne peut donner une idée exacte de Tliorreur extrême, absolue, et de la stupeur dont je fus saisi, possédé, écrasé. Mes genoux vacillèrent sous moi, poil se dressa sur — mes dénis claquèrent, — mon — Le ballon a donc ma tête. fait explosion? — Telles furent les premières idées qui se précipitèrent tumultueusement dans mon esprit. Posi- — Je tombe, — tivement, le ballon a crevé! je tombe avec la plus impétueuse, la plus incomparable vitesse! A en juger par l'immense espace déjà si rapidement parcouru, je dois rencontrer la surface de la terre dans dix minutes au plus; précipité, anéanti Mais, — dans dix minutes, je serai ! à la longue, la réflexion vint à Je fis une pause, je méditai ; mon secours. et je commençai à douter. La chose était impossible. Je ne pouvais en aucune façon être descendu aussi rapidement. Eri oulre, bien que je me rapprochasse évidemment de la surface située au-dessous de moi, ma vitesse réelle n'était nul- lement en rapport avec l'épouvantable vélocité que j'avais d'abord imaginée. Celte considération calma efficacement la perturbation de mes idées, et je réussis finalement à envisager phénomène sous son vrai point de vue. 11 fallait que ma stupéfaction m'eût privé de l'exercice de mes sens pour que je n'eusse pas vu quelle immense différence le y avait entre l'aspect de cette surface placée au-des. I 274 II ST 1 1\ ES EX F U A SOUS de moi et celai de R D I N A IRES. ma planète natale. Cette der- nière était donc au-dessus de ma tête et complètement — lune — elle-même dans toute sa gloire, s'étendait au-dessous mes pieds! sous de moi; — cachée par le ballon, tandis que la lune, la je l'avais L'étonnement et la stupeur produits dans mon esprit par cet extraordinaire changement dans la situation des choses était peut-être, après tout, ce qu'il y avait de plus étonnant et de moins explicable dans aventure. mon Car ce bouleversement, en lui-même, était non-seulement naturel et inévitable, mais depuis long- temps même je l'avais positivement prévu comme une circonstance toute simple, comme une conséquence qui devait se produire quand j'arriverais au point exact de mon parcours où l'attraction de la remplacée par l'attraction du satellite, planète serait — ou, en ter- mes plus précis, quand la gravitation du ballon vers la terre serait moins puissante que sa gravitation vers la lune» Il est vrai que je sortais d'un profond sommeil, que mes sens étaient encore brouillés, quand je me trouvai soudainement en face d'un phénomène des tous plus surprenants, — d'un phénomène que j'attendais, mais que je n'attendais pas en ce moment. La révolution elle-même devait avoir eu lieu naturellement, de la façon la plus douce et la plus graduée, et il n'est pas le moins du monde certain que, lors que j'eusse été éveillé j'eusse eu la conscience au moment où elle même s'opéra, du sens dessus dessous. — AVEi^TURE D'UN CERTAIxN IIANS PFAALL. 275 que j'eusse perçu un symptôme intérieur quelconque de l'inversion, — c'est-à-dire une incommodité, un dé- rangement quelconque, soit dans ma personne, soit dans mon appareil. 11 est presque inutile de dire qu'en revenant au sen- ma situation, et émergeant de la terreur qui avait absorbé toutes les facultés de mon âme, timent juste de mon attention s'appliqua d'abord uniquement à la contemplation de l'aspect général de la lune. Elle se développait au-dessous de moi comme une carte, — et, quoique je jugeasse qu'elle était encore à une distance assez considérable, les aspérités de sa surface se dessi- naient à mes yeux avec une netteté très-singulière dont je ne pouvais absolument pas me rendre compte. L'ab- sence complète d'océan, de mer, et même de tout lac et de toute rivière, me frappa, au premier coup d'œil, comme le signe le plus extraordinaire de sa condition géologique. Cependant, chose étrange à dire, je voyais de vastes régions planes, d'un caractère positivement alluvial, quoique la plus grande partie de l'I^émisphère visible fût couverte d'innombrables montagnes volcaniques en forme de cônes, et qui avaient plutôt l'aspect d'émi- nences façonnées par l'art que de saillies naturelles. La plus haute d'entre elles n'excédait pas trois milles trois quarts en élévation perpendiculaire; — d'ailleurs, une carte des régions volcaniques des Campi Plilegrœi donnerait à Vos Excellences une meilleure idée de leur surface générale que toute description, toujours insuf- , 27Ù lisante, HISTOIRES EXTU AOUDINAIUES. que j'essayerais d'en faire. — La plupart de ces montagnes étaient évidemment en état d'éruption, et me donnaient une idée terrible de leur furie et de leur puissance par les fulminations multipliées des pierres improprement dites météoriques, qui maintenant partaient d'en bas et filaient à côté du ballon avec une fréquence de plus en plus effrayante. 18 avril. — Aujourd'hui, j'ai trouvé un énorme dans le volume apparent de vitesse évidemment accélérée de accroissement la lune, — et la ma descente a com- mencé à me remplir d'alarmes. On se rappellera que dans le principe, quand je commençai à appliquer mes rêveries à la possibilité d'un passage vers la lune, l'hy- pothèse d'une atmosphère ambiante dont la densité devait être proportionnée au volume de la planèie avait pris une large part dans mes calculs; et cela, en dùpit de mainte théorie adverse, et même, je l'avoue, en dépit du préjugé universel contraire à l'existence d'une atmosphère lunaire quelconque. Mais, outre les idées que j'ai déjà émises relativement à la comète d'Encke et à la lumière zodiacale, ce qui me fortifiait dans mon opinion, c'étaient certaines observations de M. Shrooter, de Lilienthal. Il a observé la lune, âgée de deux jours et demi, le soir, peu de temps après le coucher du soleil, avant que la partie obscure fût visible, et il continua à la surveiller jusqu'à ce que cette partie fût devenue visible. Les deux cornes semblaient s'affiler en une sorte de prolongement très-aigu, dont l'extrémité était faiblement éclairée par les rayons solaires, alors qu'aucune parti AVENTURE D'UN CEUTAIiN HANS PFAALL. 277 de riiémisphère obscure ii'éLait visible. Peu de temps après, tout le bord sombre s'éclaira. Je pensai que ce prolongement des cornes au delà du demi-cercle prenait sa cause dans la réfraction des rayons du soleil pur l'atmosphère de la lune. Je calculai aussi que la hauteur de cette atmosphère (qui pouvait réfracter assez de lumière dans son hémisphère obscur pour pro- duire un crépuscule plus lumineux que la lumière réfléchie par la terre quand la lune est environ à 32 degrés de sa conjonction), devait être de 1,356 pieds de roi; d'après cela, je supposai que la plus grande hauteur capable de réfracter le rayon solaire était de 5,376 pieds. Mes idées sur ce sujet se trouvaient éga- lement confirmées par un passage du quatre-vingt- deuxième volume des Transaclions philosophiques, dans lequel il est dit que, lors d'une occultation des satellites de Jupiter, le troisième disparut après avoir été pendant une ou deux secondes, et que indistinct quatrième devint indiscernable en approchant le du limbe ^ 1. Ilcvélius écrit qu'il a quelquefois observé dans des cieux parfaitement clairs, où des étoiles môme de sixième et de septièn e — supposés la même hauteur môme élongation de la terre, le même télescope, grandeur brillaient visiblement, que de la lune, la excellent, bien entendu, — la lune et ses taches ne nous apparais- saient pas toujours aussi lumineuses. Ces circonstances données, que la cause du phénomène n'est ni dans notre aile télescope, ni dans la lune, ni dans l'œil (!e l'observateur, mais qu'elle doit être cherchée dans quelque cho^c (une atmosphère?) existant autour de la lune. il est évident mosphère, ni dans Cassini a constamment observé que Saturne, Jupiter et les étoiles 16 IIISTOIUES EXTUÂORDIiN AIllES. 278 C'était sur la résistance, ou, plus exactement, sur le support d'une atmosphère existant à un état de densité hypothétique, que j'avais absolument fondé mon espé- rance de descendre sain et sauf. Après tout, si j'avais une conjecture absurde, fait à attendre, je n'avais rien de mieux comme dénoûment de mon aventure, que d'être pulvérisé contre la surface raboteuse lite. Et, du satel- en somme, j'avais toutes les raisons possibles d'avoir peur. La distance où j'étais de la comparativement insignifiante, tandis que lune était le labeur exigé par le condensateur n'était pas du tout diminué, et que je ne découvrais aucun indice d'une densité croissante dans l'atmosphère. — Ce matin, à ma grande vers neuf — trouvant heures, me effroyablement près de sur19 avril. joie, la face lunaire, et dernier degré, mes appréhensions étant excitées au — le piston du condensateur a donné des symptômes évidents d'une altération de l'atmosphère. A dix heures, j'avais des raisons de croire sa densité considérablement augmentée. A onze heures, l'appareil ne réclamait plus qu'un travail très-minime; et, à midi, je me hasardai, non sans quelque hésita- tion, à desserrer le tourniquet, et, voyant qu'il n'y avait à cela fixGs, au aucun inconvénient, j'ouvris décidément la moment d'être occultes par la lune, changeaient leur forme circulaire en une forme ovale; et dans d'autres occultations il n"a saisi aucun chançiement de forme. On pourrait donc en inférer que, dans quelques cas, mais pas toujours, la lune est enveloppée d'une matière dense où sont réfractés les rayons des étoiles. — E. P. AVENTURE D'UN CERTAIN HANS PFAALL. chambre de caoutchouc, Ainsi et je 27U déshabillai la nacelle. que j'aurais du m'y attendre, une violente mi- graine accompagnée de spasmes fut la conséquence im- médiate d'une expérience si précipitée et si pleine de dangers. iMais, comme ces inconvénients et d'autres encore relatifs à la respiration n'étaient pas assez grands pour mettre ma vie en péril, je me résignai à les en- durer de mon mieux, d'autant plus que j'avais tout lieu d'espérer qu'ils disparaîtraient progressivement, chaque minute me rapprochant des couches plus denses de l'atmosphère lunaire. Toutefois, ce rapprochement s'opérait avec une im- me démontré — — que, bien que très-proba- pétuosité excessive, et bientôt certitude fort alarmante biement je ne me fusse pas trompé en comptant sur une atmosphère dont tionnelle au fut il densité la devait être propor- volume du satellite, cependant j'avais eu bien tort de supposer que cette densité, surface, serait suffisante pour supporter l'immense poids contenu dans la nacelle de pendant eût dû être surface de la terre sur l'autre planète, , même à la mon ballon. Tel cecomme à la exactement le cas, si vous supposez, sur l'une la et pesanteur réelle des corps en raison de la densité atmosphérique; mais tel n'était pas le cas; ma chute précipitée le démontrait suffisam- ment. Mais pourquoi? C'est ce qui ne pouvait s'expli- quer qu'en tenant compte de ces perturbations géologiques dont j'ai déjà posé l'hypothèse. En tout cas, je touchais presque à la planète, et je HISTOIRES EXTRAORDINAIRES. 280 tombais avec la plus terrible impétuosité. Aussi je ne perdis pas une minute; je jetai par-dessus bord tout mon lest, puis mes barriques d'eau, puis mon appareil mon sac de caoutchouc, et enfin tous condensateur et les articles contenus dans la nacelle. Mais tout cela ns servit à rien. Je tombais toujours avec une horrible rapidité, et je n'étais pas à plus d'un demi-mille surface. Comme expédient suprême, je de la me débarrassai de mon paletot, de mon chapeau et de mes bottes; je détachai du ballon la nacelle elle-même, qui n'était pas d'un poids médiocre; et, m'accrochant alors au filet avec mes deux mains, j'eus à peine le temps d'observer que tout le pays, aussi loin que mon œil pouvait atteindre, était criblé d'habitations lilliputiennes, avant de tomber, comme une balle, au cœur — même d'une cité d'un aspect fantastique et au beau milieu d'une multitude de vilain petit peuple, dont pas un individu ne prononça une syllabe ni ne se donna le moindre mal pour me prêter assistance. Ils se tenaient tous, les poings sur les hanches, comme un tas d'idiots, grimaçant d'une manière ridicule et me regardant de travers, moi et mon ballon. Je me détournai d'eux avec un superbe mépris; et, levant mes regards vers la terre que je venais de quitter, et dont je m'étais exilé pour toujours peut-être, je l'aperçus sous la forme d'un vaste et sombre bouclier de cuivre d'un diamètre de 2 degrés environ, fixe et immobile dans les cieux, et garni à l'un de ses bords d'un croissant d'or é! incelant. On n'y pouvait découvrir aucune trace de mer ni AVENTURE D'UN CERTAIN HANS PFAALL. de continent, et le tout était 281 moucheté de taches va- riables et traversé par les zones tropicales et équaloriale, comme par des ceintures. Ainsi, avec la permission de Vos Excellences, après une longue série d'angoisses, de dangers inouïs et de délivrances incomparables, j'étais enfin, dix-neuf jours après mon départ de Rotterdam, arrivé sain et sauf au terme d'un voyage, le plus extraordinaire, le plus im portant qui ait jamais été accompli, entrepris, ou même conçu par un citoyen quelconque de votre planète. Mais il me reste à raconter mes aventures. Car, en vérité, Vos Excellences concevront facilement qu'après une résidence de cinq ans sur une planète qui, déjà profondément intéressante par elle-même, l'est dou- blement encore par son intime parenté, en qualité de satellite, avec le monde habité par l'homme, je puisse entretenir avec le Collège national astronomique des correspondances secrètes d'une bien autre importance que les simples détails, si surprenants qu'ils soient, du voyage que j'ai effectué si heureusement. Telle est, en somme, la question réelle. J'ai beau- coup, beaucoup de choses à dire, et ce serait pour moi un véritable plaisir de vous les communiquer. J'ai beaucoup à dire sur le climat de cette planète; — sur — sur ses étonnantes alternatives de froid et de chaud; celte clarté solaire qui dure quinze jours, implacable et brûlante, et sur cette température glaciale, que polaire, qui remplit l'autre quinzaine; plus — sur une translation constante d'humidité qui s'opère par distille. HISTOIRES EXTRAORDINAIRES. 282 lation, comme dans le vide, du point situé au-dessous du soleil jusqu'à celui qui en est le plus éloigné; — sur la race même des habitants, sur leurs mœurs, leurs coutumes, leurs institutions politiques; sur leur orga- nisme particulier, leur laideur, leur privation d'oreilles, appendices superflus dans une atmosphère si étrange- ment modifiée; conséquemment, sur leur ignorance de l'usage et des propriétés du langage; sur la singulière méthode de communication qui remplace la parole; — sur l'incompréhensible rapport qui unit chaque citoyen de la lune à un citoyen du globe terrestre, — rapport analogue et soumis à celui qui régit également les mouvements de la planète et du satellite, et par suite duquel les existences et les destinées des habitants de l'une sont enlacées aux existences des habitants de l'autre; plaît à Vos Excellences, — et aux destinées et par-dessus par-dessus tout, tout, s'il sur les sombres et horribles mystères relégués dans les régions de l'autre hémisphère lunaire, régions qui, grâce à la concordance presque miraculeuse de la rotation du satellite sur son axe avec sa révolution sidérale autour de la terre, n'ont jamais tourné vers nous, et. Dieu merci, ne s'exposeront jamais à la curiosité des téles- copes humains. Voilà tout ce que je voudrais raconter, et beaucoup plus encore. Mais, — tout cela, pour trancher la ques- ma récompense. J'aspire à rentrer dans ma famille et mon chez moi; et, comme prix de toute communication ultérieure de ma part, en contion, je réclame AVENTURE D'UN CERTAIN HANS PFAALL. 28:1 sidération de la lumière que je puis, s'il me plaît, jeter sur plusieurs branches importantes des sciences physiques et métaphysiques, je sollicite, par l'entremise de votre honorable corps, le pardon du crime dont je me suis rendu coupable en mettant à mort mes créanciers lorsque je quittai Rotterdam. Tel est donc l'objet de la présente lettre. Le porteur, qui est un habitant de la lune, que j'ai décidé à me servir de messager sur la terre, et à qui j'ai attendra le bon donné des instructions suffisantes, plaisir de Vos Excellences, et me rapportera le pardon demandé, s'il y a moyen de l'obtenir. J'ai l'honneur d'être de Vos Excellences le très-humble serviteur, Hans Pfaall. En finissant la lecture de ce très-étrange document, le professeur Rudabub, dans l'excès de sa surprise, laissa, dit-on, tomber sa pipe par terre, Siiperbus Von Underduk, et Mynheer ayant ôté, essuyé et serré dans sa poche ses besicles, s'oublia, lui et sa dignité, au point de pirouetter trois fois sur son talon, dans la quintessence de l'étonnement et de l'admiration. — On obtiendrait la grâce; cela ne pouvait pas faire Du moins, il en fit le serment, l'ombre d'un doute. le bon professeur Rudabub, il en fit le serment avec un parfait juron, et telle fut décidément l'opinion de l'il- lustre Von Underduk, qui prit le bras de son collègue et fit, sans prononcer une parole, la plus grande partie de la route vers son domicile pour délibérer sur les HISTOIRES EXTRAORDINAIRES. 284 mesures urgentes. Cependant, arrivé à la porte de maison du bourgmestre, le la professeur s'avisa de sug- gérer que, le messager ayant jugé à propos de disparaître (terrifié sans doute jusqu'à la sionomie sauvage pardon ne avait qu'un des servirait pas habitants mort par la phy- de Rotterdam), à grand'chose, le puisqu'il n'y homme de la lune qui pût entreprendre un voyage aussi lointain. En face d'une observation aussi sensée, le bourg- mestre se rendit, et l'affaire n'eut pas d'autres suites. Cependant, il n'en fut pas de même des rumeurs et des conjectures. La lettre, ayant été publiée, donna nais- sance à une foule d'opinions et de cancans. Quelques- uns — des esprits par trop sages — poussèrent le ridi- cule jusqu'à discréditer l'affaire et à la présenter comme un pur canard. Mais je crois que le mot canard est, pour cette espèce de gens, un terme général qu'ils appliquent à toutes les matières qui passent leur intelligence. Je ne puis, quant à moi, comprendre sur quelle base ils ont fondé une pareille accusation. Voyons ce qu'ils disent : Avant tout, — que certains farceurs de Rotterdam ont de certaines antipathies spéciales contre certains bourgmestres et astronomes. Secundo, — qu'un son métier, dont petit nain bizarre, escamoteur de les deux oreilles avaient été, pour quelque méfait, coupées au ras de la tête, avait depuis quelques jours disparu de la toute voisine. ville de Bruges, qui est AVENTURE D'UN CERTAIN HANS PFAALL. TeiHio, — que les gazettes collées tout 285 autour du petit ballon étaient des gazettes de Hollande, et con- séquemment n'avaient pas pu être fabriquées dans la lune. C'étaient des papiers sales, crasseux, — très-cras- seux; et Gluk, rimprimeur, pouvait jurer sur sa Bible qu'ils avaient été imprimés à Rotterdam. Qiiarlo, — que iïans Pfaall lui-même, le vilain ivro- gne, et les trois fainéants personnages qu'il appelle ses créanciers, avaient été vus ensemble, deux ou trois jours auparavant tout au plus, dans un cabaret mal famé des faubourgs, juste comme revenaient, avec ils de l'argent plein leurs poches, d'une expédition d'outre iner. Et, — que en dernier lieu, ralement reçue, ou qui doit c'est une opinion géné- l'être, que le collège des Astronomes de la ville de Rotterdam, — aussi bien que tous autres collèges astronomiques de toutes autres parties de l'univers, sans parler des collèges et des astronomes en général, plus, ni meilleur, ni n'est nécessaire. — n'est, pour n'en pas dire plus fort, ni plus éclairé qu'il MANUSCRIT TROUVÉ DANS UNE BOUTEILLE Qui n'a plus qu'un moment à vivre rs'a plus rien à dissimuler. QuiNAULT. De mon pays et de ma famille ]e — Alys. n'ai pas graiid'- chose à dire. De mauvais procédés et l'accamidation des années m'ont rendu étranger à l'un et à Tautre. Mon patrimoine me fit bénéficier d'une éducation peu com- mune, et un tour contemplatif apte à classer d'esprit me rendit méthodiquement tout ce matériel d'in- struction diligemment amassé par une étude précoce. Par-dessus tout, les ouvrages des philosophes allemands me procuraient de grandes délices; cela ne venait pas d'une admiration malavisée pour leur éloquente folie, mais du plaisir que, grâce à mes habitudes d'analyse rigoureuse, j'avais à surprendre leurs erreurs. On m'a souvent reproché l'aridité de mon génie; un manque d'imagination m'a été imputé comme un crime, et le pyrrhonisme de mes opinions a fait de moi, en tout temps, un homme fameux. En réalité, une forte appé- HISTOIRES EXTRAORDINAIRES. 288 tence pour la philosophie physique a, je le crains, im- prégné mon esprit d'un des défauts les plus communs de ce siècle, — je veux dire de l'habitude de rapporter aux principes de cette science les circonstances môme moins susceptibles d'un pareil rapport. Par-dessus les tout, personne moins exposé que moi à n'était se laisser entraîner hors de la sévère juridiction de la vél'ité par les feux follets de la superstition. J'ai jugé à propos de donner ce préambule, dans que ne crainte la que soit considéié plutôt comme la frénésie d'une imagination indigeste l'incroyable récit à j'ai faire que comme l'expérience positive d'un esprit pour lequel les rêveries de l'imagination ont été lettre morte et nullité. Après plusieurs années dépensées dans un lointain voyage, je m'embarquai, en 18.., à Batavia, dans riche et populeuse dans l'archipel des île la de Java, pour une promenade îles de la Sonde. Je me mis en route comme passager, — n'ayant pas d'autre mobile qu'une nerveuse instabilité qui me liantaU comme un mauvais esprit. Notre bâtiment était un bateau d'environ quatre cents tonneaux, doublé en cuivre et construit à Bom- bay, en teck de Malabar. 11 était chargé laine et d'huile des Laquedives. bord du l'huile fi:in de cocotier, du de coton, de Nous avions aussi suci^e à de palmier, de de beurre bouilli, des noix de coco, et quelques oissts d'opium. L'arrimage avait été mal navire conséquemment donnait de la bande. fait, et le MANUSCRIT TROUVÉ DANS UNE BOUTEILLE. 280 Nous mîmes sous voiles avec un souffle de vent, et, pendant plusieurs jours, nous restâmes le long de la côte orientale de Java, sans autre incident pour trom- per la monotonie de notre route que la rencontre de quelques-uns des petits grabs de l'archipel où nous étions confinés. Un soir, comme j'étais appuyé sur le bastingage de la dunette, j'observai un très-singulier nuage, isolé, vers le nord-ouest. Il était remarquable autant par sa couleur que parce qu'il était le premier que nous eussions vu veillai il depuis notre départ de Batavia. Je le sur- attentivement jusqu'au coucher du soleil; alors, se répandit tout d'un coup de l'est à l'ouest, cernant l'horizon raissant Mon d'une ceinture précise de vapeur, et appa- comme une longue attention fut bientôt ligne de côte très-basse. après attirée par l'aspect rouge brun de la lune et le caractère particulier de la mer. Cette dernière subissait un changement rapide, et l'eau semblait plus transparente que d'habitude. Je pouvais distinctement voir le fond, et cependant, en jetant la sonde, je trouvai brasses. L'air était que nous étions sur quinze devenu intolérablement chaud et se chargeait d'exhalaisons spirales semblables à celles qui s'élèvent du fer chauffé. Avec la nuit, toute brise tomba, et nous fûmes pris par un calme plus complet qu'il n'est possible de le concevoir. La flamme d'une bougie brûlait à l'arrière sans le mouvement le moins sensible, et un long cheveu tenu entre l'index pouce tombait droit et sans la moindre et le oscillation. 17 HISTOIRES EXTRAORDINAIRES. 290 Néanmoins, comme le capitaine disait qu'il n'apercevait aucun symptôme de danger, et comme nous dérivions commanda de carguer On ne mit point de vigie vers la terre par le travers, il les voiles et de filer l'ancre. 4e quart, et l'équipage, qui se composait principalement de Malais, se coucha délibérément sur le pont. Je descendis dans la chambre, — non sans sentiment d'un malheur. En tômes le parfait pres- tous ces symp- réalité, me donnaient à craindre un simoun. Je parlai de mes craintes au capitaine tion à ce mais il ne fit pas atten- ; que je lui disais, et me quitta sans daigner me faire une réponse. Mon malaise, toutefois, m'empêcha de dormir, et, vers minuit, je montai sur le pont. Gomme je mettais le pied sur la dernière marche du capot d'échelle, je fus effrayé par un profond bourdon- nement semblable à celui que produit l'évolution rapide d'une roue de moulin, et, avant que j'eusse pu en vérifier la cause, je sentis que navire' le tremblait dans son centre. Presque aussitôt, un coup de jeta sur le côté, mer nous courant par-dessus nous, balaya et, tout le pont de l'avant à l'arrière. L'extrême furie du coup partie, le salut du navire. de vent fit, en grande Quoi qu'il fût absolu- ment engagé dans l'eau, comme ses mâts s'en étaient allés par -dessus bord, minute après, et, il se vacillant l'immense pression de la releva une lentement quelques instants sous tempête , finalement il se redressa. Par quel miracle échappai-je à la mort, il m'est im- MANUSCRIT TROUVE DANS UNE BOUTEILLE. 291 possible de le dire. Étourdi par le choc de l'eau, je me trouvai pris, le quand je revins à moi, entre l'étambot et gouvernail. Ce fut à grand'peine que je sur mes pieds, et, me remis regardant vertigineusement autour de moi, je fus d'abord frappé de l'idée que nous étions sur des brisants, tant était effrayant, au delà de toute imagination, le tourbillon de cette mer énorme et écu- mante dans laquelle nous étions engouffrés. Au bout de quelques instants, j'entendis la voix d'un vieux Suédois qui s'était embarqué avec nous au moment où nous ma force, et il me rejoindre à l'arrière. Nous re- quittions le port. Je le hélai de tolite ^ vint en chancelant connûmes bientôt que nous étions les seuls survivants du sinistre. Tout ce qui était sur le pont, nous exceptés, avait été balayé par-dessus bord ; le capitaine et les matelots avaient péri pendant leur sommeil, car les ca- bines avaient été inondées par la mer. Sans auxiliaires, nous ne pouvions pas espérer de pour la sécurité du navire, et faire grand'chose nos tentatives furent d'abord paralysi^es par la croyance où nous étions que nous allions sombrer d'un moment à l'autre. Notre câble avait cassé comme un fil d'emballage au premier souffle de l'ouragan; sans cela, gloutis instantanément. nous eussions été en- Nous fuyions devant la mer avec une vélocité effrayante, et l'eau nous faisait des brèches visibles. La charpente de notre arrière était excessivement endommagée, et, presque sous tous les rapports, nous avions essuyé de cruelles avaries; mais, à notre grande joie, nous trouvâmes que les pompes HISTOIRES EXTRAORDINAIRES. '292 n'étaient pas engorgées, et que notre chargement n'a- vaient pas été très- dérangé. La plus grande furie de la tempête était passée, et nous n'avions plus à craindre la violence du vent; mais nous pensions avec terreur au cas de sa totale cessation, bien persuadés que, dans notre état d'avarie, nous ne pourrions pas résister à l'épouvantable houle qui s'ensuivrait; mais cette très-juste appréhension ne semblait pas si près de se vérifier. Pendant cinq nuits et cinq jours entiers, durant lesquels nous vécûmes de quelques morceaux de sucre de palmier tirés à grand'peine du gaillard d'avant, notre coque fila avec une vitesse incalculable devant des reprises de vent qui se succédaient rapidement, et qui, sans égaler la première violence du simoun, étaient cependant plus terribles qu'aucune tempête que j'eusse essuyée jusqu'alors. Pendant les quatre premiers jours, notre route, sauf de très-légères variations, fut au sud-est quart de sud, et ainsi nous serions allés nous jeter sur la côte de la Nouvelle-Hollande. Le cinquième jour, le froid devint extrême, quoique le vent eût tourné d'un point vers le nord. Le soleil se leva avec un éclat jaune et maladif, et se hissa à quelques degrés à peine au-dessus de l'horizon, sans projeter une lumière franche. 11 n'y avait aucun nuage apparent, et cependant le vent fraîchissait, fraîchissait, et soufflait avec des accès de furie. Vers midi, ou à peu près, autant que nous en pûmes ji^ger, notre at- tention fut attirée de nouveau par la physionomie du MANUSCRIT TROUVÉ DANS UNE BOUTEILLE. 293 soleil. ler, 11 n'émettait pas de lumière, à proprement par- mais une espèce de feu sombre et triste, sans ré- flexion, comme si tous les rayons étaient polarisés. avant de se plonger dans Juste son feu central la mer grossissante, disparut soudainement, comme s'il était brusquement éteint par une puissance inexplicable. Ce n'était plus qu'une roue pâle et couleur d'argent, quand il se précipita dans l'insondable Océan. Nous attendîmes en vain l'arrivée du sixième jour; — — pour le ce jour n'est pas encore arrivé pour moi, Suédois il n'est jamais Nous fûmes dès arrivé. ensevelis dans des ténèbres de poix, si lors bien que nous n'aurions pas vu un objet à vingt pas du navire. Nous fûmes enveloppés d'une nuit éternelle que ne tempérait même pas l'éclat phosphorique de la mer auquel nous étions accoutumés sous les tropiques. Nous ob- servâmes aussi que, quoique la tempête continuât à faire rage sans accalmie, nous ne découvrions plus aucune apparence de ce ressac et de ces moutons qui nous avaient accompagnés jusque-là. Autour de nous, tout n'était qu'horreur, épaisse obscurité, un noir désert d'ébène liquide. filtrait Une terreur superstitieuse s'in- par degrés dans l'esprit du vieux Suédois, et *mon âme, quant à moi, était plongée dans une muette stupéfaction. Nous avions abandonné tout soin vire, du na- comme chose plus qu'inutile, et, nous attachant de notre mieux au tronçon du mât de misaine, nous promenions nos regards avec amertume sur l'immensité de rOcéan. Nous n'avions aucun moyen de calculer HISTOIRES EXTRAORDINAIRES. 294 temps, le jecture et nous ne pouvions former aucune con- sur notre situation. Nous étions néanmoins bien sûrs d'avoir été plus loin dans sud qu'aucun le éprouvions un des navigateurs précédents, et nous grand étonnement de ne pas rencontrer les obstacles me- ordinaires de glaces. Cependant, chaque minute naçait d'être la dernière, — chaque énorme vague se précipitait pour nous écraser. ce que j'avais imaginé La houle surpassait tout comme possible, miracle de chaque instant que nous chargement, lités de c'était un Mon camarade parlait de la légèreté de no- engloutis. tre et ne fussions pas et me rappelait les excellentes qua- notre bateau ; mais je ne pouvais m'empêcber d'éprouver l'absolu renoncement du désespoir, et je me préparais mélancoliquement à cette mort que selon moi, ne pouvait différer au delà d'une heure, rien, puisque, à chaque nœud que filait le navire, la houle de cette mer noire et prodigieuse devenait plus lugu- brement effrayante. grande que celle Parfois, à de l'albatros, une la hauteur plus respiration nous manquait, et d'autres fois nous étions pris de vertige en descendant avec une horrible vélocité dans un enfer liquide où l'air devenait stagnant, et où aucun son ne pouvait troubler les sommeils du kraken. Nous étions au fond d'un de ces abîmes, quand un cri soudain de mon compagnon éclata sinistrement dans la nuit. — Voyez voyez! me ! criait-il tout-puissant! Voyez! voyez! dans les oreilles; Dieu MANUSCRIT TROUVÉ DANS UNE BOUTEILLE. 295 Gomme il parlait, j'aperçus une lumière rouge, d'un éclat sombre et triste, qui flottait sur le versant du gouffre immense où nous étions ensevelis, et jetait à notre bord un reflet vacillant. En levant les yeux, je vis un spectacle qui glaça mon sang. A une hauteur terrifiante, juste au-dessus de nous et sur la crête même du précipice, planait un navire gigantesque, de quatre mille tonneaux peut-être. Quoique juché au sommet d'une vague qui avait bien cent fois sa hauteur, il paraissait d'une dimension beaucoup plus grande que celle d'au- cun vaisseau de ligne ou de la Compagnie des Indes. Son énorme coque était d'un noir profond que ne tempérait aucun des ornements ordinaires d'un navire. Une simple rangée de canons s'allongeait de ses sabords ouverts et renvoyait, réfléchis par leurs surfaces polies, les feux d'innombrables fanaux de combat qui se ba- lançaient dans le le gréement. Mais ce qui nous inspira plus d'horreur et d'étonnement, c'est qu'il marchait toutes voiles dehors, en dépit de cette mer surnaturelle et de cette tempête effrénée. D'abord, quand nous l'aperçûmes, nous ne pouvions voir que son avant, parce qu'il ne s'élevait que lentement du noir et horrible gouffre moment, qu'il laissait derrière pause sur ce sommet vertigineux, vrement de sa propre élévation, s'inclina, — et enfin En ce moment, maîtrisa lui. Pendant un — moment d'intense terreur, — je — il fit une comme dans l'eni- — puis trembla, — glissa sur la pente. ne sais quel sang-froid soudain mon esprit. Me rejetant autant que possible HISTOIRES EXTRAORDINAIRES. '2% vers l'arrière, j'attendis sans trembler la catastrophe qui devait nous écraser. Notre propre navire, à la longue, ne luttait plus contre la mer et plongeait de l'avant. Le choc de la masse précipitée séquemment dans cette le frappa con- partie de la charpente qui était déjà sous l'eau, et eut pour résultat inévitable de me lancer dans le gréement de l'étranger. Comme je tombais, ce navire se souleva dans un temps d'arrêt, puis vira de bord à la confusion qui s'ensuivit ; et c'est, je présume, que je dus d'échapper à l'attention de l'équipage. Je n'eus pas grand'peine à me frayer un chemin, sans être vu, jusqu'à la principale écoutille, qui était en partie ouverte, et je trouvai bientôt une occasion cale. Pourquoi fis-je propice pour me cacher dans la ainsi? je ne saurais trop le dire. Ce qui m'induisit à me cacher fut peut-être un sentiment vague de terreur qui s'était emparé tout d'a- bord de mon esprit à l'aspect des nouveaux navigateurs. Je ne me souciais pas de me confier à une race de gens qui, d'après le coup d'œil sommaire que j'avais jeté sur eux, m'avaient offert le caractère d'une indélinissable étrangeté, et tant de motifs de doute et d'appréhension. C'est pourquoi je jugeai à propos de m' arranger une cachette dans la cale. J'enlevai une partie du faux bor- dage, de manière à me ménager une retraite commode entre les énormes membrures du navire. J'avais à peine achevé ma besogne, qu'un bruit de pas dans la cale me contraignit d'en faire usage. Un homme passa à côté de ma cachette d'un pas faible et MANUSCRIT TROUVE DANS UNE BOUTEILLE. 297 mal assuré. Je ne pus pas voir son visage, mais j'eus le loisir d'observer son aspect général. Il y avait en tout le caractère de la faiblesse et de la caducité. lui Ses genoux vacillaient sous la charge des années, et tout son être en tremblait. 11 se parlait à lui-même, marmottait d'une voix basse et cassée quelques mots d'une languequeje ne pus pas comprendre, et farfouillait dans un coin où Ton avait empilé des instruments d'un aspect étrange et des cartes marines délabrées. Ses manières étaient un singulier mélange de la maus- saderie d'une seconde enfance et de la dignité solennelle d'un dieu. A la longue, il remonta sur le pont, et je ne le vis plus. Un sentiment pour lequel je ne trouve pas de mot a pris possession de mon âme, — une sensation qui n'admet pas d'analyse, qui n'a pas sa traduction dans les lexiques l'avenir du passé, et pour laquelle je crains que lui-même ne trouve pas de clef. esprit constitué — Pour un comme le mien, cette dernière consi- dération est un vrai supplice. Jamais je ne pourrai, — je sens que je ne pourrai jamais être édifié relativement à la nature de mes idées. Toutefois, il n'est pas étonnant que ces idées soient indéfinissables, puisqu'elles sont puisées à des sources si entièrement neuves. Un nouveau sentiment Il — une nouvelle entité— y a déjà bien longtemps que première fois le est ajouté à mon âme. j'ai touché pour la pont de ce terrible navire, 17. et les HISTOIRES EXTRAORDINAIRES. 298 rayons de ma destinée vont, je crois, se concentrant et s'engioutissant gens ! un dans Incompréhensibles foyer. Enveloppés dans des méditations dont je ne puis deviner nature, la remarquer. Me ils passent à côté de moi sans cacher est pure folie car ce monde-là ne veut pas voir. 11 de me ma part, n'y a qu'un in- stant, je passais juste sous les yeux du second; peu de temps auparavant, je m'étais aventuré jusque dans la cabine du capitaine lui-même, et c'est là que je me suis procuré les moyens d'écrire ceci et tout ce qui pré- cède. Je continuerai ce journal de temps en temps. est vrai que je 1! ne puis trouver aucune occasion de le transmettre au monde; pourtant, j'en veux faire l'essai. Au dernier moment, j'enfermerai le manuscrit dans une bouteille, et je jetterai le tout à la mer. Un incident est survenu qui m'a de noiTvean donné lieu à réfléchir. De pareilles choses sont-elles l'opération d'un hasard indiscipliné? Je m'étais faufilé sur le pont et m'étais étendu, sans attirer l'attention de personne, sur un amas d'enfléchures et de vieilles voiles, dans le fond de la yole. Tout en rêvant à la singularité de ma destinée, je barbouillais, sans y penser, avec une brosse à goudron, les bords d'une bonnette soigneuse- ment pliée et posée à côté de moi sur un baril. La bonnette est mairitenant tendue sur ses bouts-dehors, et les touches irréfléchies de la brosse figurent le mot DÉCOUVERTE. J'ai fait récemment plusieurs observations sur la , MANUSCRIT TROUVÉ DANS UNE BOUTEILLE. 299 Structure du vaisseau. Quoique bien armé, ce n'est pas, je crois, un vaisseau de guerre. Son gréement, sa struc- ture, tout son équipement, repoussent une supposition de cette nature. Ce qu'il n'est pas, je le perçois facile- ment; mais ce qu'il est, je crains qu'il ne me soit im- comment cela se fait, possible de le dire. Je ne sais mais, en examinant son étrange modèle et la singulière forme de ses espars, ses proportions colossales, cette prodigieuse collection de voiles, son avant sévèrement simple et son arrière d'un style suranné, parfois que la sensation d'objets qui ne me semble me sont pas il inconnus traverse mon esprit comme un éclair, et toujours à ces ombres flottantes de la mémoire est mêlé un inexplicable souvenir de vieilles légendes étrangères et de siècles très-anciens. J'ai bien regardé la charpente du navire. Elle est faite de matériaux qui me sont inconnus. H y a dans le bois un caractère qui me frappe, comme le rendant, ce me semble, impropre à l'usage auquel il a été destiné. Je veux parler de son extrême porosité, considérée indé- pendamment des dégâts faits par les vers, qui sont une conséquence de la navigation dans ces mers, de la et pourriture résultant de la vieillesse. Peut-être trouvera-t-on mon observation quelque peu subtile mais il me semble que ce bois aurait tout le caractère du chêne espagnol, si le chêne espagnol pouvait être dilaté par des moyens artificiels. En relisant la phrase précédente, il me revient à HISTOIRES EXTRAORDINAIRES. 300 l'esprit un curieux apoplithegme d'un vieux loup de mer hollandais. — Gela est positif, disait-il toujours quand on exprimait quelque doute sur sa véracité, comme il est poy a une sitif qu'il mer où le navire lui-même grossit comme le corps vivant d'un marin. 11 y a environ une heure, je me suis senti la har- diesse de me glisser dans un groupe d'hommes de l'é- quipage. Ils n'ont pas eu l'air de faire attention à moi, et, quoique je me tinsse juste au milieu d'eux, ils pa- aucune conscience de ma présence. Comme celui que j'avais vu le premier dans la cale, ils raissaient n'avoir portaient tous les signes d'une vieillesse chenue. Leurs genoux tremblaient de faiblesse ; leurs épaules étaient arquées par la décrépitude; leur peau ratatinée fris- sonnait au vent; leur voix était basse, chevrotante et cassée; leurs yeux distillaient les larmes brillantes de la vieillesse, et leurs cheveux gris fuyaient terriblement dans la tempête. Autour d'eux, de chaque côté du pont, gisaient éparpillés des instruments mathématiques d'une structure très-ancienne et tout à fait tombée en désuétude. J'ai parlé installée. un peu plus haut d'une bonnette qu'on avait Depuis ce moment, le navire, chassé par le vent, n'a pas discontinué sa terrible course droit au sud, chargé de toute sa toile disponible, depuis ses pommes de mâts jusqu'à ses bouts-dehors inférieurs, MANUSCRIT TROUVE DANS UNE BOUTEILLE. 301 et plongeant ses bouts de vergues de le perroquet dans plus effrayant enfer liquide que jamais cervelle hu- maine ait pu concevoir. Je viens de quitter le pont, ne trouvant plus la place tenable ; cependant, l'équipage ne semble pas souffrir beaucoup. C'est pour moi le miracle des miracles qu'une si énorme masse ne soit pas engloutie tout de suite et pour toujours. Nous sommes condamnés, sans doute, bord de l'éternité, à côtoyer éternellement le sans jamais faire notre plongeon définitif dans le gouffre. Nous glissons avec la prestesse de l'hirondelle de mer sur des vagues mille fois plus effrayantes qu'aucune de celles que j'ai jamais vues; et des ondes colossales élèvent leurs têtes au-dessus de nous comme des démons de l'abîme, mais comme des démons restreints aux simples menaces et auxquels il est défendu de détruire. Je suis porté à attribuer cette bonne chance perpétuelle à la seule cause naturelle qui puisse légitimer un pareil effet. Je suppose que le navire est soutenu par quelque fort courant ou remous sous-marin. J'ai vu le capitaine face à face, et dans sa propre ca- bine; mais, comme je m'y attendais, il n'a fait aucune attention à moi. Bien qu'il n'y ait rien dans sa physio- nomie générale qui révèle, pour l'œil du premier venu, quelque chose de supérieur on d'inférieur à l'homme, toutefois l'étonnement que j'éprouvai à son aspect se mêlait d'un sentiment de respect et de terreur irrésistible. 11 est à peu près de ma taille, c'est-à-dire de HISTOIRES EXTRAORDINAIRES. - 302 cinq pieds huit pouces environ. 11 est bien proportionné, bien pris dans son ensemble; mais cette constitu- tion n'annonce ni vigueur particulière, ni quoi que ce soit de remarquable. Mais c'est la singularité de l'ex- — pression qui règne sur sa face, rible, saisissante si c'est l'intense, ter- évidence de la vieillesse, si entière, absolue, qui crée dans mon esprit un sentiment, une sensation ineffable. semble porter le — Son front, quoique peu ridé, sceau d'une myriade d'années. Ses cheveux gris sont des archives du passé, et ses yeux, plus gris encore, sont des sibylles de l'avenir. Le plan- cher de sa cabine était encombré d'étranges in-folio à fermoirs de fer, d'instruments de science usés et d'anciennes cartes d'un style complètement oublié. Sa tête était appuyée sur ses mains, et d'un œil ardent et in- quiet il dévorait un papier que je pris pour une mission, et qui, royale. Il en — comme — aperçu dans se parlait à lui-même, matelot que j'avais tait tout cas, portait une la cale, com- signature le premier et marmot- d'une voix basse et chagrine quelques syllabes d'une langue étrangère; côté de lui, oreille il et, bien que je fusse tout à me semblait que sa voix arrivait à mon de la distance d'un mille. Le navire avec tout ce qu'il contient est imprégné de l'esprit des anciens âges. Les hommes de l'équipage glissent çà et là comme les ombres des siècles enterrés; dans leurs yeux vit une pensée ardente et, quand, et inquiète; sur mon chemin, leurs mains tombent dans MANUSCRIT TllOUVÉ DANS UNli BOUTEILLE. 30;J la lumière effarée des fanaux, j'éprouve quelque chose que je n'ai jamais éprouvé jusqu'à présent, quoique toute ma vie j'aie eu la folie des antiquités, et que je me sois baig:né dans l'ombre des colonnes ruinées de Balbek, de Tadmor et de Persépolis, tant qu'à la fm mon àme elle-même est devenue une ruine. Quand je regarde autour de moi, je suis honteux de mes premières terreurs. Si la tempête qui nous a poursuivis jusqu'à présent me fait trembler, ne devrais-je pas être frappé d'horreur devant cette bataille du vent et de l'Océan, dont les mots vulgaires : tourbillon et si- moun, ne peuvent pas donner la moindre idée? Le navire est littéralement enfermé dans les ténèbres d'une éternelle nuit plus ; et dans un chaos d'eau qui n' écume mais à une distance d'une lieue environ de chaque côté, nous pouvons apercevoir, indistinctement et par intervalles, de prodigieux remparts de glace qui mon- tent vers le ciel désolé et ressemblent aux murailles de l'univers! Comme je l'avais pensé, le navire est évidemment dans un courant, ainsi une — si l'on peut proprement appeler marée qui va mugissant et hurlant à travers les blancheurs de la glace, et fait entendre du côté du sud un tonnerre plus précipité que celui d'une cataracte tombant à pic. HISTOIRES EXTRAORDINAIRES. 304 Concevoir riiorreur de mes sensations est, je crois, chose absolument impossible; cependant, de pénétrer les la curiosité mystères de ces effroyables régions surplombe encore mon désespoir et suffit à me réconcilier avec le plus hideux aspect de la mort. Il est évi- dent que nous nous précipitons vers quelque entraî- nante découverte, dont la — quelque incommunicable secret connaissance implique' la mort. Peut-être ce courant nous conduit-il au pôle sud lui-même. 11 faut avouer que cette supposition, si étrange en apparence, a toute probabilité pour elle. L'équipage se promène sur le pont d'un pas tremblant et inquiet; mais il y a dans toutes les physiono- mies une expression qui ressemble plutôt à l'ardeur de l'espérance qu'à l'apathie du désespoir. Cependant nous avons toujours le vent arrière, et, comme nous portons une masse de toile, le navire s'enlève quelquefois en grand hors de la mer. reur sur horreur! — la glace Oh! hor- s'ouvre soudainement à droite et à gauche, et nous tournons vertigineusement dans d'immenses cercles concentriques, tout autour des bords d'un gigantesque amphithéâtre, dont les murs perdent leur sommet dans les ténèbres et l'espace. Mais il ne me reste que peu de temps pour rêver à ma destinée! Les cercles se rétrécissent rapidement, — nous plongeons follement dans — travers mugissement, l'étreinte billon, et, à le et le détonnement de l'Océan et du tour- le beuglement de la tempête, le na- MVNUSCRIT TROUVÉ DANS Ux\E BOUTEILLE. 305 vire tremble, — oh! Dieu! — il se dérobe, — il sombre M 1. Le Manuscrit trouvé dans une bouteille fut publié pour la première fois en 1831, et ce ne fut que bien des années plus tard que j'eus connaissance des cartes de Mercator, dans lesquelles ori rOcéan se précipiter par quatre embouchures dans le gouffre polaire (au nord) et s'absorber dans les entrailles de la terre le pôle lui-même y est figuré par un rocher noir, s'élevant à une voit ; prodigieuse hauteur. — E. A. P. UNE DESCENTR DANS LE MAELSTROM Los voies de Dieu, dans la nature comme dans l'ordre de la Provideuce, ne sont point nos voies; et les types que nous concevons n'ont aucune mesure commune avec la vastitude, la profondeur et l'incompréhensibilité de ses œuvres, qui contiennent en elles un ahlniQ plus profond que le puits de Démocrile. Joseph Glanvill. Nous avions atteint Le vieux homme élevé. le sommet du rocher le plus , pendant quelques minutes, sembla trop épuisé pour parler. — n'y a pas encore bien longtemps, — à — vous aurais guidé par aussi bien que dit-il Il fin, je ici plus jeune de mes arrivé fils. Mais, il y a trois ans, il la le m'est une aventure plus extraordinaire que n'en es- suya jamais un être mortel, ou du moins telle que ja- mais homme n'y a survécu pour la raconter, et les six mortelles heures que j'ai endurées m'ont brisé le corps et l'âme. Vous me croyez très- vieux, mais je ne le suis pas. 11 a suffi du quart d'une journée pour blanchir ces cheveux noirs comme du jais, affaiblir mes membres HISTOIRES EXTRAORDINAIRES. 308 et détendre mes nerfs au point de trembler après le moindre effort et d'être effrayé par une ombre. Savezvous bien que je puis à peine, sans attraper le vertige, regarder par-dessus ce petit promontoire. Le petit promontoire sur le bord duquel il s'était si négligemment jeté pour se reposer, de façon que la partie la plus pesante de son corps surplombait, et qu'il n'était garanti d'une chute que par le point d'appui que prenait son coude sur l'arête extrême et glissante, — le petit promontoire s'élevait à quinze ou seize cents pieds environ d'un chaos de rochers situés au-dessous de nous, — immense précipice de granit luisant et noir. Pour rien au monde je n'aurais voulu me hasarder à six pieds du bord. Véritablement, j'étais si profondé- ment agité par la situation périlleuse de mon compagnon, que je me laissai tomber tout de mon long sur le sol, m'accrochant à quelques arbustes voisins, n'osant pas même lever les yeux vers le ciel. Je m'efforçais en vain de me débarrasser de l'idée que la fureur du vent mettait en danger la base même de la montagne. 11 me fallut du temps pour me raisonner et trouver le courage de me mettre sur mon séant et de regarder au loin dans l'espace. — 11 vous faut prendre me dit le guide, le dessus sur ces lubies-là, car je vous ai amené ici pour vous faire voir à loisir le théâtre de l'événement dont je parlais tout à l'heure, et pour vous raconter toute l'his- toire avec la scène » même sous vos yeux. Nous sommes maintenant, reprit-il avec cette ma- UNE DESCENTE DANS LE MAELSÏROM. nière minutieuse qui le caractérisait, 309 nous sommes maintenant sur la côte même de Norvège, au 68« degré de latitude, dans la grande province de Nortland et dans le lugubre dont nous occupons district le de Lofoden. La montagne sommet est Helseggen, la Nua- geuse. Maintenant, levez-vous un peu; accrochez-vous au gazon, si vous sentez venir le vertige, — et regardez — c'est cela, au delà de cette ceinture de vapeurs qui nous cache la mer à nos pieds. Je regardai vertigineusement, et je vis une étendue de mer, dont la couleur d'encre tout d'abord le vasfte me rappela tableau du géographe Nubien et sa Mer des Ténèbres. C'était un panorama plus effroyable- ment désolé qu'il n'est donné à une imagination hu- maine de le concevoir. A droite et à gauche, aussi loin que l'œil pouvait atteindre, s'allongeaient, comme les remparts du monde, les lignes d'une falaise horrible- ment noire et surplombante, dont le caractère sombre était puissamment renforcé par le ressac qui montait jusque sur sa crête blanche et lugubre, hurlant et mugissant éternellement. Juste en face du promontoire sur le sommet duqiiel nous étions placés, à une distance de cinq ou six milles en mer, on apercevait une île qui avait l'air désert, ou plutôt on la devinait au moutonnement énorme des brisants dont elle était enveloppée. A deux milles environ plus près de la terre, se dressait un autre îlot plus petit, horriblement pier- reux et stérile, et entouré de groupes interrompus de roches noires. , HISTOIRES EXTRAORDINAIRES. 310 L'aspect de l'Océan, dans l'étendue comprise entre le rivage et l'île la plus éloignée, avait quelque chose d'extraordinaire. En ce moment même, il souillait du côté de la terre une si forte brise, qu'un brick, tout au large, était à la cape avec deux ris dans sa toile et que sa coque disparaissait quelquefois tout entière; et pourtant il n'y avait rien qui ressemblât à une houle faite, mais seulement, et en dépit du vent, un clapotement d'eau, bref, vif et tracassé dans tous les sens; — très- peu d'écume, excepté dans le voisinage immédiat des rochers. — L'île que vous voyez là-bas, reprit le vieux homme, est appelée par les Norvégiens Yurrgh. Celle qui est à moitié chemin est Moskoe. Celle qui est à un mille au nord est Ambaaren. Là-bas sont Islesen, Hotholm — entre — Otterholm, Flimen, Sandflesen Keildhelm, Suarven et Buckolm. Plus loin, Moskoe et Vurrgli, et Stockholm. Tels sont — mais pourquoi les vrais ai-je noms de ces endroits; jugé nécessaire de vous les nommer, je n'en sais rien, je n'y puis rien comprendre, — pas plus que vous. — Entendez-vous quelque chose? Voyez-vous quelque changement sur l'eau? Nous étions depuis dix minutes environ au haut de Helseggen, où nous étions montés en partant de l'intérieur de Lofoden, de sorte que nous n'avions pu aper- cevoir la mer que lorsqu'elle nous avait apparu tout d'un coup du sommet le plus élevé. Pendant que le homme parlait, j'eus la perception d'un bruit très-fort et qui allait croissant, comme le mugissevieux UNE DESCENTE DANS LE MAELSTROM. 311 ment d'un immense troupeau de buffles dans une prairie d'Amérique; et, au moment même, je vis ce que les marins appellent le caractère clapoteux de la mer se changer rapidement en un courant qui se faisait vers l'est. Pendant que je regardais, ce courant prit une prodigieuse rapidité. Chaque instant ajoutait à sa vitesse, — à son impétuosité déréglée. En cinq minutes, toute la mer, jusqu'à Vurrgh, fut fouettée par une indomptable furie; mais c'était entre Moskoe et la côte que dominait principalement le vacarme. Là, le vaste lit des eaux, sillonné et couturé par mille courants contraires, éclatait sou- dainement en convulsions frénétiques, bouillonnant, sifflant, pirouettant en gigantesques et innombrables tourbillons, tout entier vers l'est avec feste que — haletant, et tournoyant et se ruant une rapidité qui ne se mani- dans des chutes d'eau précipitées. Au bout de quelques minutes, le tableau subit un autre changement radical. La surface générale devint un peu plus unie, et les tourbillons disparurent un à un, pendant que de prodigieuses bandes d'écume ap- parurent là où je n'en avais vu aucune jusqu'alors. Ces bandes, à la longue, s'étendirent à une grande distance, et, se combinant entre elles, elles adoptèrent le mou- vement giratoire des tourbillons apaisés et semblèrent former le germe d'un vortex plus vaste. Soudainement, très-soudainement, celui-ci apparut et prit une existence distincte et définie, dans un cercle de plus d'un mille de diamètre. Le bord du tourbillon était marqué par une large ceinture d'écume lumineuse; mais pas HISTOIRES EXTRAORDINAIRES 312 une parcelle ne glissait dans la gueule du terrible entonnoir, dont l'intérieur, aussi loin que l'œil pouvait y plonger, était fait d'un mur liquide, poli, brillant et d'un noir de jais, faisant avec l'horizon un angle de /i5 degrés environ, tournait sur lui-même sous l'in- fluence d'un mouvement étourdissant, et projetant dans les airs une voix effrayante, moitié cri, moitié rugisse- ment, telle que la puissante cataracte du Niagara elle- même, dans ses convulsions, n'en a jamais envoyé de pareille vers le ciel. La montagne tremblait dans sa base même roc remuait. Je me jetai à plat ventre, et, , et le dans un excès d'agitation nerveuse, je m'accrochai au maigre gazon. — Ceci, dis-je enfin au vieillard, ne peut pas être autre chose que le grand tourbillon du Maelstroni. — On l'appelle quelquefois ainsi, dit-il; mais nous autres Norvégiens, nous le nommons le Moskoe-Strom, de l'île de Moskoe, qui est située à moitié chemin. Les descriptions ordinaires de ce tourbillon ne m'avaient nullement préparé à ce que je voyais. Celle de Jonas Ramus, qui est peut-être plus détaillée qu'aucune, ne donne pas la plus légère idée de la magnificence et de l'horreur du tableau, — ni de l'étrange et ravissante sensation de nouveauté qui confond le spectateur. Je ne sais pas précisément de quel point de vue ni à quelle heure l'a vu l'écrivain en question; mais ce ne peut être ni du sommet de Helseggen, ni pendant une tempête. Il y a néanmoins quelques passages de sa UNE DESCENTE DANS LE MAELSTROM. description qui peuvent être cités pour 313 détails, les quoiqu'ils soient très-insuflisants pour donner une im- pression du spectacle. — Entre Lofuden l'eau est et Moskoe, dit-il, la profondeur de de trente-six à quarante brasses; mais, de l'autre côté, du côté de Ver (il veut dire Vurrgh), cette profondeur diminue au point qu'un navire ne pourrait y chercher un passage sans courir le danger de se déchirer sur les roches, ce qui peut arriver par le temps le plus calme. Quand vient la marée, le courant se jette dans l'espace compris entre Lofoden et Moskoe avec une tumultueuse rapidité; mais le rugissement de son terrible reflux est à peine égalé par celui des plus hautes et des plus terribles cataractes; le bruit se fait entendre à plusieurs lieues, et les tourbillons ou tournants creux sont d'une telle étendue et d'une telle profondeur, que, si un navire entre dans la région de son attraction , il est inévitablement traîné au fond, et, là, déchiré en rochers; et, quand le absorbé et en- morceaux contre les courant se relâche, les débris sont rejetés à la surface. Mais ces intervalles de tranquillité n'ont lieu qu'entre le reflux et le flux, par un temps calme, et ne durent qu'un quart d'heure; puis la violence du courant revient graduellement. » Quand il bouillonne le plus et quand sa force est accrue par une tempête, cher, il est dangereux d'en appro- même d'un mille norvégien. Des barques, des yachts, des navires ont été entraînés pour n'y avoir pas pris garde avant de se trouver à portée de son attrac***** 18 HISTOIRES EXTRAORDINAIRES. 314 tioii. 11 aiT-ive assez fréquemment que des baleines viennent trop près du courant et sont maîtrisées par sa violence; et il est impossible de décrire leurs mugis- sements et leurs beuglements dans leur inutile effort pour se dégager. Une fois, un ours, essayant de passer à la nage le )) détroit entre Lofoden et Moskoe, fut saisi par le cou- rant et emporté au fond ; il rugissait si effroyablement qu'on l'entendait du rivage. De vastes troncs de pins ^et de sapins, engloutis par le courant, reparaissent brisés et déchirés, au point qu'on dirait qu'il leur a poussé des poils. Cela démontre clairement que le fond est fait de roches pointues sur lesquelles roulés çà et là. Ce courant est réglé par ont été ils le flux et le reflux de la mer, qui a constamment lieu de six en six heures. Dans Tannée 1645, le dimanche de la Sexagé- sime, de fort grand matin, il se précipita avec un tel fracas et une telle impétuosité, que des pierres se dé- tachaient des maisons de la côte... En ce qui concerne la profondeur de l'eau, je ne comprends pas comment on a pu s'en assurer dans la proximité immédiate du tourbillon. Les quarante brasses doivent avoir trait seulement aux parties du canal qui sont tout près du rivage, soit de Moskoe, soit de Lofo- den. La profondeur au centre du Moskoe-Strom doit êfre incommensurablement plus grande, et pour en acquérir la certitude, il suffit, de jeter un coup d'œil oblique dans l'abîme du tourbillon, quand on est sur le sommet le plus élevé de Helseggen. Eu plongeant UNE DESCENTE DANS LE MAELSÏROM. 315 mon regard du haut de ce pic dans le Phlégéthon hurlant, je ne pouvais m'empêcher de sourire de la simplicité avec laquelle le choses bon Jonas Ramus raconte, comme difficiles à croire, baleines; car il ses anecdotes d'ours et de me semblait que c'était chose évi- dente de soi que le plus grand vaisseau de ligne possible arrivant dans le rayon de cette mortelle attrac- tion, devait y résister aussi peu qu'une plutne à un coup de vent et disparaître tout en grand et tout d'un coup. Les explications qu'on a données du phénomène, — dont quelques-unes, je me le rappelle, me paraissaient — avaient main- suffisamment plausibles à la lecture, tenant un aspect très-différent et très-peu satisfaisant. L'explication généralement reçue est que, comme les trois petits tourbillons des îles Féroë, celui-ci « n'a pas d'autre cause que le choc des vagues montant et re- tombant, au flux et au reflux, le long d'un banc de roches qui endigue les eaux et les rejette en cataracte; et qu'ainsi, plus la marée s'élève, plus la chute est pro- fonde, et que le résultat naturel est un tourbillon ou vortex, dont la prodigieuse puissance de succion est suffisamment démontrée par de moindres exemples. )> Tels sont les termes de V Encyclopédie britannique. Kir- cher et d'autres imaginent qu'au milieu du canal du Maelstrom est un abîme qui traverse le globe et abou-tit dans quelque région très-éloignée; — le golfe de Bothnie a même été désigné une fois un peu légèrement. Cette opinion assez puérile était celle à laquelle, pendant que je contemplais le lieu, mon imagination donnait le HISTOIRES EXTRAORDliN AIKES. 316 plus volontiers son assentiment; et, comme j'en faisais part an guide, je fus assez surpris de l'entendre me dire que, bien que telle fût l'opinion presque générale des Norvégiens à ce sujet, ce n'était néanmoins pas Quant à cette idée, la sienne. incapable de la il confessa qu'il était comprendre, et je finis par être d'ac- cord avec lui; car, pour concluante qu'elle soit sur le papier, elle devient absolument inintelligible et absurde à côté du tonnerre de l'abîme. — Maintenant que vous avez bien vu le tourbillon, me dit le vieux homme, si vous voulez que nous nous glissions derrière cette roche, sous le vent, de manière qu'elle amortisse le vacarme de l'eau, je vous conterai une histoire qui vous convaincra que je dois en savoir quelque chose, du Moskoe-Strom Je ! me plaçai comme il le désirait, et il commença — Moi : et mes deux frères, nous possédions autrefois un semaque gréé en goélette, de soixante et dix tonneaux à peu près, avec lequel nous péchions habituellement parmi les îles au delà de Moskoe, près de Vurrgh. Tous les violents remous de mer donnent une bonne pêche, pourvu qu'on s'y prenne en temps opportun ait le les et qu'on courage de tenter l'aventure; mais, parmi tous hommes de la côte de Lofoden, nous trois seuls, nous faisions notre métier ordinaire d'aller aux îles, comme je vous dis. Les pêcheries ordinaires sont beaucoup plus bas vers le sud. On y peut prendre du poisson à toute heure, sans courir grand risque, et naturel- lement ces endroits-là sont préférés; mais les places de UNE DESCENTE DANS LE MAELSTROM. choix, par ici, 317 entre les rochers, donnent non-seule- ment le poisson de la plus belle qualité, mais aussi en bien plus grande abondance; si bien que nous prenions souvent en un seul jour ce que les timides dans le métier n'auraient pas pu attraper tous ensemble en une semaine. En somme, nous faisions de cela une espèce de spéculation désespérée, — le risque de la vie rem plaçait le travail, et le courage tenait lieu de capital. )) Nous abritions notre semaque dans une anse à cinq milles sur la côte au-dessus de celle-ci habitude, par le beau temps, de ; et c'était notre profiter du répit de quinze minutes pour nous lancer à travers le canal principal du Moskoe-Strom, bien au-dessus du trou, et d'aller jeter l'ancre quelque part dans la proximité d'Otterholm ou de Sandflesen, où les remous ne sont pas aussi violents qu'ailleurs. Là, nous attendions ordinairement, pour lever l'ancre et retourner chez nous, à peu près jusqu'à l'heure de l'apaisement des eaux. Mous ne nous aventurions jamais dans cette expédition sans un bon vent largue pour aller et revenir, — un — vent dont nous pouvions être sûrs pour notre retour, et nous nous sommes rarement trompés sur ce point. Deux fois, en six ans, nous avons été forcés de passer la nuit à l'ancre par suite d'un calme plat, ce qui est un cas bien rare dans ces parages; et, une autre fois, nous sommes restés à terre près d'une semaine, affa- més jusqu'à la mort, grâce à un coup de vent qui se mit à souffler peu de temps après notre arrivée et rendit le canal trop orageux pour songer à le traverser. 18. HISTOIRES EXTRAORDINAIRES. 318 Dans cette occasion, nous aurions été entraînés au large en dépit de tout (car les tourbillons nous ballottaient çà et là avec une telle violence, qu'à la fin nous avions chassé sur notre ancre faussée), si nous n'avions dérivé dans un de ces innombrables courants qui se forment, ici demain ailleurs, aujourd'hui, et et qui nous con- duisit sous le vent de Flimen, où, par bonheur, nous pûmes mouiller. » Je ne vous dirai pas la vingtième partie des dangers que nous essuyâmes dans mauvais parage, les pêcheries, — c'est un même par le beau temps, — mais nous trouvions toujours moyen de défier le Moskoe- Strom sans accident; parfois pourtant le cœur me montait aux lèvres quand nous étions d'une minute en avance ou en retard sur l'accalmie. Quelquefois, lèvent n'était pas aussi vif que nous l'espérions en la voile, et alors mettant à nous allions moins vite que nous ne l'aurions voulu, pendant que le courant rendait le se- maque plus difficile à gouverner. » Mon frère aîné avait un fils âgé de dix-huit ans, et j'avais pour mon compte deux grands garçons. Ils nous eussent été d'un grand secours dans de pareils cas, soit qu'ils eussent pris les avirons, soit qu'ils eussent péché à l'arrière; — mais, vraiment, bien que nous consen- tissions à risquer notre vie, nous n'avions pas le cœur de laisser ces jeunesses affronter le danger; — car, tout bien considéré, c'était un horrible danger, c'est la pure vérité. )) Il y a maintenant trois ans moins quelques jours UNE DESCENTE DANS LE MAELSTRO:*!. 31'J qu'arriva ce que je vais vous raconter. Celait le 10 juillet 18..., jamais, un jour que les gens de ce pays n'oublieront — car ce fut un jour où souffla la plus horrible tempête qui soit jamais tombée de la calotte des cienx. Cependant, toute la matinée et même fort avant dans l'après-midi, nous avions eu une jolie brise bien faite du sud-ouest, le soleil était superbe, si bien que le plus vieux loup de mer n'aurait pas pu prévoir ce qui allait arriver. » Nous étions passés tous les trois, mes deux frères et moi, à travers les îles à deux heures de l'après-midi environ, et nous eûmes bientôt chargé le semaque de fort beau poisson, qui trois — l'avions était plus jamais — nous l'avions remarqué tous abondant ce jour- là que nous ne vu. était Il juste sept heures à ma montre quand nous levâmes l'ancre pour retourner chez nous, de manière à faire le plus dangereux du Strom dans l'intervalle des eaux tranquilles, que nous savions avoir lieu à huit heures. » Nous partîmes avec une bonne brise à tribord, et, pendant quelque temps, nous filâmes très-rondement, sans songer réalité, le moins du monde au danger; car, en nous ne voyions pas la moindre cause d'appré- hension. Tout à coup nous fûmes masqués par une saute de vent qui venait de Helseggen, Cela était tout à fait extraordinaire, était jamais arrivée, — — c'était une et je chose qui ne nous commençais à être un peu inquiet, sans savoir exactement pourquoi. Nous fîmes arriver au vent, mais nous ne pûmes jamais HISTOIRES EXTRAORDINAIRES. 320 fendre les remous, et j'étais sur le point de proposer de retourner au mouillage, quand, regardant à rière, l'ar- nous vîmes tout l'horizon enveloppé d'un nuage singulier, couleur de cuivre, qui montait avec la plus étonnante vélocité. )) En même temps, la brise qui nous avait pris en tête tomba, et, surpris alors par im calme plat, nous déri- vâmes à la merci de tous les courants. Mais cet état de choses ne dura pas assez longtemps pour nous donner le temps d'y réfléchir. En moins d'une minute, la tem- — une minute après, entièrement chargé, — devint soudainement pête était sur nous, le ciel était et il si noir, qu'avec les embruns qui nous sautaient aux yeux nous ne pouvions plus nous voir l'un l'autre à bord. )) Vouloir décrire un pareil coup de vent, ce serait folie. Le plus vieux marin de Norvège n'en a jamais essuyé de pareil. Nous avions amené toute la toile avant que le coup de vent nous surprît; mais, dès première bord, rafale, nos deux mâts vinrent la par-dessus comme s'ils avaient été sciés par le pied, — le grand mât emportant avec lui mon plus jeune frère qui s'y était accroché par prudence. » Notre bateau était bien le plus léger joujou qui eut jamais glissé sur la mer. Il avait un pont effleuré avec une seule petite écoutille à l'avant, et nous avions toujours eu pour habitude de la fermer solidement en traversant le Strom, bonne précaution dans une mer clapoteuse. Mais, dans cette circonstance présente, nous aurions sombré du premier coup, — car, pendant DESCENTE DANS LE xMAELSTROM. UiNE 321 quelques instants, nous fûmes littéralement ensevelis Gomment mon frère aîné échappa-t-il à la sous l'eau. mort? je ne puis le dire, je n'ai jamais pu me l'expli- quer. Pour ma part, à peine avais-je lâché la misaine, que je m'étais jeté sar le pont à plat ventre, contre l'étroit plat-bord de l'avant, et les les pieds mains accro- chées à un boulon, auprès du pied du mât de misaine. Le pur instinct m'avait fait agir ainsi, — c'était indu- ditablement ce que j'avais de mieux à faire, j'étais trop )) — car ahuri pour penser. Pendant quelques minutes, nous fûmes complè- tement inondés, comme je vous tout ce temps, je retins le disais, et, pendant ma respiration et me cram- ponnai à l'anneau. Quand je sentis que je ne pouvais pas rester ainsi plus longtemps sans être suffoqué, je me dressai sur mes genoux, tenant toujours bon avec mes mains, et je dégageai ma tête. Alors, notre petit bateau donna de lui-même une secousse, juste comme un chien qui sort de l'eau, et se leva en partie au- dessus de la mer. Je m'efforçais alors de secouer de mon mieux la stupeur qui m'avait envahi et de recouvrer suffisamment mes esprits pour voir ce avait à faire, quand je sentis sissait le bras. C'était quelqu'un qui qu'il y me sai- mon frère aîné, et mon cœur en sauta de joie, car je le croyais parti par-dessus bord; — mais, un moment après, toute en horreur, oreille, )) il cette joie se changea quand, appliquant sa bouche à mon vociféra ce simple mot : Le Moskoe-Strom ! Personne ne saura jamais ce que furent en ce mo HISTOIRES EXTRAORDINAIRES. 322 ment mes pensées. Je frissonnai de la tête aux pieds, comme pris du plus violent accès de fièvre. prenais Je com- suffisamment ce qu'il entendait par ce seul — je savais bien ce qu'il voulait me faire en- mot, tendre! Avec le vent qui nous poussait maintenant, nous étions destinés au tourbillon du Strom, et rien ne pouvait nous sauver » ! Vous avez bien compris qu'en traversant le canal de Strom, nous faisions toujours notre route bien audessus du tourbillon, me, et encore même par le temps le plus cal- avions-nous bien soin d'attendre et d'épier le répit de la marée; mais, maintenant, nous courions droit sur pareille tempête! « rons juste au le gouffre lui-même, et avec une A coup sûr, pensai-je, nous y se- moment de l'accalmie^ il y a là encore un petit espoir. » Mais, une minute après, je me maufou pour rêver d'une espé- dissais d'avoir été assez rance quelconque. Je voyais parfaitement que nous étions condamnés, eussions-nous été ne sais combien de canons )) un vaisseau de je ! En ce moment, la première fureur de la tempête était passée, ou peut-être ne la sentions-nous pas autant parce que nous fuyions devant; mais, en tout cas, la mer, que le vent avait d'abord maîtrisée, plane et écu- meuse, se dressait maintenant en véritables montagnes. Un changement ciel. singulier avait eu lieu aussi dans le Autour de nous, dans toutes les directions, il était toujours noir commedela poix, mais presque au-dessus de nous il s'était fait une ouverture circulaire, — un — UNE DESCENTE DANS LE MAELSTROM. ciel clair, — clair comme je ne l'ai jamais vu, — bleu brillant et foncé, dissait la 323 — d'un et à travers ce trou resplen- lune avec un éclat que je ne pleine lui avais jamais cdnnu. Elle éclairait toutes choses autour de nous avec Dieu ! la plus grande netteté, quelle scène à éclairer » Je fis — mais, grand ! un ou deux efforts pour parler à mon frère ; mais le vacarme, sans que je pusse m'expliquer com- ment, était accru à un tel point, que faire je ne pus lui entendre un seul mot, bien que je criasse dans son oreille de toute la force de mes poumons. Tout -à coup il secoua la )) devint pâle comme la mort, et comme pour me dire Écoute tête, leva un de ses doigts ! : D'abord, je ne compris pas ce qu'il voulait dire, mais bientôt une épouvantable pensée se fit jour en moi. Je tirai ma montre de mon gousset. Elle ne mar- chait pas. Je regardai le cadran au clair de la lune, et je fondis en larmes en la jetant au loin Elle s'était arrêtée à sept passer' le répit de la était )) marée, Jieures! et le dans l'Océan. Nous avions tourbillon laissé du Strom dans sa pleine furie! Quand un navire est bien construit, proprement équipé et pas trop chargé, les lames, par une grande brise, et quand il est au large, semblent toujours étrange à un homme de terre, s'é- — ce qui paraît — ce qu'on appelle, chapper de dessous sa quille, très- et en langage de bord, chevaucher {riding.) Cela allait bien, tant que nous grimpions lestement sur la houle; mais, actuellement, une mer gigantesque venait nous HISTOIRES EXTRAORDINAIRES. 324 prendre par notre arrière et nous enlevait avec elle, — haut, haut, — comme pour nous pousser jusqu'au ciel. si Je n'aurais jamais cru qu'une lame pût monter haut. Puis nous descendions en faisant une courbe, une glissade, un plongeon, qui me donnait la nausée et le vertige, comme si je tombais en rêve du haut d'une immense montagne. j'avais jeté M^ais, du haut de la lame, un rapide coup d'œil autour de moi, — et ce seul coup d'œil avait suffi. Je vis exactement notre position en une seconde. Le tourbillon de Moskoe- Strom était à un quart de mille environ, droit devant nous, mais il ressemblait aussi peu au Moskoe-Strom de tous les jours que ce tourbillon que vous voyez maintenant ressemble à un remous de moulin. Si je n'avais pas su où nous étions et ce que nous avions à attendre, je n'aurais pas reconnu l'endroit. Tel que je le vis, je fermai involontairement les yeux d'horreur; mes paupières se collèrent comme dans un spasme. )) Moins de deux minutes après, nous sentîmes tout à coup la vague s'apaiser, et nous fûmes enveloppés d'écume. Le l^ateau fit un brusque demi-tour par bâbord, et partit dans cette nouvelle direction comme Au même foudre. perdit dans tel que la instant, le rugissement de l'eau se une espèce de clameur aiguë, — un son vous pouvez le concevoir en imaginant les sou- papes de plusieurs milliers de steamers lâchant à la fois leur vapeur. Nous étions alors dans la ceinture moutonneuse qui cercle toujours le tourbillon ; et je UNE DESCENTE DANS LE MAELSTROM. 325 croyais naturellement qu'en une seconde nous allions plonger dans le gouffre, au fond duquel nous ne pouvions pas voir distinclement, en raison de la prodi- gieuse vélocité avec laquelle nous y étions entraînés. Le bateau ne semblait pas plonger dans l'eau, mais la raser, comme une bulle d'air qui voltige sur la surface de la lame. Nous avions le tourbillon à tribord, et à bâbord se dressait le vaste Océan que nous venions de quitter. Il s'élevait comme un mur gigantesque se tor- dant entre nous et l'horizon. » Cela peut paraître étrange; mais alors, quand nous gueule même de l'abîme, je me sentis plus de sang-froid que quand nous en approchions. fûmes dans la Ayant fait mon deuil de toute espérance, je fus délivré d'une grande partie de cette terreur qui m'avait d'abord écrasé. Je suppose que c'était le désespoir qui roi- dissait mes nerfs. » Vous prendrez peut-être cela pour une fanfaronnade, mais ce que je vous dis est la vérité : je commençai à songer quelle magnifique chose c'était de mourir d'une pareille manière, et combien il moi de à était sot m'occuper d'un aussi vulgaire intérêt que ma conser- vation individuelle, en face d'une si prodigieuse manifestation de la puissance de Dieu. Je crois que je rougis de honte quand cette idée traversa mon esprit. Peu d'instants après, je fus possédé de la plus ardente curiosité relativement au tourbillon lui-même. Je sentis positivement le dèslr d'explorer ses profondeurs, même au prix du sacrifice que ***** j'allais faire; mon principal j9 HISTOIRES EXTRAORDINAIRES. 326 chagrin était de penser que je ne pourrais jamais raconter à mes vieux camarades les mystères que j'allais connaître. C'étaient là, sans doute, de singulières pensées pour occuper l'esprit d'un reille extrémité, que lors les — et j'ai homme dans une pa- souvent eu l'idée depuis évolutions du bateau autour du gouffre m'avaient un peu étourdi la tête. Il )) y eut une autre circonstance qui contribua à me rendre maître de moi-même; ce fut la complète cessation du vent, qui ne pouvait plus notre situation actuelle : nous atteindre dans — car, comme vous pouvez en juger par vous-même, la ceinture d'écume est considérablement au-dessous du niveau général de l'Océan, et ce dernier nous dominait crête d'une haute et noire êtes jamais trouvé maintenant comme la montagne. Si vous ne vous en mer par une grosse tempête, vous ne pouvez vous faire une idée du trouble d'esjorit occasionné par l'action simultanée du vent et des embruns. Cela vous aveugle, vous étourdit, vous étrangle et vous ôte toute faculté d'action ou de réflexion. Mais nous étions maintenant grandement soulagés de tous ces embarras, mort, à — comme ces misérables condamnés à qui on accorde dans leur prison quelques petites faveurs qu'on leur refusait tant que l'arrêt n'était pas prononcé. )) il Combien de fois fîmes-nous le tour de cette ceinture, m'est impossible de le dire. Nous courûmes tout au- tour, pendant une heure à peu près; nous volions plutôt que nous ne flottions, et nous nous rapprochions . UNE DESCENTE DANS LE MAELSTROM. toujours de plus en plus du centre 3'27 du tourbillon, et toujours plus près, toujours plus près de son épou- vantable arête intérieure. » Pendant tout ce temps, je n'avais pas lâché le boulon Mon frère était à l'arrière, se tenant à une petite barrique vide, solidement attachée sous Téchauguette, derrière l'habitacle; c'était n'eût pas été balayé le quand du bord qui seul objet le coup de temps nous avait surpris. )) Comme nous approchions de la margelle de ce puits mouvant, il lâcha le baril et tâcha de saisir l'anneau, que, dans l'agonie de sa terreur, il s'efforçait d'arracher de mes mains, et qui n'était pas assez large pour nous donner sûrement prise à tous deux. Je n'ai jamais éprouvé de douleur plus profonde que quand je le vis tenter une pareille action, — quoique je visse bien qu'alors il était insensé et que la pure frayeur en avait fait )) un fou furieux. Néanmoins, je ne cherchai pas place. Je savais bien qu'il appartiendrait m'en grande allai au l'anneau; je lui baril difficulté à de à lui disputer la importait fort peu à qui laissai l'arrière. 11 le boulon, n'y avait et pas opérer cette manœuvre; car le se- maque filait en rond avec assez d'aplomb et assez droit sur sa quille, poussé quelquefois çà et là par les immenses houles et les bouillonnements du tourbillon. A peine m'étais-je arrangé dans ma nouvelle position, que nous donnâmes une violente embardée à tribord, et que nous piquâmes la tête la première dans l'abîme. IlISTOinES liXTUAORDINAIRES. 328 Je murmurai une rapide prière à Dieu, et je pensai que tout était fini. » Gomme je subissais l'effet douloureusement nau- séabond de la descente, je m'étais instinctivement cramponné au baril avec plus d'énergie, et j'avais fermé les yeux. ouvrir, Pendant quelques secondes, je n'osai pas les — m'attendant à une destruction instantanée et m'étonnant de ne pas déjà en être aux angoisses su- prêmes de l'immersion. Mais les secondes s'écoulaient; je vivais encore. La sensation de chute avait cessé, et mouvement du navire ressemblait beaucoup à ce qu'il était déjà, quand nous étions pris dans la ceinture le d'écume, à l'exception que maintenant nous donnions davantage de la bande. Je repris courage ^t regardai une fois encore le tableau. » Jamais je n'oublierai les sensations d'effroi, d'hor- reur et d'admiration que j'éprouvai en jetant les yeux autour de moi. Le bateau semblait suspendu conmie par magie, à mi-chemin de sa chute, sur intérieure d'un entonnoir d'une la surface vaste circonférence, d'une profondeur prodigieuse, et dont les parois, ad- mirablement polies, auraient pu être prises pour de l'ébène, sans l'éblouissante vélocité avec laquelle elles pirouettaient et Tétincelante et horrible clarté qu'elles répercutaient sous les rayons de la pleine lune, qui, de ce trou circulaire que j'ai déjà décrit, ruisselaient en un fleuve d'or et de splendeur le long des murs noirs et pénétraient jusque dans les plus intimes profondeurs de l'abîme. UNE DESCENTE DANS LE MAELSTROM. )) 320 D'abord, j'étais trop troublé pour observer n'importe quoi avec quelque exactitude. L'explosion générale de cette magnificence terrifique était tout ce que je pou- vais voir. Néanmoins, quand je revins un peu à moi, mon regard se dirigea instinctivement vers le fond. Dans cette direction, je pouvais plonger ma vue sans obstacle à cause de la situation de notre semaque qui. était suspendu sur la surface inclinée du gouffre; il courait toujours sur sa quille, c'est-à-dire que son pont formait un plan parallèle à celui de l'eau, qui faisait comme un talus incliné à plus de /i5 degrés, de sorte que nous avions l'air de nous soutenir sur notre côté. Je ne pouvais m'empêcher de remarquer, toutefois, que je n'avais guère plus de peine à me retenir des mains et des pieds, dans cette avions été sur un plan situation, que si nous horizontal; et cela tenait, je suppose, à la vélocité avec laquelle nous tournions. )) Les rayons de la lune semblaient chercher le fm fond de l'immense gouffre; cependant, je ne pouvais rien distinguer nettement, à cause d'un épais brouillard qui enveloppait toutes choses, et sur lequel planait un magnifique arc-en-ciel, semblable à ce pont étroit et vacillant que les musulmans affirment être le seul pas> sage entre le Temps et l'Éternité. Ce brouillard ou cette écume était sans doute occasionné par le conflit des grands murs de f entonnoir, quand ils se rencontraient et se brisaient au fond; — quant au hurlement qui montait de ce brouillard vers le ciel, je n'essayerai pas de le décrire. HISTOIRES EXTRAORDINAIRES. 33J )) Notre première glissade dans l'abîme, à partir de la ceinture d'écume, nous avait portés à une grande dis- tance sur la pente; mais postérieurement notre des- cente ne s'effectua pas aussi rapidement, à beaucoup près. Nous filions toujours, toujours circulairement, non plus avec un mouvement uniforme, mais avec des «élans qui parfois ne nous projetaient qu'à une centaine de yards, et d'autres fois nous faisaient accomplir une évolution complète autour du tourbillon. tour, nous nous rapprochions est vrai, » Je A chaque du gouffre, lentement, il mais d'une manière très-sensible. regardai au large sur le vaste désert d'ébène qui nous portait, et je m'aperçus que notre barque n'était pas le seul objet qui fût tombé dans l'étreinte du tourbillon. Au-dessus et au-dessous de nous, on voyait des débris de navires, de gros morceaux de charpente, des troncs d'arbres, ainsi que bon petits, tels que des pièces nombre d'articles plus de mobilier, des malles brisées, des barils et des douves. J'ai déjà décrit la curiosité surnaturelle qui s'était substituée à mes pri- me sembla qu'elle augmentait à mesure que je me rapprochais de mon épouvantable mitives terreurs. Il destinée. Je commençai alors à épier avec un étrange intérêt les nombreux objets qui flottaient en notre com- pagnie. Il fallait que j'eusse le délire, — car je trouvais même une sorte d'amusement à calculer les vitesses relatives de leur descente )) vers le tourbillon d'écume. — Ce sapin, me surpris-je une fois à dire, sera cer- tainement la première chose qui fera le terrible pion- UNE DESCENTE DANS LE MAELSïROM. — geon et qui disparaîtra; et je fus fort 331 désappointé de voir qu'un bâtiment de commerce hollandais avait pris les devants et s'était engouffré le premier. A la longue, après avoir fait quelques conjectures de cette — ce — de mon invariable mécompte, — me jeta dans un nature, et m'être toujours trompé, fait, le fait ordre de réflexions qui firent de nouveau trembler mes membres et battre mon cœur encore plus lourdement. Ce n'était pas une nouvelle terreur qui m'affectait n ainsi, mais l'aube d'une espérance bien plus émou- vante. 'Cette espérance surgissait en partie de la mé- moire, en partie de l'observation présente. Je me rappelai l'immense variété d'épaves qui jonchaient la côte de Lofoden, et qui avaient toutes été absorbées et re- vomies par le Moskoe-Strom. Ces articles, pour la plus grande partie, étaient déchirés de la manière la plus extraordinaire, — éraillés, écorchés, au point qu'ils avaient l'air d'être tout garnis de pointes et d'esquilles. — Mais en je me rappelais distinctement alors qu'il y avait quelques-uns qui n'étaient pas défigurés tout. Je ne pouvais cette du maintenant me rendre compte de différence qu'en supposant que les fragments écorchés fussent les seuls qui eussent été complètement absorbés, — les autres étant entrés dans le tourbillon à une période assez avancée de la marée, ou, après y être entrés, étant, par une raison ou par une autre, descendus assez lentement pour ne pas atteindre le fond avant le retour du flux ou du reflux, Je concevais qu'il était — suivant possible, dans les le cas. deux cas, HISTOIRES EXTRAORDINAIRES. 332 qu'ils eussent remouté, en tourbillonnant de nouveau jusqu'au niveau de l'Océan, sans subir le sort de ceux qui avaient été entraînés de meilleure heure ou absorbés plus rapidement. aussi trois observations importantes » Je fis — règle générale, — plus mière, que, gros, plus leur descente était les rapide; : la pre- corps étaient — seconde, la que, deux masses étant données, d'une égale étendue. Tune sphérique et l'autre de n'importe quelle autre formeyla. supériorité de vitesse dans la descente était pour la sphère ; — la troisième, — que, de deux masses d'un volume égal, l'une cylindrique et l'autre de n'importe quelle autre forme, le cylindre était absorbé le plus lentement. » Depuis ma délivrance, j'ai eu à ce sujet quelques conversations avec un vieux maître d'école du district ; et c'est de lui que j'ai appris l'usage des mots cylindre et sphère. cation Il m'a expliqué — que ce que — mais j'ai j'avais observé oublié l'expliétait la consé- quence naturelle de la forme des débris flottants, et il m'a démontré comment un cylindre, tournant dans un tourbillon, présentait plus de résistance à sa succion et était attiré avec plus de difficulté qu'un corps d'une autre forme quelconque et d'un volume égal '. » Il y avait une circonstance saisissante qui donnait une grande force à ces observations, anxieux de les vérifier i. : c'était et me rendait qu'à chaque révolution Archimède, De occidentibus in fluklo. — E. A. P. UNE DESCENTE DANS LE MAELSTROM. 33) nous passions devant un baril ou devant une vergue ou un mât de navire, et que la plupart de ces objets, nageant à notre niveau quand j'avais ouveit les yeux pour la première fois sur les merveilles du tourbillon, étaient maintenant situés bien au-dessus de nous et semblaient n'avoir guère bougé de leur position première. » Je n'hésitai pas plus longtemps sur ce que j'avais à faire. Je résolus de m'attacher avec confiance à la bar- rique que je tenais toujours embrassée, de larguer le cable qui la retenait à la cage, et de me jeter avec à la mer. Je m'efforçai d'attirer par signes l'attention de mon frère sur les barils flottants auprès desquels nous passions, et je fis tout ce qui était en mon pouvoir pour lui faire comprendre ce que j'allais tenter. Je crus à la longue qu'il avait deviné l'eût ou mon dessein; — mais, qu'il ne l'eût pas saisi, il secoua la tête avec déses- poir et refusa de quitter sa place près du boulon. Il m'était impossible de m'emparer de lui; la conjoncture ne permettait pas de délai. Ainsi, avec une amère angoisse, je l'abandonnai à sa destinée; je m'attachai moi-même à la barrique avec le câble qui l'amarrait à l'échauguette, et, sans hésiter un moment de plus, je me précipitai avec dans la mer. » Le résultat fut précisément ce que j'espérais. c'est Comme moi-même qui vous raconte cette histoire, comme vous voyez que j'ai échappé, connaissez déjà le mode de salut — et comme — vous que j'employai et pouvez dès lors prévoir tout ce que j'aurais de plus à 19. 33Î HISTOIRES EXTRAORDINAIRES. VOUS dire, — j'abrégerai mon récit et j'irai droit à la conclusion. )) Il s'était écoulé une heure environ depuis que j'avais quitté le bord du semaque, quand, étant descendu à une vaste distance au-dessous de moi, il fit coup sur coup trois ou quatre tours précipités, et, emportant mon frère bien-aimé, piqua de Pavant décidément et pour toujours, dans le chaos d'écume. Le baril auquel j'étais attaché nageait presque à moitié chemin de la distance qui séparait le fond du gouffre de l'endroit oi^i je m'étais précipité par-dessus bord, changement eut lieu dans le quand un grand caractère du tourbillon. La pente des parois du vaste entonnoir se fit de moins en moins escarpée. Les évolutions du tourbillon devinrent graduellement de moins en moins rapides. Peu à peu l'écume et l'arc-en-ciel disparurent, et le fond du gouffre sembla s'élever lentement. )) Le ciel était clair, le vent était tombé, et la pleine lune se couchait radieusement à Louest, quand je me retrouvai à la surface de l'Océan, juste en vue de la côte de Lofoden, et au-dessus de l'endroit oi^i était na- guère le tourbillon du Moskoe-Strom. C'était l'heure de l'accalmie, — mais la mer se vagues énormes par suite de la soulevait toujours eri tempête. Je fus porté violemment dans le canal du Strom et jeté en quelques minutes à la côte, parmi repêcha, danger avait disparu, vait les pêcheries. — épuisé de fatigue; — le et, Un bateau me maintenant que le souvenir de ces horreurs m'a- rendu muet. Ceux qui me tirèrent à bord étaient UNE DESCENTE DANS LE MAELSTROM. 33a mes vieux camarades de mer et mes compagnons de chaque jour, — mais ils ne me reconnaissaient pas plus qu'ils n'auraient reconnu un voyageur revenu du monde des esprits. Mes cheveux, qui la veille étaient d'un noir de corbeau, étaient aussi blancs que vous les voyez maintenant. Ils dirent aussi que toute l'expression de ma physionomie histoire, — ils était changée. Je leur contai ne voulurent pas y croire. — mon Je vous la raconte, à vous, maintenant, et j'ose à peine espérer que vous y ajouterez plus de foi que les plaisants pêcheurs de Lofoden. LA VERITE SUR LE CAS DE M. VALDEMAU Que le cas extraordinaire de M. Valdemar ait excité une discussion, il n'y a certes pas lieu de s'en étonner. C'eût été un miracle qu'il n'en fût pas ainsi, culièrement dans de telles circonstarîces. — parii- Le désir de toutes les parties intéressées à tenir l'affaire secrète, au moins pour le présent ou en attendant l'opportunité d'une nouvelle investigation, et nos efforts pour y réussir ont laissé s'est place à un récit tronqué ou exagéré qui propagé dans le public, et qui, présentant l'af- faire sous les couleurs les plus désagréablement fausses, est naturellement devenu la source d'un grand dis- crédit. est maintenant devenu nécessaire que je donne les faits, autant du moins que je les comprends moi- Il même. Succinctement les voici : Mon attention, dans ces trois dernières années, avait été à plusieurs reprises attirée vers le magnétisme; et, y a environ neuf mois, cette pensée frappa presque soudainement mon esprit, que, dans la série des expé- il HISTOIRES EXTRAORDINAIRES. 338. riences faites jusqu'à présent, il y avait une très-remar- quable et très-inexplicable lacune — personne n'avait : encore été magnétisé in articiUo mortis. Restait à savoir, d'abord, si dans un pareil état existait chez le patient une réceptibilité quelconque de l'influx magné- tique; en second lieu, si, dans le cas d'affirmative, elle était atténuée ou augmentée par la circonstance; troi- pour combien de sièmement, jusqu'à quel point et temps mort pouvaient être les empiétements de arrêtés par l'opération. rifier, la y avait d'autres points à vé- Il mais ceux-ci excitaient le plus ma curiosité, particulièrement le — dernier, à cause du caractère im- mensément grave de ses conséquences. En cherchant autour de moi un sujet au moyen duquel je pusse éclaircir ces points, je fus amené à jeter •les yeux sur mon ami, M. Ernest Valdemar, le compi- lateur bien connu de la Bibliotlieca forensica, et auteur (sous le pseudonyme d'issachar Marx) des traductions polonaises de Wallenslein et de Gargantua. M. Valdemar, qui résidait généralement à Harlem (New-York) depuis l'année 1839, est ou était particulièrement remarquable par l'excessive maigreur de sa personne, — ses mem- bres inférieurs ressemblant beaucoup à ceux de John Randolph, — et aussi par la blancheur de ses favoris qui faisaient contraste avec sa chevelure noire, que chacun prenait conséquemment pour une perruque Son tempérament était singulièrement nerveux et en faisait un excellent sujet tiques. Dans pour les expériences magné- deux ou trois occasions, je l'avais amené LA VÉRITÉ SUR L!-: CAS DE M. VALDEMAR. à dormir sans grande difficulté ; mais je fus 339 désap- pointé quant aux autres résultats que sa constitution particulièr3 m'avait naturellement fait espérer. Sa vo- lonté n'était jamais positivement ni entièrement sou- mise à mon influence, et relativement à \ai clairvoyance je ne réussis à faire avec lui rien sur quoi l'on pût faire fond. J'avais toujours attribué mon insuccès sur ces points au dérangement de sa santé. Quelques mois avant l'époque où je fis sa connaissance, les médecins l'avaient déclaré atteint d'une phthisie bien, caracté- risée. C'était à vrai dire sa coutume de parler de sa fin prochaine avec beaucoup de sang-froid, comme d'une chose qui ne pouvait être ni évitée ni regrettée. Quand ces idées, que j'exprimais tout à l'heure, me vinrent pour la première fois, il était très-naturel que je pensasse à M. Valdemar. Je connaissais trop bien la solide philosophie de l'homme pour redouter quelques scrupules de sa part, et il n'avait point de parents en Amérique qui pussent plausiblement intervenir. Je lui parlai franchement de la chose; et, à ma grande surma prise, il parut y prendre un intérêt très-vif. Je dis à grande surprise, car, quoiqu'il eût toujours gracieuse- mes expériences il n'avait jamais témoigné de sympathie pour mes études. Sa ment livré sa personne à , maladie était de celles qui admettent un calcul exact relativement à l'époque de leur dènoûment; et il fut finalement convenu entre nous qu'il m'enverrait cher- cher vingt-quatre heures avant le terme marqué par les médecins pour sa mort. HISTOIRES EXTRAORDINAIRES. 340 Il y a maintenant sept mois passés que je reçus do M. Valdemar le billet suivant « » : Mon cher P..., Vous pouvez aussi bien venir maintenant. D... et F... s'accordent à dire que je n'irai pas, demain, au delà de minuit; et je crois qu'ils ont calculé juste, ou bien peu s'en faut. » Valdemar. » Je recevais ce billet une demi-heure après qu'il m'était écrit, et, la en quinze minutes au plus, j'étais dans chambre du mourant. Je ne l'avais pas vu depuis dix jours, et je fus effrayé de la terrible altération que ce court intervalle avait produite en Sa face était lui.. d'une couleur de plomb; les yeux étaient entièrement éteints, et l'amaigrissement était les si remarquable, que pommettes avaient crevé la peau. L'expectoration était excessive; le pouls à peine sensible. néanmoins d'une manière facultés spirituelles et physique. 11 fort la parlait distinctement, chambre, il conservait toutes ses une certaine quantité de force — prenait sans aide quelques drogues palliatives, — dans Il singulière était et, quand j'entrai occupé à écrire quelques noies sur un agenda. Il était soutenu dans son lit par lui donnaient Après avoir serré la main de Valdemar, je pris ces des oreillers. Les docteurs D... et F... leurs soins. messieurs à part et j'obtins un compte rendu minutieux LA VERITE SUR LE CAS DE M. VALDEMAR. de l'état 341 du malade. Le poumon gauche était depuis dix-huit mois dans un état semi-osseux ou cartilagi- neux, et conséquemment tout à fait impropre à toute fonction vitale. Le droit, dans s'était aussi ossifié, sinon en sa région totalité, supérieure, du moins par- tiellement, pendant que la partie inférieure n'était plus qu'une masse de tubercules purulents, se pénétrant les uns les antres. 11 existait plusieurs perforations pro- fondes, et en un certain point il y avait adhérence per- manente des côtes. Ces phénomènes du lobe droit étaient de date comparativement récente. L'ossification avait marché avec une rapidité très-insolite, — un mois auparavant on n'en découvrait encore aucun symptôme, — et l'adhérence n'avait été remarquée que dans ces trois derniers jours. Indépendamment de la phthisie, on soupçonnait un anévrisme de l'aorte, mais sur ce point les symptômes d'ossification rendaient impossible tout diagnostic exact. L'opinion des deux médecins était que M. Valdemar mourrait le lendemain dimanche vers minuit. Nous étions au samedi était sept , et il heures du soir. En quittant le chevet du moribond pour causer avec moi, les docteurs D... et F... lui avaient dit un suprême adieu. Ils n'avaient pas l'intention de revenir; mais, à ma requête, ils consentirent à venir voir le patient vers dix heures de la nuit. Quand ils furent partis, je causai librement avec M. Valdemar de sa mort prochaine, et plus particuliè- rement de l'expérience que nous nous étions proposée. HiSTOIRES EXTRAOUDINAIRES. 342 montra toujours plein de bon vouloir; il témoi- se Jl gna même un vif désir de cette expérience et me pressa de commencer tout de suite. Deux domestiques, un homme et une femme, étaient là pour donner leurs soins; mais je ne me sentis pas tout à fait libre de m'engager dans une tâche d'une telle gravité sans autémoignages plus rassurants que ceux que pour- tres raient produire ces gens-là en cas d'accident soudain. Je renvoyais donc l'opération à huit heures, quand l'ar- rivée d'un étudiant en médecine, avec lequel un peu lié, M. Théodore L...., me tira j'étais définitivement d'embarras. Primitivement j'avais résolu d'attendre les médecins suite, ; mais je fus induit à commencer tout de d'abord par les sollicitations de M. Valdemar, en second lieu par la conviction que je n'avais pas un instant à perdre, car il s'en allait évidemment. M. L.... fut assez bon pour accéder au désir que j'exprimai qu'il prît des notes de tout ce qui surviendrait ; et c'est d'après son procès-verbal que je décalque pour ainsi dire mon récit. Quand je n'ai pas condensé, j'ai copié mot pour mot. M. environ huit heures moins cinq, quand, pre- était Il nant la main du L...., c'était son patient, je le priai de confirmer à aussi distinctement qu'il formel désir, à Valdemar, que lui le pourrait, que je fisse une expérience magnétique sur lui, dans de telles conditions. 11 (( répliqua faiblement, mais Oui, je désire être magnétisé; très-distinctement » : ajoutant immédiate- LA VÉRITÉ SUR LE CAS DE M. VALDEMAR. ment après crains bien que vous n'ayez différé « Je : 3î3 trop longtemps. » Pendant qu'il parlait, j'avais commencé les passes que j'avais déjà reconnues les plus efficaces pour l'endormir. Il évidemment influencé par le premier fut mouvement de ma main qui traversa son front; mais, quoique je déployasse toute ma puissance, aucun autre effet sensible ne se manifesta jusqu'à dix heures dix minutes, quand les médecins D... et F... arrivèrent au mots mon comme ils n'y faisaient aucune objection, rendez-vous. Je leur expliquai en peu de dessein; et, disant que le patient était déjà dans sa période d'agonie, je continuai les sans hésitation, changeant toutefois passes latérales en passes longitudinales, et con- centrant tout mon regard juste dans l'œil du mori- bond. Pendant ce temps, son pouls devint imperceptible, et sa respiration obstruée et marquant lin intervalle d'une demi-minute. Cet état dura un quart, d'heure, presque sans chan- gement. A l'expiration de cette période, néanmoins, un soupir naturel, quoique horriblement profond, chappa du sein du moribond, flante cessa, c'est-à-dire s'é- et la respiration ron- que son ronflement ne fut plus sensible; les intervalles n'étaient pas diminués. Les extrémités du patient étaient d'un froid de glace. A onze heures moins cinq minutes, j'aperçus des symptômes non équivoques de l'influence magnétique. Le vacillement vitreux de l'œil s'était changé en cette HISTOIRES EXTRAORDINAIRES. 344 expression pénible do regard en dedans qui ne se voit jamais que dans les cas de somnambulisme, quelle est impossible il ques passes latérales rapides, je pières, comme et à la- de se méprendre; avec quelpalpiter les pau- fis quand le sommeil nous prend, et, en insistant un peu, je les fermai tout à fait. Ce n'était pas assez pour moi, et je continuai mes exercices vigou- reusement et avec la plus intense projection de volonté, jusqu'à ce que j'eusse complètement paralysé les membres du dormeur, après les avoir placés dans une po- apparence commode. Les jambes étaient tout sition en à fait allongées; les bras à peu près étendus, et repo- sant sur le lit à une distance médiocre des reins. La tête était très-légèrement élevée. Quand j'eus fait tout cela, il était minuit sonné, et je priai ces messieurs d'examiner la situation de M. Val- demar. Après quelques expériences, qu'il était ils reconnurent dans un état de catalepsie magnétique ex- traordinairement parfaite. La curiosité des deux médecins était grandement excitée. Le docteur D... résolut tout à coup de passer toute la nuit auprès du patient, pendant que le docteur F... prit congé de nous en promettant de revenir au petit jour; M. L... et les gardes- malades restèrent. Nous laissâmes M. Valdemar absolument tranquille jusqu'à trois heures du matin; alors, je m'approchai de lui et le trouvai exactement dans le même état que quand le docteur F... était étendu était parti, — c'est-à-dire dans la même position ; qu'il que le pouls était LA VÉRITÉ SUR LE CAS DE M. VALDEMAR. 345 imperceptible, la respiration douce, à peine sensible, — excepté par les yeux l'application d'un miroir aux lèvres; fermés naturellement, et les membres aussi rigides et aussi froids que du marbre. Toutefois, l'ap- parence générale n'était certainement pas celle de la mort. En approchant de M. Valdemar, je lis une espèce de demi-effort pour déterminer son bras droit à suivre le mien dans les mouvements que je décrivais doucement çà et là au-dessus de sa personne. Autrefois, quand j'avais tenté ces expériences avec le patient, elles n'a- vaient jamais pleinement réussi, et assurément je n'espérais guère mieux réussir cette fois-, mais, à mon giand étonnement, son bras suivit très- doucement, quoique les indiquant faiblement, toutes les directions que le mien lui assigna. Je me déterminai à essayer quelques mots de conversation. — Monsieur Valdemar, 11 dis-je, dormez-vous? ne répondit pas, mais j'aperçus un tremblement sur ses lèvres, et je fus obligé de répéter ma question une seconde et une troisième fois. A la troisième, tout son être fut agité d'un léger frémissement; les paupières se soulevèrent d'elles-mêmes voiler une ligne blanche du globe ; comme pour déles lèvres remuè- rent paresseusement et laissèrent échapper ces mots dans un murmure à peine intelligible — Oui; je dors : maintenant. Ne m'éveillez pas! — Laissez-moi mourir ainsi! Je tâtai les membres et les trouvai toujours aussi ri- HISTOIRES EXTRAORDINAIRES. 346 gides. Le bras droit, comme tout à l'heure, obéissait à la direction de ma main. Je questionnai de nouveau le somnambule. — Vous sentez-vous toujours mal à la poitrine, mon- sieur Valdemar ? La réponse ne fut pas immédiate; moins accentuée que la première — Mal — non, — ? je elle fut encore : meurs. Je ne jugeai pas convenable de le tourmenter davan- tage pour le moment, et il ne se dit, il ne se fit rien de nouveau jusqu'à l'arrivée du docteur F..., qui précéda un peu le lever du soleil, et éprouva un étonne- ment sans bornes en trouvant le patient encore vivant. Après avoir tâté le somnambule et lui avoij^ pouls du appliqué un miroir sur les lèvres, il me pria de lui parler encore. — Monsieur Valdemar, dormez-vous toujours? Gomme précédemment, quelques minutes s'écoulèrent avant la réponse; et, durant l'intervalle, le ribond sembla A ma question rallier toute son énergie pour répétée pour quatrième la mo- parlei-. fois, répondit très-faiblement, presque inintelligiblement — Oui, toujours; — je dors, C'était alors l'opinion, cins, — je il : meurs. ou plutôt le désir des méde- qu'on permît à M. Valdemar de rester sans être troublé dans cet état actuel de calme apparent, jus- qu'à ce que la mort survînt; et cela devait avoir lieu, — on fut unanime là-dessus, — dans un délai de cinq minutes. Je résolus cependant de lui parler encore LA VÉRITÉ SUR LE CAS DE M. VALDEMAR. 347 une fois, et je répétai simplement ma question précédente. Pendant que je parlais, il se fit un changement mar- qué dans la physionomie du somnambule. Les yeux foulèrent dans leurs orbites, lentement découverts par les paupières qui remontaient; la peau prit un ton gé- néral cadavéreux, ressemblant moins à du parchemin qu'à du papier blanc; et les deux taches hectiques circirculaires, qui jusque-là étaient vigoureusement fixées dans le centre de chaque joue, s'éteignirent tout d'un coup. Je me sers de cette expression, parce que la sou- daineté de leur disparition gie me fait penser à une bou- soufflée plutôt qu'à toute supérieure, en autre chose. La lèvre même temps, se tordit en remontant au-dessus des dents que tout à l'heure elle couvrait entièrement, pendant que la mâchoire inférieure tombait avec une saccade qui put être entendue, laissant la bouche toute grande ouverte, et découvrant en plein la langue noire et boursouflée. Je présume que tous les témoins étaient familiarisés avec les horreurs d'un lit de mort; mais l'aspect de M. Valdemar en ce moment était tellement hideux, ception, que ce fut hideux au delà de toute con- une reculade générale loin de la région du lit. Je sens maintenant que je suis arrivé à un point de mon récit où le lecteur croyance. Cependant, 11 mon n'y avait plus dans révolté me refusera toute devoir est de continuer. M. Valdemar le plus faible HISTOIRES EXTRAORDINAIRES. 348 symptôme de vitalité; et, concluant qu'il était mort, nous le laissions aux soins des gardes-malades, quand un fort mouvement de vibration se manifesta dans la langue. Gela dura pendant une minute peut-être. A l'expiration de cette période, des mâchoires distendues et immobiles jaillit — une voix une voix, serait folie d'essayer telle que ce de la décrire. 11 y a cependant deux ou trois épithètes qui pourraient quées comme des à peu près : lui être appli- ainsi, je puis dire que le son était âpre, déchiré, caverneux; mais le hideux total n'est pas définissable, par la raison que de pareils sons n'ont jamais hurlé dans Toreille de l'humanité. II y avait cependant deux particularités qui pensai alors, et je le pense encore, — peuvent — je le être jus- tement prises comme caractéristiques de l'intonation, et qui sont propres à donner quelque idée de son étrangeté extra-terrestre. En premier lieu, la voix semblait parvenir à nos oreilles, — aux miennes du moins, — comme d'une très-lointaine distance ou abîme souterrain. (je crains, faire de quelque En second lieu, elle m'impressionna en vérité, qu'il ne me soit impossible de me comprendre), de la tières glutineuses même manière que les ma- ou gélatineuses affectent le sens du toucher. J'ai le parlé à la fois de son et de voix. Je veux dire que son était d'une syllabisation distincte, et même terri- blement, effroyablement distincte. M. V-àldemar parlait, évidemment pour répondre avais à la question que je lui adressée quelques minutes auparavant. Je lui LA VÉRITÉ SUR LE CAS DE M. VALDEMAU. :U9 avais demandé, on s'en souvient, s'il dormait toujours. Il disait maintenant ; — Oui, — non, — j'ai dormi, — et maintenant, — maintenant, je suis mort. Aucune des personnes présentes n'essaya de nier ni même de réprimer l'indescriptible, la frissonnante horreur que ces quelques mots ainsi prononcés étaient si bien faits pour créer. M. L..., l'étudiant, s'évanouit. Les gardes-malades s'enfuirent immédiatement de la chambre, et il fut impossible de les y ramener. Quant à mes propres impressions, je ne prétends pas les rendre intelligibles pour le lecteur. Pendant près d'une heure, nous nous occupâmes en silence (pas un mot ne fut prononcé) à rappeler M. L.... à la vie. Quand il fut revenu à lui, nous reprîmes nos investigations sur l'état de M. Valdemar. Il était resté à tous égards tel que je dernier lieu, à l'exception que le l'ai décrit en miroir ne donnait plus aucun vestige de respiration. Une tentative de sai- gnée au bras resta sans succès. Je dois mentionner aussi que ce membre n'était plus soumis à ma volonté. Je m'efforçai en vain de lui faire suivre la direction de ma main. La seule indication réelle de l'influence magnétique se manifestait maintenant dans le mouvement vibratoire de la langue. Chaque une question à M. Valdemar, effort pour répondre, il fois que j'adressais semblait qu'il mais que sa volition ne fît un fût pas suffisamment durable. Aux questions faites par une autre personne que moi il paraissait absolument insen20 HISTOIRES EXTRAORDINAIRES. 350 ' sible, — quoique j'eusse tenté de mettre chaque membre de la société en rapport magnétique avec lui. Je crois que j'ai cessaire pour bule dans maintenant relaté tout ce qui est né- comprendre faire période. cette du somnam- l'état JNous nous procurâmes d'autres infirmiers, et, à dix heures, je sortis de la maison, en compagnie des deux médecins et de M. L... Dans l'après-midi, nous revînmes tous voir le patient. Son état était absolument le même. Nous eûmes alors une discussion sur l'opportunité et la possibilité de l'éveiller; mais nous fûmes bientôt d'accord en ceci qu'il n'en pouvait résulter aucune utilité. 11 était évi- dent que jusque-là, la mort, ou ce que l'on définit habituellement par le mot mort, avait été l'opération magnétique. Il arrêtée par nous semblait clair à tous qu'éveiller M. Valdemar, c'eût été simplement assurer sa minute suprême, ou au moins accélérer sa désorganisation. — — nous nous Depuis lors jusqu'à la fin de la semaine dernière, un intervalle de sept mois à peu près, réunîmes journellement dans la maison de M. Valdemar, accompagné de médecins et d'autres amis. Pendant tout ce temps, le somnambule resta exactement tel que je l'ai décrit. La surveillance des infirmiers était continuelle. Ce fut vendredi dernier que nous résolûmes finale- ment de faire l'expérience du réveil, ou du moins d'essayer de l'éveiller; et c'est le résultat, déplorable peut-être, de cette dernière tentative, qui adonné nais- LA VÉRITÉ SUR LE CAS DE M. VALDEMAR. 351 sance à tant de discussions dans les cercles privés, à tant de bruits dans lesquels je ne puis de voir le m'empêcher résultat d'une crédulité populaire injusti- fiable. Pour arracher M. Valdemar à tique, je fis magné- la catalepsie usage des passes accoutumées. Pendant quelque temps, elles furent sans résultat. Le premier symptôme de retour à la vie fut un abaissement partiel de l'iris. Nous observâmes comme un fait très-remar- quable que cette descente de l'iris était accompagnée du flux très-abondant les d'une liqueur jaunâtre (de dessous paupières) d'une odeur acre et fortement désa- gréable. On me suggéra alors d'essayer d'influencer le bras du patient, comme par le passé. J'essayai, je ne pus. Le docteur F... exprima le désir que je lui adressasse une question. Je le lis de la manière suivante : — Monsieur Valdemar, pouvez-vous nous expliquer quels sont maintenant vos sensations ou vos désirs? 11 y eut un retour immédiat des cercles hectiques sur les joues ; la langue trembla ou plutôt roula violemment dans la bouche (quoique les mâchoires et les lèvres demeurassent toujours immobiles), et à la longue la même horrible voix que j'ai décrite fit éruption : — — Pour l'amour de Dieu! — vite! — — éveillez-moi! — ou bien, faites-moi dormir, — vous que je mort! vite! vite! vite ! J'étais Je dis suis totalement énervé, et pendant une minute je restai indécis sur ce que j'avais à faire. Je fis d'abord HISTOIRES EXTRAORDINAIRES. 352 un effort pour calmer le patient; mais, cette totale vacance de ma volonté ne me permettant pas d'y réussir, je fis l'inverse et m'efforçai aussi vivement que possible de le réveiller. Je vis bientôt que cette tentative aurait un plein succès, — ou du moins je me figurai bientôt que mon succès serait complet, — et je suis sûr que chacun dans la chambre s'attendait au réveil du som- nambule. Quant à ce qui arriva en réalité, aucun être humain n'aurait jamais pu s'y attendre; c'est au delà de toute possibilité. Comme je faisais rapidement les passes magnétiques à travers les cris de littéralement explosion « Mort! mort! » qui faisaient sur la langue et non sur les — tout son corps, — d'un seul coup, lèvres du — dans l'espace d'une minute, même moins, — déroba, — s'émietta, — se poitrnf absolument sous sujet, se et mes mains. Sur le lit, devant tous une masse dégoûtante minable putréfaction. et les témoins, gisait quasi liquide, — une abo- REVELATION MAGNÉTIQUE Bien que les ténèbres du doute enveloppent encore toute la théorie positive du magnétisme, ses fou- droyants effets sont maintenant presque universellement admis. Ceux qui doutent de ces de purs effets sont doutêurs de profession, une impuissante et peu honorable caste. Ce serait absolument perdre son temps aujourd'hui, que de s'amuser à prouver que l'homme, par un pur exercice de sa volonté, peut impressionner suffisamment son semblable pour dans une le jeter condition anomale, dont les phénomènes ressemblent littéralement à ceux de la mort, ou du moins leur res- semblent plus qu'aucun des phénomènes produits dans une condition anomale connue que dure cet état, la personne le temps ainsi influencée n'em- ; que, tout ploie qu'avec effort, et conséquemment avec titude, les peu d'ap- organes extérieurs des sens, et que néan- moins elle perçoit, avec une perspicacité singulièrement subtile et par un canal mystérieu-x, des objets situés au delà de la portée des organes physiques; que, de plus, ses facultés fient d'une intellectuelles s'exaltent et se forti- manière prodigieuse; que ses sympathies avec la personne qui agit sur elle sont profondes; ei 20. 354 HISTOIRES EXTRAORDINAIRES. que finalement sa susceptibilité des impressions magnétiques croît en proportion de leur fréquence, en même temps que les phénomènes particuliers obtenus s'étendent et se prononcent davantage et dans la même proportion. Je dis qu'il serait superflu de démontrer ces faits divers, où est contenue la loi générale du magné- tisme, et qui en sont les ti-aits principaux. Je n'infligerai donc pas aujourd'hui à mes lecteurs une démonstration i\ ussi parfaitement oiseuse. Mon dessein , quant à présent, est en vérité d'une tout autre nature. Je sens le besoin, en dépit de tout un monde de préjugés, de raconter, sans commentaires, mais dans tous ses détails, un. très-remarquable dialogue qui eut lieu entre un som- nambule et moi. J'avais depuis longtemps l'habitude de magnétiser la personne en question, M. Vankirk, et la susceptibilité vive, l'exaltation du sens magnétique, s'étaient déjà manifestées. Pendant plusieurs mois, M. Vankirk avait dont les effets les plus cruels avaient été diminués par mes beaucoup souffert d'une phthisie avancée, passes, et, dans la nuit du mercredi, 15 courant, je fus appelé à son chevet. Le malade souffrait des douleurs vives dans la région du cœur et respirait avec une grande diflîculté, ayant tous les symptômes ordinaires d'un asthme. Dans des spasmes semblables, il avait généralement trouvé du soulagement dans des applications de moutarde aux centres nerveux; mais, ce soir-là, il y avait eu recours en vain. RÉVÉLATION MAGNÉTIQUE. Quand j'entrai dans chambre, sa il 355 me salua d'un gracieux sourire, et, quoiqu'il fût en proie à des douleurs physiques aiguës, il me parut absolument calme quant au moral. — Je vous ai envoyé chercher cette nuit, dit-il, non pas tant pour m' administrer un soulagement physique me que pour satisfaire relativement à de certaines m'ont récemment causé impressions psychiques qui beaucoup d'anxiété et de surprise. Je n'ai pas besoin de vous dire combien j'ai été sceptique jusqu'à présent sur le sujet de l'immortalité de l'âme. Je ne puis pas vous nier que, dans cette âme que j'allais niant, a toujours existé comme un demi-sentiment assez vague de sa propre existence. Mais ce demi-sentiment jamais élevé à l'état ne s'est ma de conviction. De tout cela raison n'avait rien à faire. Tous mes efforts pour éta- là-dessus une enquête logique n'ont abouti qu'à blir me laisser plus me suis sceptique qu'auparavant. Je avisé d'étudier Cousin; je l'ai étudié dans ses propres ouvrages aussi bien que dans ses échos européens et américains. Charles J'ai eu entre Elwood de profonde attention. à l'autre; pure mais logique les sont les mains, par exemple, Brownson. Je l'ai Je l'ai avec le une trouvé logique d'un bout portions qui ne malheureusement piimordiaux du héros incrédule du résumé, lu sont pas de la les arguments livre. Dans son me parut évident que le raisonneur n'avait il pas même réussi à se convaincre lui-même. du livre a visiblement oublié le La fin commencement, HISTOIRES EXTRAORDINAIRES. 356 comme Trinculo son gouvernement. Bref je pas longtemps à m'apercevoir que, si être intellectuellement convaincu ne mortalité, il tions qui ont été listes doit de sa propre im- sera jamais par les pures abstrac- le si longtemps la manie des mora- anglais, français et allemands. Les abstractions elles amusement un peuvent être mais ne et une gymnastique, prennent pas possession Tant que nous serons sur cette terre, j'en suis persuadé, consentir, la de l'esprit. philosophie, nous sommera toujours en vain de considérer les qualités peut ne fus l'homme comme des êtres. La — mais l'âme, — mais volonté l'intellect, jamais. Je répète donc que j'ai seulement senti à moitié, et que je n'ai jamais cru intellectuellement. Mais, dernièrement, il y eut en moi un certain renforcement de sentiment, qui prit une intensité assez grande pour ressembler à un acquiescement de la raison, au point que je trouve fort difficile de distinguer entre les deux. Je crois avoir le droit d'attribuer à l'influence simplement cet effet magnétique. Je ne saurais expliquer ma pensée que par une hypothèse, à savoir que l'exaltation magnétique me rend apte à concevoir un système de raisonnement qui dans mon existence anomale me convainc, mais qui, par une complète analogie avec le phénomène magnétique, ne son effet, jusqu'à s'étend pas, excepté par mon existence normale. Dans l'état somnambuliqne, il y a simultanéité et contemporanéité entre le raisonnement et la conclusion, entre la cause et RÉVÉLATION MAGNÉTIQUE. son effet. Dans mon état naturel, la 357 cause s'éva- nouissant, l'effet seul subsiste, et encore peut-être fort affaibli. Ces considérations m'ont induit à penser que l'on pourrait tirer quelques bons i^ésultats d'une série de questions bien dirigées, proposées à mon intelligence dans l'état magnétique. Vous avez souvent observé la profonde connaissance de soi-même manifestée par le somnambule et la vaste science les points relatifs à l'état qu'il déploie sur tous magnétique. De cette con- naissance de soi-même on pourrait tirer des instructions suffisantes pour la rédaction rationnelle d'un catéchisme. Naturellement, je consentis à faire cette expérience. Quelques passes plongèrent M. Vankirk dans le sommeil magnétique. Sa respiration devint immédiatement plus aisée, et il ne parut plus souffrir aucun malaise physique. La conversation suivante s'engagea. — V dans le dialogue représentera le somnambule, et P, ce sera moi. P. Êtes-vous endormi? V. Oui, — non. Je voudrais bien dormir plus profon- dément. P. (après quelques nouvelles passes). Dormez-vous bien, maintenant? V. Oui. P. Comment supposez-vous que finira votre maladie actuelle? HISTOIRES EXTRAORDINAIRES. 358 V {après une longue hèsUalion cl pariant comme avec effort). J'en mourrai. P. Cette idée de mort vous affïige-t-elle? • V {avec vivacité). Non, non! P. Cette perspective vous réjouit-elle? V. Si j'étais éveillé, j'aimerais mourir. Mais mainte- nant il n'y a pas lieu de le désirer. L'état magnétique est assez près de la mort pour me contenter. P. Je voudrais bien une nette, explication un peu plus monsieur Vankirk. V. Je le voudrais bien aussi; d'effort que je mais cela demande plus ne me sens capable d'en faire. Vous ne me questionnez pas convenablement. P. Alors, que faut-il vous demander? V. le commen- le commen- Vous savez bien que le commencement est Dieu. 11 faut que vous commenciez par cement. P. Le commencement ! Mais où est-il, cement? V. {Ceci fut dit sur un ton bas, ondoyant, et avec tous les signes de la plus profonde vénération.) P. Qu'est-ce donc que Dieu? V {hésitant quelques minutes). Je ne puis pas le dire. P. Dieu n'est-il pas un esprit? V. Quand j'étais éveillé, je savais ce que vous en- tendiez par esprit. Mais maintenant, cela ne me semble — — une qualité plus qu'un mot, beauté, tel, par exemple, que enfin. P. Dieu n'est-il pas immatériel? - vérité, RÉVÉLATION MAGNETIQUE. — 359 c'est un simple mot. Ce qui n'est pas matière n'est pas, — à moins V. Il n'y a pas d'immatérialité; que les qualités ne soient des êtres. P. Dieu est-il donc matériel? V. Non. {Cette réponse m'abasourdit.) P. Alors, qu'est-il? une longue pause, et en marmottant) V. [après vois, — à dire. je le vois, — mais {Autre pose esprit, car il existe. Je le . c'est une chose très-difficile également longue.) 11 n'est pas n'est pas non plus matière, comme Il vous l'entendez. Mais il y a des gradations de matière dont l'homme n'a aucune connaissance, la plus dense entraînant la plus subtile, la plus subtile pénétrant la plus dense. L'atmosphère, par exemple, met en mou- vement le principe électrique, pendant que le principe électrique pénètre l'atmosphère. Ces gradations de matière augmentent en raréfaction et en subtilité jusqu'à ce que nous arrivions à une matière imparticulée, — sans molécules, — indivisible, — une; e.t ici la loi d'impulsion et de pénétration est modifiée. La matière suprême ou imparticulée non-seulement pénètre êtres, mais met tous les êtres en mouvement, les — et ainsi elle est tous les êtres en un, qui est elle-même. Cette matière est Dieu. Ce que les à personnifier dans le mot pensée, hommes cherchent c'est la matière en mouvement. P. Les métaphysiciens maintiennent que toute action se réduit à mouvement l'origine de celui-là. et pensée, et que celle-ci est HISTOIRES EXTRAORDINAIRES. 360 Oui; je vois maintenant la confusion d'idées. Le V. mouvement est l'action de l'esprit, non de la pensée. La matière imparticulée, ou Dieu à l'état de repos, est, autant que nous pouvons le concevoir, ce que les hom- mes appellent esprit. Et cette faculté d'automouvement — équivalente en effet à la volonté humaine — est dans la matière imparticulée le résultat de son unité et de son omnipotence; comment, je ne le sais pas, et maintenant je vois clairement que je ne le saurai jamais; mais la matière imparticulée, mise en mouve- ment par une loi ou une qualité contenue en elle, est pensante. P. Ne pouvez-vous pas me donner une précise de ce que vous entendez idée plus par matière impar- ticulée? V. Les matières dont l'homme a connaissance échap- pent aux sens, à mesure que l'on monte l'échelle. Nous avons, par exemple, un métal, un morceau de bois, une goutte d'eau, l'atmosphère, un gaz, le calorique, l'électricité, Péther lumineux. Maintenant, nous appe- lons toutes ces choses matière, et nous embrassons toute matière dans une délinition générale; mais, en dépit de tout ceci, il n'y a pas deux idées plus essen- tiellement distinctes que celle que nous attachons au métal, et celle que nous attachons à l'éther lumineux. Si nous prenons ce dernier, nous sentons une presque irrésistible tentation de le classer avec l'esprit ou avec le néant. La seule considération qui nous retient est notre conception de sa constitution atomique. Et encore, REVELATION MAGNÉTIQUE. 361 même, avons-nous besoin d'appeler à notre aide et de nous remémorer notre notion primitive de l'atome, ici c'est-à-dire de quelque chose possédant dans une infinie exiguïté la solidité, la tangibilité, la pesanteur. Suppri- mons l'idée de la constitution atomique, et il nous sera impossible de considérer l'élher comme une entité, ou au moins comme une matière. Faute d' un meilleur mot, nous pourrions l'appeler esprit. Maintenant, montons d'un degré au delà de l'éther lumineux, concevons une matière qui soit à l'éther, quant à la raréfaction, ce que l'éther est au métal, et nous arrivons enfin, en dépit de tous les dogmes de l'école, à une masse unique, tière imparticulée. Car, bien mettre une infinie petitesse — à une ma- que nous puissions addans les atomes eux- mêmes, supposer une infinie petitesse dans les espaces qui les séparent est une absurdité. Il y aura un point, — il y aura un degré de raréfaction, où, si les atomes sont en nombre sufiisant, les espaces s'évanouiront, et où la masse sera absolument une. Mais la considération de la constitution atomique étant maintenant mise de côté, la nature de cette masse glisse inévitablement dans notre conception de l'esprit. 11 est clair, toutefois, qu'elle est tout aussi matière est qu'il est aussi impossible qu'auparavant. Le vrai de concevoir l'esprit que d'imaginer ce qui n'est pas. Quand nous nous flattons d'avoir enfin trouvé cette conception, nous avons sim- plement donné le change à notre intelligence par la considération de la matière infiniment raréfiée. P. 11 me semble qu'il y a une insurmontable objec- 362 HISTOIRES EXTRAOKDINÂIHES. lion à cette idée de cohésion absolue, — et c'est la très- faible résistance subie par les corps célestes dans leurs révolutions à travers l'espace, — résistance qui existe à un degré quelconque, cela est aujourd'hui démontré, — mais à un degré si faible, qu'elle a échappé à la sa- gacité de Newton lui-même. Nous savons que la résis- tance des corps est surtout en raison de leur densité. L'absolue cohésion est l'absolue densité; là où il n'y a pas d'intervalles, il ne peut pas y avoir de passage. Un éther absolument dense constituerait un obstacle plus efiicace à la marche d'une planète qu'un éther de dia- mant ou de fer. y. Vous m'avez fait cette objection avec une aisance qui est à peu près en raison de son apparente irréfutabilité. — Une étoile marche; qu'importe que l'étoile passe à travers l'éther ou l'éther à travers elle? Il n'y a pas d'erreur astronomique plus inexplicable que celle qui concilie le retard connu des comètes avec l'idée de leur passage à travers l'éther; car, quelque raréfié qu'on suppose l'éther, il fera toujours obstacle à toute révolution sidérale, dans une période singulièrement plus courte que ne l'ont admis tous ces astronomes qui se sont appliqués à glisser sournoisement sur un point qu'ils jugeaient insoluble. Le retard réel est d'ailleurs à peu près égal à celui qui peut résulter du frottement de l'éther dans son passage incessant à travers l'astre. La force de retard est donc double, d'abord momentanée et complète en elle-même, et en second lieu inhni- ment croissante* RÉVÉLATION MAGNÉTIQUE. P. Mais dans tout cela, 363 — dans cette identification de la pure matière avec Dieu, n'y a-t-il rien d'irrespec- tueux? {Je fas forcé de répéter celle question pour que le somnambule put complètement saisir ma pensée.) Y. Pouvez-vous dire pourquoi la matière est moins respectée que l'esprit? Mais vous oubliez que la matière dont je parle est, à tous égards et surtout relative- ment à ses hautes propriétés, la véritable intellifjence ou esprit des écoles et en ces même temps la matière de mômes écoles. Dieu, avec tous les pouvoirs attri- bués à l'esprit, n'est que la perfection de la matière. P. Vous affirmez donc que la matière imparticulée en mouvement est pensée? V. En général, ce mouvement est la pensée univer- selle de l'esprit universel ; cette pensée crée; toutes les choses créées ne sont que les pensées de Dieu. P. Vous dites en général. : y. Oui, l'esprit universel est Dieu; pour les nouvelles individualités, la matière est nécessaire. P. Mais vous parlez maintenant d'esprit et de ma- tière comme les métaphysiciens. y. Oui, pour éviter la confusion. Quand je dis esprit, j'entends la matière imparticulée ou suprême; sous le nom de matière, je comprends toutes les autres espèces. P. Vous disiez : pour les nouvelles individualités la matière est nécessaire. F. Oui, car l'esprit existant incorporellement, c'est Dieu. Pour créer des êtres individuels pensants, il était nécessaire d'incarner des portions de l'esprit divin. HISTOIRES EXTRAOKDINAIKES. 3ti4 C'est ainsi que l'homme est individualisé; dépouillé du vêtement corporel, il serait Dieu. Maintenant, le mou- vement spécial des portions incarnées de imparticulée, c'est la la matière pensée de l'homme, comme le mouvement de l'ensemble est celle de Dieu. P. Vous dites que, dépouillé de son corps, Thomjne sera Dieu? V. {Apres quelque hèsUation). Je n'ai pas pu dire cela, c'est une absurdité. P. {Consultant ses notes). Vous avez affirmé que, dépouillé du vêtement corporel, l'homme serait Dieu. y. Et cela est vrai. L'homme ainsi dégagé serait Dieu, il serait désindividualisé pouillé, — du moins il ; mais il ne peut être ainsi dé- ne le sera jamais ; — autrement, il nous faudrait concevoir une action de Dieu revenant sur elle-même, une action futile et sans but. L'homme est une créature; les créatures sont les pensées de Dieu, et c'est la nature d'une pensée d'être irrévocable. P. Je ne comprends pas. Vous dites que l'homme ne pourra jamais rejeter son corps. V. Je dis qu'il ne sera jamais sans corps. P. Expliquez-vous. F. Il y a deux corps : le rudimentaire et le complet, correspondant aux deux conditions de la chenille et du papillon. Ce que nous appelons mort n'est que la méta- morphose douloureuse; notre incarnation actuelle est progressive, préparatoire, temporaire ; notre incarnation future est parfaite, finale, immortelle. La vie finale est le but suprême. RÉVÉLATION MAGNÉTIQUE. 365 P. Mais nous avons une notion palpable de la méta- morphose de la chenille. F. Nous, certainement, mais non la chenille. La ma- tière dont notre corps rudimentaire est composé est à la portée des organes de ce même corps, ou, plus dis- tinctement, nos organes rudimentaires sont appropriés à la matière dont est fait le corps rudimentaire, mais non à celle dont le corps suprême est composé. Le corps ultérieur ou suprême échappe donc à nos sens rudi- mentaires, et nous percevons seulement la coquille qui tombe en dépérissant et se détache de la forme intérieure, et non la forme intime elle-même; mais cette forme intérieure, aussi bien que la coquille, est appréciable pour ceux qui ont déjà opéré la conquête de la vie ultérieure. P. Vous avez dit souvent que l'état magnétique res- semblait singulièrement à la mort. Comment cela? Y. Quand je dis qu'il ressemble à la mort, j'entends qu'il ressemble à la vie ultérieure, car, lorsque je suis ndagnétisé, les sens de vacance , ma vie rudimentaire sont en et je perçois les choses extérieures directe- ment, sans organes, par un agent qui sera à mon ser- vice dans la vie ultérieure ou inorganique. P. Inorganique? y. Oui. Les organes sont des mécanismes par lesquels l'individu est mis en rapport sensible avec certaines catégories et formes de la matière, à l'exclusion des autres catégories et des autres formes. Les organes de l'homme sont appropriés à sa condition rudimentaire, HISTOIRES EXTRAORDINAIRES. 306 et à elle seule. Sa condilion ultérieure, étant inorga- nique, est propre à une compréhension infinie de toutes choses, une seule exceptée , volonté de Dieu, c'est-à-dire tière imparticulée. — qui est le la nature de la mouvement de la ma- Vous aurez une idée distincte du corps définitif en le concevant tout cervelle; cela, il n'est pas mais une conception de cette nature vous rappro- chera de l'idée de sa constitution réelle. Un corps lumi- neux communique une vibration à Téther chargé de transmettre la lumière; cette vibration en engendre de semblables dans la rétine, lesquelles en communiquent de semblables au nerf optique; le nerf les traduit au cerveau, et le cerveau à la matière imparticulée qui le pénètre; le mouvement de cette dernière est la pensée, et sa première vibration, c'était la perception. Tel est le mode par lequel l'esprit de la vie rudimentaire comextérieur, et ce monde exté- munique avec le monde rieur est, dans la vie rudimentaire, limité par l'idio- syncrasie des organes. Mais, dans la vie ultérieure, monde extérieur communique avec le inorganique, le corps entier, — qui affinité est d'une substance ayant quelque avec le cerveau, comme je vous l'ai dit, — sans autre intervention que celle d'un éther infiniment plus subtil que Téther lumineux; et le corps tout entier vibre à l'unisson avec cet éther et met en mouvement la matière imparticulée dont il est pénétré. C'est donc à l'absence d'organes idiosyncrasiques qu'il faut attri- buer la perception quasi illimitée de la vie ultérieure. Les organes sont des cages nécessaires où sont enfer- RÉVÉLATION MAGNÉTIQUE. mes les êtres rudimentaires jusqu'à ce 307 qu'ils soient garnis de toutes leurs plumes. P. Vous parlez d'êtres rudimentaires, y a-t-il d'autres êtres rudimentaires pensants que l'homme? L'incalculable agglomération F. de matière subtile dans les nébuleuses, les planètes, les soleils, et autres corps qui ne sont ni nébuleuses, ni soleils, ni planètes, a pour unique destination de servir d'aliment aux or- ganes idiosyncrasiques d'une infinité d'êtres rudimentaires; mais, sans cette nécessité de la vie rudimen- laire, acheminement à la vie définitive, de pareils mondes n'auraient pas existé; chacun de ces mondes est occupé par une variété distincte niques, rudimentaires, pensantes; de créatures orga- dans toutes, les organes varient avec les caractères généraux de l'habitacle. A la mort ou métamorphose, ces créatures, jouis- sant de la vie ultérieure, de l'immortalité, et connaissant tous les secrets, excepté V unique, opèrent tous leurs actes et se meuvent dans tous les sens par un pur effet de leur volonté les étoiles ; elles habitent, — non plus qui nous paraissent les seuls mondes palpa- bles et pour la commodité desquelles nous croyons stupidement que l'espace a été créé, mais l'espace lui- même, cet infini dont l'immensité véritablement substantielle absorbe les étoiles comme des ombres et pour l'œil des anges les efface comme des non-entités. P. Vous dites que, sans la nécessité de la vie rudimentaire, les astres n'auraient pas été créés. Mais pourquoi cette nécessité? HISTOIRES EXTRAORDINAIRES. 368 V. Dans la vie inorganique, aussi bien que générale- ment dans la matière inorganique, il n'y a rien qui puisse contredire l'action d'une loi simple, unique, qui est la Volition divine. La vie et la matière organiques, — complexes, substantielles multiple, et gouvernées par une loi — ont été constituées dans le but de créer un empêchement. P. Mais encore , — où était la nécessité de créer cet empêchement? V. tice, Le résultat de la loi inviolée est perfection, jus- bonheur négatif. Le résultat de la loi violée est douleur positive. Grâce aux imperfection, injustice, empêchements apportés par le nombre, la complexité ou la substantialité des lois de la vie et organiques, la violation de la loi de la matière devient jusqu'à un certain point praticable. Ainsi la douleur, qui est im- possible dans la vie inorganique, est possible dans l'or- ganique. P. Mais en vue de quel résultat satisfaisant la possibilité de la douleur a-t-elle été créée? V. Toutes choses sont bonnes ou mauvaises par com- paraison. plaisir, Une analyse démontrera que suffisante le dans tous les cas, n'est que le contraste de la peine. Le plaisir positif est une pure idée. Pour être heureux jusqu'à un certain point, ayons souffert jusqu'au frir serait il il faut que nous même point. Ne jamais souf- équivalent à n'avoir jamais été heureux. Mais est démontré que dans la vie inorganique la peine ne peut pas exister; de là la nécessité de la peine dans REVELATION MAGNETIQUE. organique. La douleur de la vie la vie m) primitive sur la terre est la seule base, la seule garantie du bonheur dans la vie ultérieure, dans le ciel. P. Mais encore il y a une de vos expressions que je ne puis absolument pas comprendre : l'immensité véri- tablement substantielle de rinflni. probablement parce que vous n'avez pas C'est V. une notion sufîisamment générique de l'expression substance elle-même. Nous ne devons pas la considérer comme une qualité, mais comme un sentiment ; c'est perception, dans les êtres pensants, de l'appropria- la tion de la matière à leur organisation. Il y a bien des choses sur la terre qui seraient néant pour les habitants de Vénus, bien des choses visibles et tangibles dans Vénus, dont nous l'existence. Mais, les anges, — sommes incompétents à apprécier pour les êtres inorganiques, la totalité de la — pour matière imparticulée est substance, c'est-à-dire que, pour eux, la totalité de ce que nous appelons espace est la plus véritable substantialité. Cependant, les astres, pris au point de vue ma- tériel, échappent au sens angélique dans la même pro- portion que la matière imparticulée , prise au point de vue immatéj-iel, échappe aux sens organiques. Comme le somnambule, d'une voix faible, prononçait ces derniers mots, j'observai dans sa physionomie une singulière expression qui m'alarma un peu et me décida à le réveiller immédiatement. Je ne l'eus pas plus tôt fait, qu'il tomba en arrière sur son oreiller et 21. 370 HISTOIRES EXTRAOUDINAIRES. expira, avec un brillant sourire qui illuminait tous ses traits. Je remarquai que moins d'une minute après son corps avait l'immuable rigidité de la pierre; son front était d'un froid de glace, tel sans doute je l'eusse trouvé après une longue pression de la main d'Azraël. Le somnambule pendant la dernière partie de son dis, cours, m'avait-il donc parlé ombres ? du fond de la région des LES SOUVENIRS DE M. AUGUSTE BEDLOE Vers la fin de Tannée 1827, pendant que je demeurais près de Charlottesville, dans la Virginie, je fis par hasard la connaissance de M. Auguste Bedloe. Ce jeune gentleman était remarquable à tous égards et excitait en moi une curiosité et un intérêt profonds. Je jugeai impossible de me rendre compte de son être tant physique que moral. Je ne pus obtenir sur sa famille au- cun renseignement positif. D'où venait-il? je ne le sus jamais bien. Même relativement à son âge, quoique je l'aie appelé un jeune gentleman, il y avait quelque chose qui m'intriguait au suprême degré. Certainement il semblait jeune, et même il affectait de parler de sa jeunesse; cependant, il y avait des moments oi\ je n'aurais guère hésité à le supposer âgé d'une centaine d'années. Mais c'était surtout son extérieur qui avait un aspect tout à fait particulier. ment grand et mince; — Il se voûtant était membres excessivement longs et émaciés; large et bas ; singulière- beaucoup; — — les le front — une complexion absohmient exsangue; HISTOIRES EXTRAORDINAIRES. 372 — sa bouche, large et flexible, et ses dents, quoique saines, plus irrégulières que je n'en vis jamais dans au- cune bouche humaine. L'expression de son sourire, toutefois, n'était nullement désagréable, comme on pourrait le supposer; mais elle n'avait aucune espèce de nuance. C'était une profonde mélancolie, une tristesse sans phases et sans intermittences. Ses yeux étaient d'une largeur anomale et ronds comme ceux d'un chat. Les pupilles elles-mêmes subissaient une contraction et une dilatation proportionnelles à l'accroissement et à la di- minution de la lumière, exactement comme on l'a observé dans les races félines. Dans les moments d'excitation, les prunelles devenaient brillantes à un degré presque inconcevable et semblaient émettre des rayons lumineux d'un éclat non mais intérieur, réfléchi, comme fait un flambeau ou le soleil ; toutefois, dans leur condition habituelle, elles étaient tellement ternes, inertes et nuageuses, qu'elles faisaient penser aux yeux d'un corps enterré depuis longtemps. Ces particularités personnelles semblaient lui causer beaucoup d'ennui, et il y faisait continuellement allu- dans un style semi-explicatif, sion semi-justificatif, qui, la première fois que je l'entendis, m'impressionna très-péniblement. Toutefois, je m'y accoutumai bientôt, et mon déplaisir se dissipa. Il semblait avoir l'in- tention d'insinuer, plutôt que d'affirmer positivement, que physiquement il n'avait pas toujours été ce qu'il était; qu'une longue série d'attaques l'avait réduit névralgiques d'une condition de beauté personnelle LES SOUVENIRS DE M. AUGUSTE BEDLOE. 373 non commune à celle que je voyais. Depuis plusieurs années, il recevait les soins d'un médecin nommé Templeton, — un vieux gentleman âgé de soixante — première avait pour peut-être, tré à qu'il et dix ans, fois la Saratoga, et des soins duquel il tira rencon- dans ce temps, ou crut tirer un grand secours. Le résultat fui que Bedloe, qui était riche, fit un arrangement avec le docteur Templeton, par lequel ce dernier, en échange d'une généreuse rémunération annuelle, consentit à consacrer exclusivement son temps et son expérience médicale à soulager le malade. Le docteur Templeton avait voyagé dans les jours de sa jeunesse, et était devenu à Paris un des sectaires les plus ardents des doctrines de Mesmer. C'était unique- ment par le moyen des remèdes magnétiques qu'il avait réussi à soulager les douleurs aiguës de son et ce malade; succès avait très-naturellement inspiré à ce der- nier une certaine confiance dans les opinions qui ser- vaient de base à ces remèdes. D'ailleurs, le comme tous les enthousiastes, avait travaillé de son docteur, mieux à faire de son pupille un parfait prosélyte, et finalement il réussit si bien, qu'il décida le patient à se soumettre à de nombreuses expériences. Fréquemment répétées, elles amenèrent un résultat qui, depuis long- temps, est devenu assez commun pour n'attirer que peu ou point l'attention, mais qui, à l'époque dont je parle, s'était très-rarement manifesté en Amérique. Je veux dire qu'entre s'était établi le docteur Templeton et Bedloe peu à peu un rapport magnétique très- HISTOIRES EXTRAORDINAIRES. 374 distinct et très-fortement accentué. Je n'ai pas toutefois l'intention d'affirmer que ce rapport s'étendît au delà des limites de la puissance somnifère; mais cette puis- sance elle-même avait atteint une grande intensité. A la première tentative faite pour produire le sommeil magnétique, le disciple de Mesmer échoua complètement. A la cinquième ou sixième, il ne réussit que trèsimparfaitement, et après des efforts opiniâtres. Ce fut seulement à la douzième que le triomphe fut complet. Après celle-là, la volonté du patient succomba rapide- ment sous celle du médecin, si bien que, lorsque je fis pour la première arrivait presque le sommeil instantanément par un pur acte de fois leur volition de l'opérateur, connaissance, même quand le malade n'avait pas conscience de sa présence. C'est seulement maintenant, ont été en l'an 18/i"5, quand de semblables miracles journellement par attestés des milliers d'hommes, que je me hasarde à citer cette apparente impossibilité comme un fait positif. Le tempérament de Bedloe était au plus haut degré sensitif, excitable, enthousiaste. Son imagination, sin- gulièrement vigoureuse et créatrice, tirait sans doute une force additionnelle de l'usage habituel de l'opium, qu'il consommait en grande quantité, et sans lequel l'existence lui eût été impossible. C'était son habitude d'en prendre une bonne dose immédiatement après son déjeuner, chaque matin, — ou plutôt immédiatement après une lasse de fort café, car dans l'avant-midi, — il ne mangeait rien et alors il partait seul, ou seule- LES SOUVENIRS DE M. AUGUSTE BEDLOE. 375 ment accompagné d'un chien, pour une longue promenade à travers la chaîne de sauvages et lugubres hauteurs qui courent à l'ouest et au sud de Charlottesville, et qui sont décorées ici du nom de Ragged Mountains^. Par un jour sombre, chaud et brumeux, vers la fin de novembre, et durant l'étrange interrègne de saisons que nous appelons en Amérique l'été indien, M. Bedloe partit, suivant son habitude, pour les montagnes. Le jour s'écoula, et il ne revint pas. Vers huit heures du soir, étant sérieusement alarmés par cette absence prolongée, nous allions nous mettre sa recherche, à quand il reparut inopinément, ni mieux ni plus mal portant, et plus animé que de coutume. Le récit qu'il nements qui singuliers fit l'avaient de son expédition et des évéretenu fut en vérité des plus : — Vous vous rappelez, dit-il, qu'il était environ neuf heures du matin quand je quittai Charlottesville. Je dirigeai et, immédiatement mes pas vers la montagne, vers dix heures, j'entrai dans une gorge qui était entièrement nouvelle pour moi. Je suivis toutes les sinuosités de cette passe avec beaucoup d'intérêt. — Le théâtre qui se présentait de tous côtés, quoique ne méritant peut-être pas l'appellation de sublime, portait en soi un caractère indescriptible, et pour moi délicieux, de lugubre désolation. solument vierge. La solitude semblait ab- Je ne pouvais \. Montagnes déchirées; m' empêcher de croire une branche des Montagnes bleues, Reul Bidge, partie orientale des AUeghanys. — C. B. — HISTOIRES EXTRAORDINAIRES. 370 que les gazons verts et les roches grises que je foulais n'avaient jamais été foulés par un pied humain. L'en- du ravin est trée si complètement cachée, inaccessible, excepté à travers une de fait et série d'accidents, pas du tout impossible que je fusse en qu'il n'était véiité le premier aventurier, — le premier et le seul qui eût jamais pénétré ces solitudes. )) L'épais et singulier brouillard ou fumée qui dis- tingue l'été indien et qui s'étendait alors pesamment sur , tous les objets, approfondissait sans doute les impressions vagues que ces objets créaient en moi. Cette brume poétique était si dense, que je ne pouvais jamais voir au delà d'une douzaine de yards de ma route. Ce comme il chemin était excessivement sinueux, et, était impossible de voir le soleil, j'avais perdu toute idée de la direction dans laquelle je marchais. Cependant, l'opium avait produit son effet accoutumé, qui est d monde extérieur d'une intensité revêtir tout le Dans térêt. trèfle, brin d'herbe, — dans dans l'éclat du vent, forêt, tremblement d'une le couleur d'un le feuille, — dans la — dans bourdonnement d'une les vagues odeurs qui la forme d'un abeille, d'une goutte de rosée, — dans — dans ; d'in- le — soupir venaient de la — se produisait tout un monde d'inspirations, une procession magnifique et bigarrée de pensées désordonnées et rapsodiques. » Tout occupé par ces rêveries, je marchai plusieurs heures, durant lesquelles le brouillard s'épaissit autour de moi à un degré tel, que je fus réduit cà chercher LES SOUVENIRS DE M. AUPxUSTE BEDLOE. 377 mon chemin à tâtons. Et alors un indéfinissable malaise s'empara veuse et — une espèce de moi, d'irrilation ner- de tremblement. Je craignais d'avancer, de peur d'être précipité dans quelque abîme. Je me souvins aussi d'étranges histoires sur ces Ragged Mountains, et de races d'hommes bizarres et sauvages qui habitaient leurs bois et leurs cavernes. Mille pensées vagues me pressaient et me déconcertaient, — pensées que leur vague rendait encore plus douloureuses. Tout à coup mon attention fut arrêtée par un fort battement de tambour. » Ma stupéfaction, naturellement, fut extrême. Un tambour, dans ces montagnes, était chose inconnue. Je n'aurais pas été plus surpris par le son de la trompette de l'Archange. Mais une nouvelle et bien plus extraordinaire cause d'intérêt et de perplexité se manifesta. J'entendais cliquetis, s'approcher un bruissement sauvage, comme d'un et à l'instant trousseau de grosses clefs, un — même un homme à moitié nu, au visage basané, passa devant moi en poussant un cri aigu. Il ma personne, que je sentis le chaud de son haleine sur ma figure. Il tenait dans une main passa si près de un instrument composé d'une série d'anneaux de fer et les A peine secouait vigoureusement en courant. avait-il disparu dans le brouillard, que, haletante derrière lui, la gueule ouverte et les yeux étincelants, s'élança une énorme bête. Je ne pouvais pas prendre sur son espèce )) : me mé- c'était une hyène. La vue de ce monstre soulagea plutôt qu'elle n'aug- HISTOIRES EXTRAORDINAIRES. 378 menta mes terreurs; — car j'étais bien sûr maintenant que je rêvais, et je m'efforçai, je m'excitai moi-même à réveiller ma conscience. Je marchai délibérément et lestement en avant. Je me frottai les yeux. Je criai trèshaut. Je me pinçai les membres. Une petite source ma vue, je m'y arrêtai, et je m'y s'étant présentée à lavai les mains, la tête et le cou. Je crus sentir se dis- siper les sensations équivoques qui menté jusque-là. Il m'avaient tour- me parut, quand je me relevai, que j'étais un nouvel homme, et je poursuivis ferme- ment et complaisamment ma route inconnue. » A la longue, tout à fait lourdeur la oppressive épuisé par l'exercice et par de l'atmosphère, je m'assis sous un arbre. En ce moment parut un faible rayon de soleil, et l'ombre des feuilles de l'arbre tomba sur le gazon, légèrement mais suffisamment définie. Pen- dant quelques minutes, je fixai cette ombre avec éton- nement. Sa forme me comblait de stupeur. Je levai les yeux. L'arbre était un palmier. )) Je me levai précipitamment et tation terrible, — car l'idée désormais suffisante. Je vis, parfait dans un état d'agi- que je rêvais n'était plus — je sentis que j'avais le gouvernement de mes sens, — et ces sens ap- portaient maintenant à mon âme un monde de sensations nouvelles et singulières. La chaleur devint tout d'un coup intolérable. Une étrange odeur chargeait la brise. — Un murmure profond et continuel, comme celui qui s'élève d'une rivière abondante, mais coulant régulièrement, vint à mes oreilles, entremêlé du bour- LES SOUVENIRS DE M. AUGUSTE BEDLOE. 370 donnement particulier d'une multitude de voix humaines. Pendant que j'écoutais, avec un étonnement qu'il » est bien inutile de vous décrire, un fort et bref coup de vent enleva, comme une baguette de magicien, le brouillard qui chargeait la terre. )) Je me trouvai au pied d'une haute montagne domi- nant une vaste plaine, à travers laquelle coulait une majestueuse rivière. Au bord de cette rivière s'élevait une ville d'un aspect oriental, telle que nous en voyons dans les Mille et une Nuits, mais d'un caractère encore plus singulier qu'aucune de celles qui y sont décrites. De ma position, qui était bien au-dessus du niveau de la ville, je pouvais apercevoir tous ses recoins et tous ses angles, comme s'ils eussent été dessinés sur une carte. Les rues paraissaient innombrables et se croi- saient irrégulièrement dans toutes les directions, mais ressemblaient moins à des rues qu'à de longues allées contournées, et fourmillaient littéralement d'habitants. Les maisons étaient étrangement pittoresques. De chaque côté, c'étaitune véritable débauche de balcons, de vérandas, de minarets, de niches et de tourelles fantastiquement découpées. Les bazars abondaient; les plus riches marchandises s'y variété et une profusion infinies la plus déployaient avec une : soies, mousselines, éblouissante coutellerie, diamants et bijoux des plus magnifiques. A côté de ces choses, on voyait de tous côtés des pavillons, des palanquins, des litières où se trouvaient de magnifiques dames sévèrement HISTOIRES EXTRAORDINAIRES. 3S0 • voilées, des éléphants fastiieiisement caparaçonnés, des idoles grotesquement taillées, des tambours, des ban- nières et des gongs, des lances, des casse-tête dorés et argentés. Et parmi la foule, la clameur, la la confusion générales, parmi un million noirs et mêlée et d'hommes jaunes, en turban et en robe, avec la barbe flottante, circulait une multitude innombrable de bœufs saintement enrubannés, pendant que des kîgions de singes malpropres et sacrés grimpaient, jacassant et piaillant, après les corniches des mosquées, ou se suspendaient aux minarets et aux tourelles. Des rues fourmillantes aux quais de rivière la descendaient d'innombrables escaliers qui conduisaient à des bains, pendant que la rivière elle-même semblait avec peine se frayer un passage à travers les vastes flottes de bâ- timents surchargés qui tourmentaient sa surface en tout sens. Au delà des murs de la ville s'élevaient fré- quemment, en groupes majestueux, le palmier et le cocotier, avec d'autres arbres d'un grand âge, gigan- tesques et solennels; et çà et là on pouvait apercevoir un champ de riz, la hutte de une citerne, un temple une gracieuse fille isolé, solitaire une cruche sur sa tête, vers chaume d'un paysan, un camp de gypsies, ou prenant sa route, avec les bords de la magni- fique rivière. )) Maintenant, sans doute, vous direz que mais nullement. Ce que je voyais, dais, — ce que je sentais, — ce que vait rien en soi de l'idiosyncrasie je rêvais; — ce que j'entenje pensais n'a- non méconnaissable LES SOUVENIRS DE M. AUGUSTE BEDLOE. du rêve. Tout se tenait D'abord, doutant logiquement et si j'étais 381 faisait corps. réellement éveillé, je me soumis à une série d'épreuves qui me convainquiient bien vite que je l'étais réellement. Or, quand quel- qu'un rêve, et que dans son rêve il soupçonne rêve, le soupçon et le dormeur Ainsi, Novalis qu'il ne manque jamais de se confirmer, est presque immédiatement réveillé. ne se trompe pas en disant que nous sommes près de nous réveiller quand nous rêvons que nous rêvons. Si la vision s'était offerte à moi je l'eusse être purement que un rêve; mais, se présentant comme je Tai dit, et suspectée et vériliée comme elle le fut, je suis forcé de la classer parmi d'autres — En telle soupçonnée d'être un rêve, alors elle eût pu cela, je ji'afïirme phénomènes. pas que vous ayez tort, remarqua le docteur Templeton. Mais poursuivez. Vous vous levâtes, et vous descendîtes dans la cité. — me levai, continua Bedloe regardant le docteur avec un air de profond étonnement; je me levai, comme vous dites, et descendis dans la cité. Sur ma route, je tombai au milieu d'une immense poJe pulace qui encombrait chaque avenue, se dirigeant toute dans le même sens, et montrant dans son action la plus violente je ne animation. Très-soudainement, et sous sais quelle pression inconcevable, je me sentis profondément pénétré d'un intérêt personnel dans ce qui allait arriver. Je croyais sentir que j'avais un rôle important à jouer, sans comprendre exactement quel il était. Contre la foule qui m'environnait j'éprouvai 382 HISTOIRES KXTKAOUDIiN AIRES. toutefois un profond sentiment d'animositc. Je m'ar- rachai du milieu de cette cohue, et rapidement, par un chemin circulaire, j'arrivai à la ville, et j'y entrai. Elle était en proie au tumiilte et à la plus violente dis- corde. Un petit détachement d'hommes ajustés moitié à l'indienne, moitié à l'européenne, et commandés par des gentlemen qui portaient un uniforme en partie anglais, soutenait pulace un combat très-inégal contre la po- fourmillante des avenues. Je rejoignis cette faible troupe, je me saisis des et je frappai désespoir. au hasard avec armes d'un officier tué, la férocité nerveuse du Nous fûmes bientôt écrasés par le nombre et contraints de chercher un refuge dans une espèce de kiosque. Nous nous y barricadâmes, et nous fûmes moment en sûreté. Par une meurtrière, pour le près du sommet du kiosque, j'aperçus une vaste foule dans une agitation furieuse, entourant et assaillant un beau palais qui dominait la Alors, par une fenêtre rivière. supérieure du palais, descendit un personnage d'une apparence efféminée, au moyen d'une corde faite avec les turbans de ses domestiques. Un bateau était tout près, dans lequel il s'échappa vers le bord opposé de la rivière. » Et alors un nouvel objet prit possession de mon âme. J'adressai à mes compagnons quelques paroles précipitées, mais énergiques, rallier quelques-uns à et, ayant réussi à en mon dessein, je fis une sortie furieuse hors du kiosque. Nous nous précipitâmes sur la foule qui l'assiégeait. Ils s'enfuirent d'abord de- LES SOUVENIRS DE M. AUGUSTE liEDLOE. 383 vaut nous. Ils se rallièrent, combattirent comme des enragés, et firent une nouvelle Cependant, retraite. nous avions été emportés loin du kiosque, perdus étions étouffées et nous et embarrassés dans des rues étroites, par de hautes maisons, dans le fond des- quelles le soleil n'avait jamais envoyé sa lumière. La populace se pressait impétueusement sur nous, nous harcelait avec ses lances, et nous accablait de ses volées de Ces dernières étaient remarquables flèches. et ressemblaient en quelque sorte au kriss tortillé des — imitant rampe, — longues et noires, avec Malais; le mouvement d'un serpent qui une pointe empoi- sonnée. L'une d'elles me frappa à la tempe droite. Je pirouettai, je tombai. Un mal instantané et terrible s'empara de moi. Je m'agitai, pirer, — je — je m'efforçai de res- mourus. — Vous ne vous obstinerez plus sans doute, dis-je en souriant, à croire que toute votre aventure n'est pas un rêve? Êtes-vous décidé à soutenir que vous êtes mort? Quand prononcé ces mots, j'eus je m'attendais à quelque heureuse saillie de Bedloe, en manière de réplique; mais, à mon grand étonnement, il hésita, trembla, devint terriblement pâle, et garda le silence. Je levai les yeux sur Templeton. Il se tenait droit et roide sur sa chaise; — ses dents claquaient, et ses yeux s'élançaient de leurs orbites. — Continuez, rauque. dit-il enhn à Bedloe d'une voix HISTOIRES EXTRAORDINAIRES. 384 — Pendant quelques minutes, poursuivit ce dernier, ma seule impression, — ma seule sensation, — fut de la nuit et du non-être, avec la conscience de celle la mort. A la longue, il me sembla qu'une secousse violente et soudaine comme l'électricité traversait mon àme. Avec cette secousse vint le sens de rélasticité et de la lumière. Quant à cette dernière, je la sentis, je me sembla que je m'élevais de terre; mais je ne possédais pas ma présence ne la vis pas. En un instant, il corporelle, visible, audible ou palpable. La foule s'était Le tumulte avait cessé. La ville était compa- retirée. rativement calme. Au-dessous de moi gisait mon corps, avec la flèche dans ma tempe, toute la tête grandement enflée et défigurée. Mais toutes ces choses, je les sentis, — je ne les vis pas. Je ne pris d'intérêt à rien. Et même le cadavre me semblait un objet avec lequel je n'avais rien de commun. Je n'avais aucune volonté, mais il me sembla que j'étais mis en mouvement et que je m'envolais légèrement hors par le de l'enceinte de la ville même circuit que j'avais pris pour y entrer. Quand j'eus atteint, dans la montagne, l'endroit du ravin oi^i j'avais rencontré l'hyène, veau un choc j'éprouvai de nou- comme celui d'une pile galvanique; le sentiment de la pesanteur, celui de substance, rentrèrent en moi. Je moi-même, mon propre redevins individu, et je dirigeai vivement mes pas vers logis; — mais vivante de la ne puis le mon passé n'avait pas perdu l'énergie réalité, contraindre — mon et maintenant intelligence, encoie je même pour LES SOUVEiMUS DE Liiie M. ALGUSTE BEDLOE. 385 minute, à considérer tout cela comme un songe. — Ce n'en était pas un, dit Templeton, avec un air de profonde solennité ; mais il serait de dire difficile quel autre terme définirait mieux le cas en question. Supposons que l'àme de Thomme moderne est sur le bord de quelques prodigieuses découvertes psychiques. Contentons-nous de cette hypothèse. Quant au reste, j'ai quelques éclaircissements à donner. Voici une peinture à l'aquarelle que je vous aurais déjà montrée si un indéfinissable sentiment d'horreur ne m'en avait pas empêché jusqu'à présent. Nous regardâmes Je n'y vis la peinture qu'il nous présentait. aucun caractère bien extraordinaire; mais son effet sur Bedloe fut prodigieux. A peine l'eut-il re- gardée, qu'il faillit s'évanouir. Et cependant, ce n'était un qu'un portrait à la miniature, portrait merveil- leusement fini, à vrai dire, de sa propre physionomie SI originale. Du moins, telle fut ma pensée en la regar- dant. — Vous apercevez pleton; elle est là, la date de la peinture, dit à peine 1780. C'est dans cette année faite. C'est le portrait d'un — à qui visible, que — peinture fut — un M. Oldeb, je m'attachai très-vivement à Calcutta, durant l'administration de Warren Hastings. Je n'avais alors que vingt ans. Quand je vous fois, dans ce coin, cette ami défunt, Tem- vis pour la première monsieur Bedloe, à Saratoga, ce fut la miraculeuse similitude qui existait entre vous et le portrait qui me détermina à vous aborder, à rechercher votre amitié 22 liiSTOiatlS 380 EXTKAOKDliNAlHES. et à amener ces arrangements qui firent de moi votre compagnon perpétuel. En agissant ainsi, j'étais poussé en partie, et peut-être principalement, par les sou- venirs pleins de regrets du défunt, mais d'une autre part aussi par une curiosité inquiète à votre endroit, et qui n'était pas dénuée d'une certaine terreur. » Dans votre récit de la vision qui s'est présentée à vous dans les montagnes, vous avez décrit, avec le plus minutieux détail, la ville indienne de Bénarès, sur Les rassemblements, les combats, la Rivière-Sainte. le massacre, c'étaient les épisodes réels de l'insur- rection de Gheyte-Sing, qui eut lieu en 1780, alors que Hastings courut les plus grands dangers pour sa vie. L'homme qui s'est échappé par la corde faite de turbans, c'était Cheyte-Sing lui-même. La troupe du kiosque était composée de cipayes et d'officiers anglais, Hastings à leur tête. Je faisais partie de cette troupe, et je fis tous mes efforts pour empêcher cette impru- dente et fatale sortie de l'officier qui tomba dans la bagarre sous la flèche empoisonnée d'un Bengali. Cet officier était mon plus cher ami. C'était Oldeb. Vous verrez par ce manuscrit, — ici le narrateur produisit un livre de notes, dans lequel quelques pages paraissaient d'une date toute fraîche, vous pensiez ces choses au j'étais — que, pendant que milieu de la montagne, occupé ici, à la maison, à les décrire sur le papier. Une semaine environ après cette conversation, l'article suivant parut dans un journal de Charlottesville ; LES SOUVENIRS DE M. AUGUSTE BEDLOE. u C'est pour la 387 nous un devoir douloureux d'annoncer mort de M. Auguste Bedlo, un gentleman que ses manières charmantes et ses nombreuses vertus avaient depuis longtemps rendu cher aux citoyens de Gharlottesville. M. » B., depuis quelques années, souffrait d'une névralgie qui avait souvent menacé d'aboutir fatalement; mais elle ne peut être regardée que comme la cause indirecte de sa mort. La cause immédiate caractère spécial. qu'il singulier dans fit les jours, il contracta et Dans d'un fut une excursion Ragged Mountains, il y a quelques un léger rhume avec de la fièvre, qui fut suivi d'un grand mouvement du sang à la tête. Pour le soulager, le docteur Templeton eut recours à la saignée locale. Des sangsues aux furent appliquées tempes. Dans un délai effroyablement court, le malade mourut, et l'on s'aperçut que, dans le bocal qui contenait les sangsues, avait été introduite par hasard une de ces sangsues vermiculaires venimeuses qui se rencontrent çà et là dans les étangs circonvoisins. Cette bête se fixa d'elle-même sur une petite artère de la tempe droite. Son sangsue médicinale extrême fit ressemblance avec la que la méprise fut découverte trop tard. « N.-B. — La sangsue venimeuse de Charlottesville peut toujours se distinguer de la sangsue médicinale par sa noirceur, et spécialement par ses tortillements, ou mouvements vermiculaires, qui ressemblent beau- coup à ceux d'un serpent. » HISTOIRES EXTR AORDINATIIKS. 388 Je me trouvais avec l'éditeur du journal en question, et nous causions de ce singulier accident, vint à l'idée de lui demander quand il me pourquoi l'on imprimé le nom du défunt avec rorthographe — : avait Bedlo. Je présume, dis-je, que vous avez quelque autorité pour l'orthographier ainsi; j'ai toujours cru que le nom devait s'écrire avec un e à la fin. — Autorité? non, répliqua-t-il. C'est une simple erreur du typographe. Le nom est Bedloe avec un e; c'est connu écrit — de tout le monde, et je ne peut donc se faire Il même, comme je jamais vu murmurai-je en moi- mes talons, qu'une que toutes les fictions; qu'est-ce que Bedlo sans e, si ce — car n'est Oldeb retourné? homme me dit que c'est une faute typogra- cet phique , tournais sur vérité soit plus étrange VA l'ai autrement. ! MORELLA Lui-même, par lui-m^me, avec lui-même, homogène éternel. Platon. Ce que j'éprouvais relativement à mon amie Morella était une profonde fait mais très-singulière affection. Ayant sa connaissance par hasard, il y a nombre d'an- nées, mon âme, dès notre première rencontre, brûla de feux qu'elle n'avait jamais connus; — mais ces feux n'étaient point ceux d'Éros, et ce fut pour mon esprit un amer tourment que la conviction croissante que je ne pourrais jamais définir leur caractère insolite, ni régulariser leur intensité errante. Cependant, nous nous convînmes, et la destinée nous fit nous unir à l'autel. Jamais je ne parlai de passion, jamais je ne songeai à l'amour. Néanmoins, elle fuyait la société, et, s'at tachant à moi seul, elle me étonné, c'est un bonheur; — rendit heureux. Être et rêver, n'est-ce pas un bonheur aussi? L'érudition de Morella était profonde. père le Comme j'es- montrer, ses talents n'étaient pas d'un ordre secondaire ; la puissance de son esprit était gigantes22. HISTOIRES EXTRAORDINAIRES. 390 que. Je le sentis, et, dans mainte occasion, je devins son écolier. Toutefois, je m'aperçus bientôt que Morella, en raison de son éducation étalait devant moi bon raisons que je devinrent aussi la comme Técume de allemande. Ces livres, pour des ne pouvais concevoir, étude constante et favorite qu'à la Presbour^% nombre de ces écrits mystiques qui sont généralement considérés la première littérature faite à ; mienne, — il et, si faisaient son avec le temps ils ne faut attribuer cela simple mais très-efïicace influence de l'habi- tude et de l'exemple. En toutes ces choses, si je ne me trompe, ma raison n'avait presque rien à faire. Mes convictions, ou je ne me connais plus moi-même, n'étaient en aucune façon basées sur l'idéal, et on n'aurait pu découvrir, à moins que je ne m'abuse grandement, aucune teinture du mysticisme de mes lectures, soit dans mes actions, soit dans mes pensées. Persuadé de cela, je m'abandonnai aveuglément à la direction de ma femme, avec un cœur imperturbé dans études. Et alors, le et j'entrai labyrinthe de ses — quand, me plongeant dans des pages maudites, je sentais un esprit maudit qui s'allu- mait en moi, — Morella venait, posant sa main froide sur la mienne et ramassant dans les cendres d'une philosophie morte quelques graves et singulières paroles qui, par leur sens bizarre, s'incrustaient dans ma mé- moire. Et alors, pendant des heures, je m'étendais rê- veur à son côté, et je me plongeais dans la musique de sa voix, — jusqu'à ce que cette mélodie à la longue s'in- MORELLA. fectât de terreur; — et une ombre tombait sur mon âme, et je devenais pâle, ment à ces sons 391 et je frissonnais intérieure- trop extra-terrestres. Et ainsi, la jouissance s'évanouissait soudainement dans l'horreur, et l'idéal du beau devenait l'idéal de la hideur, comme vallée de la Hinnom est devenue la Géhenne. Il est inutile d'établir le caractère exact des problèmes qui, jaillissant des volumes dont j'ai parlé, furent pen- dant longtemps presque le seul objet de conversation entre Morella et moi. Les gens instruits dans ce que l'on peut appeler la morale théologique les concevront facilement, et ceux qui sont illettrés n'y comprendraient que peu de chose en tout cas. L'étrange panthéisme de Fichte, la Palingénésie modifiée des Pythagoriciens, et, par-dessus tout, la doctrine de Videntité telle qu'elle est présentée par Schelling, étaient généralement les points de discussion qui offraient le plus de charmes à l'imaginative Morella. Cette identité, dite personnelle, M. Locke, je crois, la fait judicieusement consister dans la permanence de l'être rationnel. En tant que par personne nous entendons une essence pensante, douée de raison, et en tant qu'il existe une conscience qui — cette con— qui nous tous être ce que nous appedes autres lons nous-mème, — nous distinguant accompagne toujours la pensée, c'est elle, science, fait ainsi êtres pensants, et nous donnant notre identité personnelle. Mais le principlum individuatlonis, — la notion de cette identité qui, à la mort, est, ou n'est pas perdue à jamais, fut pour moi, en tout temps, un problème HISTOIRES EXTRAORDINAIRES. 392 du plus intense intérêt, non-seulement à cause de nature inquiétante et embarrassante de ses la consé- quences, mais aussi à cause de la façon singulière et agitée dont en parlait Morella. Mais, en vérité, le temps était maintenant arrivé où mystère de la nature de ma femme m'oppressait comme un charme. Je ne pouvais plus supporter l'atle touchement de ses doigts pâles, ni le timbre profond de sa parole musicale, ni l'éclat de ses yeux mélancoliques. Et elle savait tout cela, aucun reproche; faiblesse elle mais ne m'en faisait semblait avoir conscience de ma ou de ma folie, et, tout en souriant, elle ap- pelait cela la Destinée. Elle semblait aussi science de la cause, à moi inconnue, avoir con- de l'altération graduelle de mon amitié; mais elle ne me donnait au- cune explication et ne faisait aucune allusion à la nature de cette cause. Morella toutefois n'était qu'une femme, et elle dépérissait journellement. A la longue, une tache pourpre se fixa immuablement sur sa joue, et les veines bleues de son front pâle devinrent proé- minentes. Et ma nature se fondait parfois en pitié; mais, un moment après, je rencontrais l'éclair de ses yeux chargés de pensées, vait mal gard a et et alors mon âme se trou- éprouvait le vertige de celui dont le re- plongé dans quelque lugubre et insondable abîme. Dirai-je que j'aspirais, avec un désir intense et dé- vorant, au moment de la mort de Morella? Cela fut ainsi; mais le fragile esprit se cramponna à son habi- MORl-lLLA. 393 tacle d'argile pendant bien des jours, bien des semai- nes et bien des mois fastidieux, si bien qu'à la fin mes nerfs torturés remportèrent la victoire sur ma raison ; je devins et furieux de tous ces retards, et avec un cœur de démon je maudis les jours, et les heures, et minutes amères qui semblaient s'allonger et s'al- les longer sans cesse, à mesure que sa noble vie déclinait, comme les ombres dans l'agonie du jour. Mais, un soir d'automne, comme l'air dormait immobile dans le ciel , Morella m'appela à son chevet. Il y avait un voile de brume sur toute la terre, et un chaud embrasement sur les ceux, et, à voir les splendeurs d'octobre dans le feuillage de la forêt, on eût dit qu'un bel arc-en-ciel s'était laissé choir du firmament. — Voici le jour le plus beau des jours, dit-elle quand j'approchai, des jours pour vivre ou pour mourir. C'est un beau jour pour les fils de la terre et de ah ! plus beau encore pour les mort filles du la vie , ciel et — de la ! Je baisai son front, et elle continua : — vais mourir, cependant — Morella! — n'ont jamais t'aurait été ces jours où permis de m'aimer; — mais que, dans tu Je je vivrai. été, Ils il la vie, celle abhorras, dans la mort tu l'adoreras. — Morella! — répète que Je je vais mourir. gage de cette affection Mais en moi est un — ah quelle mince affection ! — que vous avez éprouvée pour moi, Morella. ! Et, quand HISTOIRES EXTRAORDINAIRES. 394 mon esprit partira, l'enfant vivra, — ton enfant, mon enfant à moi, Morella. Mais tes jours seront des jours pleins de chagrin, — de ce chagrin qui est la plus du- rable des impressions, comme le cyprès est le plus vi- vace des arbres; car les heures de ton bonheur sont passées, et la joie ne se cueille pas deux fois dans une vie, comme les roses de Passtum deux fois dans une année. Tu ne joueras plus avec le temps l'homme de Téos ; le le jeu de myrte et la vigne te seront choses inconnues, et partout sur la terre tu porteras avec toi ton suaire, comme le musulman de la Mecque. — Morella! m'écriai-je , Morella! comment sais-tu cela? Mais elle retourna son visage sur l'oreiller; un léger tremblement courut sur ses membres, elle mourut, et je n'entendis plus sa voix. — Cependant, comme elle l'avait prédit, son enfant, auquel en mourant elle avait donné naissance, et qui ne respira qu'après que la mère eut cessé de respirer, son enfant, une fille, vécut. Et elle — grandit étrange- ment en taille et en intelligence, et devint la parfaite ressemblance de celle qui était partie, et je l'aimai d'un plus fervent amour que je ne me serais capable d'en éprouver pour aucune habitante do cru la terre. Mais, avant qu'il fût longtemps, le ciel de cette pure affection s'assombrit, et la mélancolie, et l'horreur, et l'angoisse, y défilèrent en nuages. J'ai dit que l'enfant grandit étrangement ^n taille et en intelligence. MORÉLLA. 395 Étrange, en vérité, fut le rapide accroissement de sa nature corporelle, — mais terribles, oh ! terribles fu- rent les tumultueuses pensées qui s'amoncelèrent sur moi son , pendant que je surveillais être intellectuel. développement de le Pouvait-il en être autrement, quand je découvrais chaque jour dans les conceptions de l'enfant la puissance adulte et les facultés de la femme? —quand les leçons de l'expérience tombaient des lèvres de l'enfance? — quand je voyais à chaque instant la sagesse et les passions de la maturité jaillir de cet œil noir et méditatif? Quand, dis-je, tout cela frappa sible à — quand il fut imposmes sens épouvantés, mon âme de se le dissimuler plus longtemps, — à mes facultés frissonnantes de repousser cette cerde s'étonner que des soupçons titude, — y a-t-il lieu d'une nature terrible et inquiétante se soient glissés dans mon esprit, ou que mes pensées se soient repor- tées avec horreur vers les contes étranges et les péné- trantes théories de la défunte Morella? J'arrachai à la curiosité du monde un être que la destinée me com- mandait d'adorer, et, dans la rigoureuse retraite de mon intérieur, je veillai avec une anxiété mortelle sur tout ce qui concernait la créature aimée. Et comme les années se déroulaient, et comme cha- que jour je contemplais son saint, son doux, son éloquent visage, et comme j'étudiais ses santes^ chaque jour je découvrais de formes mûris- nouveaux points de ressemblance entre l'enfant et sa mère, la mélancolique et la morte. Et, d'instant en instant, ces ombres HISTOIRES EXTRAORDINAIRES. 3% de ressemblance s'épaississaient, toujours plus pleines, plus définies, plus inquiétantes et plus affreusement terribles dans leur aspect. Car, que son sourire ressem- blât au sourire de sa mère, je pouvais l'admettre; mais cette ressemblance était une identité qui me donnait le frisson; — que ses yeux ressemblassent de Morella, je devais le supporter; mais aussi à ceux pé- ils nétraient trop souvent dans les profondeurs de mon âme avec l'étrange et intense pensée de Morella ellemême. Et dans le contour de son front élevé, et dans les boucles de sa chevelure soyeuse, et dans ses doigts pâles qui s'y plongeaient d'habitade, et dans le timbre grave et musical de sa parole, et par-dessus tout, oh! par-dessus tout, — dans les — phrases et les expres- sions de la morte sur les lèvres de Taimée, de la vivante, je trouvais un aliment pour une horrible pensée dévorante, — pour un ver qui ne voulait pas mourir. Ainsi passèrent deux lustres de sa vie, et toujours ma fille restait sans nom sur la terre. Mon enfant et mon amour étaient les appellations habituellement dictées par l'affection paternelle, et la sévère réclusion de son existence s'opposait à toute autre relation. Le nom de Morella était mort avec elle. De la mère, je n'avais jamais parlé à la fille; — il m'était impossible d'en parler. En réalité, durant la brève période de son existence, cette dernière n'avait reçu aucune impres- sion du monde extérieur, excepté celles qui avaient pu 'lui être fournies dans les étroites limites de sa retraite. MORELLA. A la longue, s'offrit à tation, 397 cependant, la cérémonie du baptême mon esprit, dans cet état d'énervation et d'agicomme l'heureuse délivrance des terreurs de ma destinée. Et, aux fonts baptismaux, j'hésitai sur le choix d'un nom. Et une foule d'épithètes de sagesse et de beauté, de noms dernes, de tirés des temps anciens et mo- mon pays et des pays étrangers, vint se presser sur mes lèvres, et une multitude d'appellations charmantes de noblesse, de bonheur et de bonté. Qui m'inspira donc alors d'agiter le souvenir de la morte enterrée? Quel démon me poussa à soupirer un son dont le simple souvenir faisait toujours refluer mon sang par torrents des tempes au cœur? Quel méchant du fond des abîmes de mon âme, quand, esprit parla sous ces voûtes obscures et dans le silence de la nuit, chuchotai dans l'oreille du saint homme les syllabes je (i Morelïa »? Quel être, plus que démon, convulsa les traits de mon enfant et les couvrit des teintes de la mort, quand, tressaillant à ce nom à peine perceptible, elle tourna ses yeux limpides du sol vers le ciel, et, tom- bant prosternée sur les dalles noires de notre caveau de famille, répondit Ces simples mots : Me voilà! tombèrent distincts, froidement, tranquillement distincts, dans mon oreille, et, de là, comme du plomb fondu, roulèrent en sifflant dans ma cervelle. Les années, les années peuvent passer, mais le souvenir la de cet instant, — jamais! Ah! les fleurs et vigne n'étaient pas choses inconnues pour moi; — mais l'aconit et le cyprès m'ombragèrent nuit et jour. ***** 1)3 HISTOIRES EXTRAORDINAIRES. 398 Et je perdis tout sentiment du temps et des lieux, et les étoiles de ma destinée disparurent du ciel, et dès lors la terre devint ténébreuse, et toutes les figures ter- restres passèrent près de tigeantes, et rella! moi comme des ombres vol- parmi elles je n'en voyais qu'une, — Mo- Les vents du firmament ne soupiraient qu'un son à mes oreilles, et le clapotement de la mer mur- murait incessamment rut, et, : a Morella! » Mais elle mou- de mes propres mains je la portai à sa tombe, et je ris d'un amer et long rire, quand, dans le caveau la seconde, je ne découvris aucune trace oi^i je déposai de la première — Morella. LIGEIA Etil y a là dedans la volonté, qui ne meurt Qui donc connaît les mystères de la vo- pas. ainsi que sa vigueur? Car Dieu n'est qu'une grande volonté pénétrant toutes choses lonté, par l'intensité qui lui est propre. L'homme ne cède aux anges et ne se rend entièrement à la mort que par l'infirmité de sa pauvre volonté, Joseph Glanvill. Je ne puis pas nie rappeler, sur mon âme, comment, quand, ni même où je fis pour la première fois con- naissance avec lady Ligeia. De longues années se sont écoulées depuis lors, et une grande souffrance a affaibli ma mémoire. Ou peut-être ne puis-je plus maintenant me rappeler ces points, parce qu'en vérité le caractère de ma bien-aimée, sa rare instruction, son genre de beauté, si singulier et si placide, et la pénétrante et subjuguante éloquence de sa profonde parole musicale, ont fait leur nière ai si patiente, chemin dans mon cœur d'une masi constante, si furtive, que je n'y pas pris garde et n'en ai pas eu conscience. Cependant, je crois que je la rencontrai pour la pre- mière fois, et plusieurs fois depuis lors, dans une vaste et antique ville délabrée sur les bords du Rhin. Quant HISTOIRES EXTRAORDINAIRES. 400 famille, à sa — très-certainement elle m'en a parlé. Qu'elle fût d'une dale excessivement ancienne, je n'en fais aucun doute. — Ligeia! Ligeia! — Plongé dans des éludes qui par leur nature sont plus propres que toute autre à amortir les impressions du monde extérieur, — il me suffit de ce mot si doux, pour ramener devant les yeux de — Ligeia! — ma pensée l'image de celle qui n'est plus. Et maintenant, pendant que j'écris, il me revient, comme une h.eur, que je n'ai jamais sa le nom de famille de celle qui fut mon amie et ma fiancée, qui mon compagnon d'études, devint mon cœur. Était-ce par suite de ma Ligeia, était-ce une preuve de la force de mon affection, que je ne pris aucun renseignement sur ce point? Ou plutôt et enfin l'épouse de quelque injonction était-ce — folâtre de — une offrande bizarre un caprice à moi, et romantique sur l'autel du culte le plus passionné? Je ne me rappelle le fait que confusément; — faut-il donc s'étonner si j'ai entièrement oublié les circonstances qui lui donnèrent naissance ou qui l'accompagnèrent? Et, en vérité, si jamais l'esprit de roman, — si jamais la \}k\eAshtophet de l'idolâtreÉgypte, aux ailes ténébreuses, ont présidé, comme on augure, Il est dit, — très-sûrement néanmoins un aux mariages de ils sinistre ont présidé au mien. sujet très-cher sur lequel ma mémoire n'est pas en défaut, c'est la personne de Ligeia. Elle était d'une grande taille, dans un peu mince, et même les derniers jours très-amaigrie. J'essayerais en vain de dépeindre la majesté, l'aisance tranquille de sa LTGEIA. démarche et 401 l'incompréhensible légèreté de son pas; elle venait et s'en allait Je , l'élasticité comme une ombre. ne m'apercevais jamais de son entrée dans mon cabinet de travail que par la chère musique de sa voix douce et profonde, quand elle posait sa main de marbre sur mon épaule. Quant à la beauté de la figure, aucune femme ne l'a jamais égalée. C'était l'éclat d'un rêve d'opium, une vision aérienne et ravissante, plus étran- gement céleste que les rêveries qui voltigent dans les âmes assoupies des filles de Délos. Cependant, ses traits n'étaient pas jetés dans ce moule régulier qu'on nous a faussement enseigné à révérer dans les ouvrages clas- siques du paganisme, dit lord a II n'y a pas de beauté exquise, Verulam, parlant avec justesse de toutes les formes et de tous les genres de beauté, sans une certaine étrangcté dans les proportions. » Toutefois, bien que je visse que les traits de Ligeia n'étaient pas d'une régularité classique, quoique je sentisse que sa beauté était véritablement exquise et fortement pénétrée de cette étrangeté, je me suis efforcé en vain de découvrir cette irrégularité et de poursuivre jusqu'en son gîte ma perception de l'étrange. J'examinais le contour du front mot est — un front irréprochable, — combien ce froid appliqué à une majesté aussi divine! — la peau rivalisant avec le plus haut et pâle, pur ivoire, la largeur imposante, le calme, la gracieuse proéminence des régions au-dessus des tempes, et puis cette chevelure d'un noir de corbeau , lustrée , luxuriante , -naturelle- ment bouclée et démontrant toute la force de l'exprès- HISTOIRES EXTRAORDINAIRES. 402 sion homérique les lignes dans : chevelure d'hyacinthe. Je considérais délicates du nez, et nulle autre part que gracieux médaillons les hébraïques je n'avais c'était ce même même surface unie et superbe, cette même contemplé une semblable perfection jet, cette ; tendance presque imperceptible à l'aquilin, ces mêmes narines harmonieusement arrondies et révélant un esprit libre. Je regardais la charmante bouche : c'était là qu'était le triomphe de toutes les choses célestes ; le tour glorieux de la lèvre supérieure, un peu courte, l'air doucement, voluptueusement reposé de l'infé- rieure, les fossettes qui se jouaient et la couleur qui parlait, les dents, réfléchissant comme une espèce d'éclair chaque rayon de la lumière bénie qui tombait sur elles dans ses sourires sereins et placides, mais toujours radieux et triomphants. J'analysais la forme du menton, et, là aussi, je trouvais la grâce dans la largeur, la douceur et la majesté, la plénitude et la spiri- grecques tualité , ce contour révéla qu'en rêve à Cléomènes, thènes ; et puis je que fils le dieu Apollon ne de Cléomènes d'A- regardais dans les grands yeux de Ligeia. Pour les yeux, je ne trouve pas de modèles dans la plus lointaine antiquité. Peut-être bien était-ce dans les yeux de ma bien-aimée que se cachait le mystère dont parle lord Verulam : ils étaient, je crois, plus grands que les yeux ordinaires de l'humanité; mieux fendus que les plus beaux yeux de gazelle de la tribu de la vallée de Nourjahad; mais ce n'était que par in- LIGEIÂ. 403 tervalles, dans des moments d'excessive animation, que cette particularité devenait singulièrement frappante. Dans ces moments-là, sa beauté était — du moins, — beauté elle apparaissait telle à ma pensée enflammée la de la fabuleuse houri des Turcs. Les prunelles étaient du noir le plus brillant et surplombées par des cils de jais très-longs; ses sourcils, d'un dessin régulier, avaient la get6 légèrement ir- même couleur; toutefois, Vélran- que je trouvais dans les yeux était indépendante de leur forme, de leur couleur et de leur éclat, et devait décidément être attribuée à V expression. Ah mot qui n'a ! pas de sens ! un pur son ! vaste latitude où se retranche toute notre ignorance du spirituel! L'expression des yeux de Ligeia!... Combien de longues heures ai-je médité dessus! combien de fois, durant toute une nuit d'été, me suis-je efforcé de les sonder! Qu'était donc ce je ne sais quoi, ce quelque chose plus profond que le puits de Démocrite, qui gisait au fond des pupilles de ma bien-aimée? Qu'était cela?... J'étais possédé de la passion de le découvrir. Ces yeux! ces larges, ces brillantes, ces divines prunelles! elles étaient devenues pour moi les étoiles jumelles de Léda, et, moi, j'étais pour elles le plus fervent des astrologues. Il n'y a pas de cas parmi les nombreuses et incom- préhensiblesa nomalies de la science psychologique, qui soit plus saisissant, plus excitant je crois, dans les que celui, écoles, — où, dans nos — négligé, efforts pour ramener dans notre mémoire une chose oubliée depuis longtemps, nous nous trouvons souvent sm^ le bord HISTOIRES EXTRAORDINAIRES. 404 même do souvenir, sans pouvoir toutefois nous souvenir. Et ainsi que de fois, dans mon ardente analyse des yeux de Ligeia, ai-je senti s'approcher connaissance de leur expression! — la complète Je l'ai sentie s'ap- procher, mais elle n'est pas devenue tout à fait mienne, et à la longue elle a disparu entièrement! Et étrange, oh! plus étrange des mystères! j'ai trouvé dans les le objets les plus communs du monde une série d'analo- pour cette expression. Je veux dire qu'après l'é- gies poque où la beauté de Ligeia passa dans mon esprit et s'y installa comme dans un reliquaire , je puisai dans plusieurs êtres du monde matériel une sensation ana- logue à celle qui se répandait sur moi, en moi, sous l'influence de ses larges et lumineuses prunelles. Ce- pendant, je n'en suis pas moins incapable de définir ce sentiment, de l'analyser, ou même d'en avoir une perception nette. Je l'ai reconnu quelquefois, je le répète, à l'aspect d'une vigne rapidement grandie, dans la con- templation d'une phalène, d'un papillon, d'une chrysalide, d'un courant d'eau précipité. Je l'ai trouvé dans l'Océan, dans la chute d'un météore; je l'ai senti dans les regards de quelques personnes extraordinairement âgées. Il y a dans le ciel une ou deux étoiles, plus par- ticulièrement une étoile de sixième grandeur, double et changeante, de la qu'on trouvera près de la grande étoile Lyre, qui, vues au télescope, m'ont donné un sentiment analogue. Je m'en suis senti rempli par certains sons d'instruments à cordes, et quelquefois aussi par des passages de mes lectures. Parmi d'innombrables Lin ET A. 40a exemples, je me rappelle fort bien quelque chose dans un volume de Joseph Glanvill, qui, peut-être simple- — qui ment à cause de sa bizarrerie, jours inspiré le même sentiment la : a sait? — m'a tou- Et il y a là dedans volonté qui ne meurt pas. Qui donc connaît les mys- volonté, ainsi que sa vigueur? car Dieu tères de n'est qu'une grande volonté pénétrant toutes choses la par l'intensité qui lui est propre ; l'homme ne cède aux anges et ne se rend entièrement à la mort que par l'infirmité de sa pauvre volonté. » Par la suite des temps et par des réflexions subséquentes, je suis parvenu à déterminer un certain rapport éloigné entre ce passage du philosophe anglais el une partie du caractère de Ligeia. Une intensUè singulière dans la pensée, dans l'action, dans la parole, était peut-être en elle le résultat ou au moins l'indice de cette gigantesque puissance de volition qui, durani nos longues relations, eût pu donner d'autres et plus positives preuves de son existence. De toutes les femmes que j'ai connues, elle, la toujours placide Ligeia, à l'extérieur si calme, était la proie la plus déchirée par les tumultueux vautours de la cruelle passion. Et je ne pouvais évaluer cette passion que par la miraculeuse expansion de cesyeuxqui me ravissaient et m'effrayaient en même temps, par la mélodie presque magique, la modulation, la netteté et la placidité de sa voix pro- fonde, et par la sauvage énergie des étranges paroles qu'elle prononçait habituellement, et dont l'effet étail doublé par le contraste de son débit. 23. IIISTOIRKS EXTRAORDINAIRES. 400 J'ai parlé de l'instruction de Ligeia; elle était im- mense, telle que jamais je n'en vis de pareille dans une femme. Elle connaissait à fond les langues classiques, et, aussi loin que s'étendaient mes propres con- naissances dans les langues modernes de l'Europe, je ne l'ai jamais prise en faute. Véritablement, sur n'importe quel thème de l'érudition académique si vantée, si admirée, uniquement à cause qu'elle est plus abs- truse, ai-je jamais trouvé Ligeia en fau'e? trait unique de la nature de Combien ce ma femme, seulement dans cette dernière période, avait frappé, subjugué mon attention! J'ai dit que son instruction dépassait mais où d'aucune femme que j'eusse connue, — celle est l'homme qui a traversé avec succès tout le vaste champ des sciences morales, physiques et mathématiques? Je ne vis pas alors ce que maintenant je perçois clairement, que les connaissances de Ligeia étaient gi- gantesques, étourdissantes; cependant, j'avais une conscience suffisante de son infinie supériorité pour me résigner, avec la confiance d'un écolier, à me laisser guider par elle à travers le monde chaotique des investigations métaphysiques dont je m'occupais avec ar- deur dans les premières années de notre mariage. Avec quel vaste triomphe, avec quelles vives délices, avec quelle espérance éthéréenne sentais-je, — ma Ligeia penchée sur moi au milieu d'études si peu frayées, si s'élargir par degrés cette admirable peu connues, — perspective, cette longue avenue, splendide et vierge, par laquelle je devais enfin arriver au terme d'une sa- LIGEIA. 407 gesse trop précieuse et trop divine pour n'èlre pas interdite ! Aussi, avec quelle poignante douleur ne vis-je pas, au bout de quelques années, mes espérances si bien fondées prendre leur vol et s'enfuir! Sans Ligeia, je n'étais qu'un enfant tâtonnant dans la nuit. Sa présence, ses leçons, pouvaient seules éclairer d'une lumière vi- vante les mystères du transcendantalisme dans lesquels nous nous étions plongés. Privée du lustre rayonnant de ses yeux, toute cette littérature, ailée et dorée naguère, devenait maussade, saturnienne et lourde comme le plomb. Et maintenant, ces beaux yeux éclairaient de plus en plus rarement les pages que je déchiffrais. Ligeia tomba malade. Les étranges yeux flam- boyèrent avec un éclat trop splendide; les pâles doigts prirent la couleur de la mort, la couleur de la cire transparente; les veines bleues de son grand front palpitèrent impétueusement au courant de la plus douce émotion : je vis qu'il lui fallait mourir, et je luttai désespérément en esprit avec l'affreux Azraël. Et les efforts de cette femme passionnée furent, à mon grand étonnement, encore plus énergiques que les miens. Il y avait certes dans sa sérieuse nature de quoi me faire croire que pour elle la mort viendrait sans son monde de terreurs. Mais il n'en fut pas ainsi; les mots sont impuissants pour donner une idée de la férocité de résistance qu'elle déploya dans sa lutte avec l'Ombre. Je gémissais d'angoisse à ce lamentable spectacle. J'aurais voulu la calmer, j'aurais voulu la HISTOIRES EXTRAORDINAIRES. 408 raisonner; mais, dans l'intensité de son sauvage désir de vivre, — de vivre, — de rien que vivre — toute , consolation et toutes raisons eussent été le comble de la folie. Cependant, jusqu'au dernier moment, au milieu des tortures et des convulsions de son sauvage esprit, l'apparente placidité de sa conduite ne se démentit pas. Sa voix devenait plus douce, fonde, — mais je — devenait plus pro- ne voulais pas m'appesantir sur le sens bizarre de ces mots-prononcés avec tant de calme. Ma cervelle tournait quand je prêtais l'oreille en extase à cette mélodie surhumaine, à ces ambitions et à ces aspirations que l'humanité n'avait jamais connues jusqu'alors. Qu'elle m'aimât, je n'en pouvais douter, et il m'était aisé de deviner que, dans une poitrine telle que la sienne, l'amour ne devait pas régner comme une passion ordinaire. Mais, dans la mort seulement, je compris toute la force et toute l'étendue de son affection. Pendant de longues heures, ma main dans la sienne, elle épanchait devant le moi le trop-plein d'un cœur dont dévouement plus que passionné montait jusqu'à l'i- Comment avais-je mérité la béatitude d'enComment avais-je mérité d'être damné à ce point que ma bien-aimée me fût dolâtrie. tendre de pareils aveux? enlevée à l'heure où elle m'en octroyait la jouissance? Mais il ne m'est pas permis de m'étendre sur ce sujet. Je dirai seulement que dans l'abandonnement plus que féminin de Ligeia à un amour, hélas! non mérité, accordé tout à fait gratuitement, je reconnus enfin le LIGEIA. 400 principe de son ardent, de son sauvage regret de cette vie qui fuyait maintenant si rapidement. C'est cette ardeur désordonnée, cette véhémence dans son désir de la vie, la — et de rien que la vie, — que je n'ai pas puissance de décrire; les mots me manqueraient pour l'exprimer. Juste au milieu de la nuit pendant laquelle elle mourut, elle m'appela avec autorité auprès d'elle, et me fit répéter certains vers composés par elle peu de jours aiipar<ivant. Je lui obéis. Ces vers, les voici Voyez ! c'est nuit de gala Depuis ces dernières années désolées! Une multitude d'anges, ailés, ornés De voiles, et noyés dans les larmes, Est assise dans un tiiéâtre, pour voir Un drame d'espérance et de craintes. Pendant que l'orchestre soupire par intervalles La musique des sphères. Des mimes, faits à l'image du Dieu très-haut, Marmottent et marmonnent tout bas Et voltigent de côté et d'autre; Pauvres poupées qui vont et viennent Au commandement de vastes êtres sans forme Qui transportent la scène çà et là, Secouant de leurs ailes de condor L'invisible Malheur! Ce drame bigarré Il ! oh ! à coup sûr, ne sera pas oublié, Avec son Fantôme éternellement pourchassé Par une foule qui ne peut pas le saisir, A travers un siècle qui toujours retourne : HISTOIRES EXTRAORDINAIRES. 410 Sur lui-même, exactement au môme point! Et beaucoup de Folie, et encore plus de Péché Et d'Horreur font l'âme de l'intrigue ! Mais voyez à travers la cohue des mimes, Une forme rampante fait son entrée! Une chose rouge de sang qui vient en se tordant De la partie solitaire de la scène! Elle se tord! elle se tord! — Avec des angoisses mortelles Les mimes deviennent sa pâture, Et les séraphins sanglotent en voyant les dents du ver Mâcher des caillots de sang humain. Toutes les lumières s'éteignent, — toutes, toutes! Et sur chaque forme frissonnante. Le rideau, vaste drap mortuaire. Descend avec la violence d'une tempête, — \'.t les anges, tous pâles et blêmes, Se levant et se dévoilant, affirment Que ce drame est une tragédie qui s'appelle l'Homme, Et dont le héros est le ver conquérant. — Dieu! cria presque Ligeia, se dressant, sur ses pieds et éleiidanl ses bras vers le ciel dans un mouve- ment spasmodique, comme je finissais de réciter ces — ces choses s'accomirrémissiblement? — Ce conquérant ne jamais vaincu? — Ne sommes-nous pas une vers, ô Dieu! ô Père céleste! pliront-elles sera-t-il partie et une parcelle de loi? Qui donc connaît les mystères de la volonté ainsi que sa vigueur? L'homme ne cède aux anges et ne se rend entièrement à la mort que par rinhrmité de sa pauvre volonté. Et alors, comme épuisée par l'émotion, elle laissa retomber ses bras blancs, et retourna solennellement LIGEIA. 411 à son lit de mort. Et, comme elle soupirait ses derniers soupirs, il s'y mêla sur ses lèvres comme indistinct. Je tendis Toreille, et je reconnus de nou- veau la conclusion du passage de Glanvill cède im murmure : Vhomme ne aux anges et 'de se rend entièrement à la mort que par rinfuinUè de sa pauvre volonté. Elle mourut; et moi, anéanti, pulvérisé par la dou- leur, je ne pus pas supporter plus longtemps l'affreuse désolation de ma demeure dans cette sombre cité dé- labrée au bord du Rhin. Je ne manquais pas de ce que le monde appelle la fortune. Ligeia m'en avait apporté beaucoup plus que n'en comporte la destinée or- plus, dinaire des mortels. Aussi, après quelques mois per- dus dans un vagabondage fastidieux et sans but, je me jetai dans une espèce de retraite dont je fis l'acquisi- — une abbaye dont — dans une des parties tion, je ne veux pas dire le nom, les plus incultes et les moins fréquentées de la belle Angleterre. La sombre et triste grandeur du bâtiment, l'aspect presque sauvage du domaine, mélancoliques et vénérables souvenirs qui les s'y rattachaient, étaient à l'unisson du sentiment de complet abandon qui m'avait exilé dans cette lointaine et solitaire région. térieur de intact et le murs, je Cependant, tout en laissant à l'ex- l'abbaye son caractère primitif presque verdoyant délabrement qui tapissait ses me mis avec une perversité enfantine, et peut-être avec une faible espérance de distraire mes chagrins, à déployer au dedans des magnificences plus que royales. Je m'étais, depuis l'enfance, pénétré d'un HISTOIRES EXÏR AO 412 grand goût pour ces folies, DI N AIRES. P. et maintenant elles me revenaient comme un radotage de la douleur. Hélas! je sens qu'on aurait pu découvrir un commencement de folie dans ces splendides et fantastiques draperies, dans ces solennelles sculptures éizyptiennes, dans ces corniches et ces ameublements bizarres, dans les extra- vagantes arabesques de ces tapis tout fleuris J'étais devenu ses liens, — d'or ! uq esclave de l'opium, il me tenait dans mes travaux et mes plans avaient mes rêves. Mais je ne m'arrêterai et tous pris la couleur de pas au détail de ces absurdités. Je parlerai seulement de cette chambre, maudite à jamais, où dans un mo- ment d'aliénation mentale je conduisis à l'autel et pris pour épouse, — après l'inoubliable Ligeia — lady ! Rowena Trevanion de Tremaine, à la blonde chevelure et aux yeux bleus. Il n'est pas un détail d'architecture ou de la décora- tion de cette chambre nuptiale qui ne soit maintenant présent à mes yeux. Où donc la hautaine famille de la mue par la soif de fiancée avait-elle l'esprit, quand, l'or, elle passer le permit à une seuil d'un fille si tendrement chérie de appartement décoré de cette étrange façon? J'ai dit que je me rappelais minutieuse- ment les détails de cette chambre, bien que ma triste mémoire perde souvent des choses d'une rare importance; et pourtant il n'y avait pas dans ce luxe fantastique de système ou d'harmonie qui pût s'imposer au souvenir. La chambre faisait partie d'une haute tour de cette — LIGEIA. abbaye, fortifiée 413 comme un château; elle était d'une forme pentagone et d'une grande dimension. Tout le côté sud du pentagone était occupé par une fenêtre immense unique, faite d'une glace de Venise, d'un seul morceau et d'une couleur sombre, de sorte que les rayons du soleil ou de la lune qui la traversaient jetaient sur les objets intérieurs une lumière sinistre. Au-dessus de cette énorme fenêtre se prolongeait le treillis d'une vieille vigne qui grimpait sur les murs massifs de la tour. Le plafond, de chêne presque noir, était excessivement élevé, façonné en voûte et curieusement sillonné d'ornements des plus bizarres et des plus fantastiques, d'un semi-gothique, semi-druidique. style Au fond de cette voûte mélancolique, au centre même, était suspendue, par une seule chaîne d'or faite de longs anneaux, une vaste lampe de même métal en forme d'encensoir, conçue dans le goût sarrasin et brodée de perforations capricieuses, à travers lesquelles on voyait courir et se tortiller avec la vitalité d'un serpent les lueurs continues d'un feu versicolore. Quelques rares ottomanes et des candélabres d'une forme orientale occupaient différents endroits, et le lit aussi, — le lit nuptial, — était dans le style indien, bas, sculpté en bois d'ébéne massif, et surmonté d'un baldaquin qui avait cun des angles de l'air d'un drap mortuaire. A cha- la chambre que sarcophage de granit rois en face se dressait un gigantes- noir, tiré des tombes des de Louqsor, avec son antique couvercle chargé de sculptures immémoriales. Mais c'était dans HISTOIRES EXTRAORDINAIRES. 414 la tenture de l'appartement, hélas! taisie capitale. qu'éclatait la fan- Les murs, prodigieusement hauts, delà même de toute proportion, — au — étaient tendus du haut jusqu'en bas d'une tapisserie lourde et d'apparence massive qui tombait par vastes nappes, rie faite avec la — tapisse- môme matière qui avait été employée pour le tapis du parquet, les ottomanes, le lit d'ébène, le baldaquin du lit cachaient en partie et les somptueux rideaux qui Cette matière était un la fenêtre. tissu d'or des plus riches, tacheté, par intervalles irré- guliers, de figures arabesques, d'un pied de diamètre environ, qui enlevaient sur noir de jais. Mais ces caractère arabesque que seul point de vue. le fond leurs dessins d'un figures ne participaient du quand on les examinait à un Par un procédé aujourd'hui fort commun, et dont on retrouve la trace dans la plus lointaine antiquité, elles étaient faites de manière à chan- ger d'aspect. Pour une personne qui entrait dans la chambre, elles avaient l'air de simples monstruosités ; mais, à mesure qu'on avançait, ce caractère disparaissait graduellement, et, pas à pas, le visiteur changeant de place se voyait entouré d'une procession continue de formes affreuses, comme celles qui sont nées de la superstition du Nord, ou les sommeils celles qui se dressent dans coupables des moines. L'effet fantasma- gorique était grandement accru par l'introduction artificielle ture, d'un fort courant d'air continu derrière la ten- — qui donnait au tout une hideuse animation. et inquiétante LIGEIA. Telle était la 415 demeure, telle était la chambre nuptiale où je passai avec la dame de Tremaine les heures im- pies du premier mois de notre mariage, — et je les passai sans trop d'inquiétude. Que ma femme redoutât mon humeur farouche, qu'elle m'évitât, qu'elle ne m'aimât que très-médiocre- ment, — je ne pouvais pas me le dissimuler; mais cela me faisait presque plaisir. Je la haïssais d'une haine qui appartient moins à l'homme qu'au démon. Marné- moire se retournait, — oh! avec quelle intensité de re- gret!— vers Ligeia, l'aimée, l'auguste, la belle, la morte. Je faisais des orgies de souvenirs; je me délectais dans sa pureté, dans sa sagesse, dans sa haute nature éthé- réenne, dans son amour passionné, idolàtrique. Maintenant, mon esprit brûlait pleinement et largement d'une flamme plus ardente que n'avait été la sienne. — car habituellement sous l'empire du poison, — l'enthousiasme de mes rêves opiacés, Dans j'étais je criais son nom à haute voix durant le silence de la nuit, et, le jour, dans les retraites ombreuses des vallées, comme si, par l'énergie sauvage, la passion solennelle, l'ardeur dévorante de ma passion pour la défunte je pouvais la ressusciter dans les sentiers de cette vie qu'elle avait abandonnés; pour toujours? était-ce \ra\ment possible? Au commencement du second mois de notre mariage, lady Rowena fut attaquée d'un mal soudain dont elle ne se releva que lentement. La (lèvre qui la con- sumait rendait ses nuits pénibles, et, dans l'inquiétude d'un demi-sommeil, elle parlait de sons et de mouve- HISÏOIRHS EXTRAORDINAIRES. 410 ments qui se produisaient çà et là dans la chambre de la tour, et que je ne pouvais vraiment attribuer qu'au dérangement de ses idées ou peut-être aux influences fantasmagoriques de la chambre. A la longue, elle entra en convalescence, et finalement elle se rétablit. Toutefois, il ne s'était écoulé qu'un laps de temps fort court quand une nouvelle attaque plus violente la rejeta sur son lit de douleur, et, depuis cet accès, sa constitution, qui avait toujours été faible, ne put jamais se relever complètement. Sa maladie montra, dès cette époque, un caractère alarmant et des rechutes plus alarmantes encore, qui défiaient; toute la science et tous les efforts de ses médecins. A mesure qu'augmentait ce mal chronique qui, dès lors sans doute, s'était trop bien emparé de sa constitution pour en être arraché par des mains humaines, je ne pouvais m'empêcher de remarquer une irritation nerveuse croissante dans son tempérament et une excitabilité telle, que les causes les plus vulgaires lui étaient des sujets de peur. Elle parla encore, et plus souvent alors, niâtreté, des bruits, avec plus d'opi- — des légers bruits, — et des mouvements insolites dans les rideaux, dentelle avait, disait-elle, déjà souffert. Une nuit, — vers la fin de septembre, — elle attira mon attention sur ce sujet désolant avec une énergie plus vive que de coutume. Elle venait justement de se réveiller d'un sommeil agité, et j'avais épié, avec un sentiment moitié d'anxiété, moitié de vague terreur, le jeu de sa physionomie amaigrie. J'étais assis au che- LIGKIA. vet du lit 417 d'ébène, sur un des divans indiens. Elle se dressa à moitié, et me parla à voix basse, dans un chuchotement anxieux, de sons qu'elle venait d'entendre, mais que je ne pouvais pas entendre, vements qu'elle venait d'apercevoir, — de mou- mais que ne Le vent courait activement der- pouvais apercevoir. rière les tapisseries, et je m'appliquai à lui démontrer — ce que, je le confesse, je tièrement, — que ces soupirs à peine articulés ne pouvais pas croire en- changements presque insensibles dans les mur n'étaient que je les effets naturels et ces figures du du courant d'air habituel. Mais une pâleur mortelle qui inonda sa face me prouva que mes efforts pour la rassurer seraient inutiles. Elle semblait s'évanouir, et je n'avais pas domestiques à ma portée. Je de me souvins de l'endroit où avait été déposé un flacon de vin léger ordonné par les médecins, et je traversai vivement la chambre pour me le procurer. Mais, comme je passais sous la lumière de la lampe, deux circonstances d'une nature saisissante attirèrent mon attention. J'avais senti que quel- que chose de palpable, quoique invisible, avait frôlé légèrement ma personne, et je vis sur le tapis d'or, au centre même du riche rayonnement projeté par l'en- censoir, une ombre,— une ombre faible, indéfinie, d'un aspect angélique, — telle qu'on peut se figurer l'ombre d'une Ombre. Mais, comme j'étais en proie à une dose exagérée d'opium, je ne fis que peu d'attention à ces choses, et je n'en parlai point à Kowena. Je trouvai le vin, je traversai de nouveau la cham- m HISTOIRES EXTUAOUDINAIHES. bre, et je remplis un verre que je portai aux lèvres de ma femme défaillante. Cependant, elle était un peu remise, et elle prit le verre elle-même, pendant que je me laissais tomber sur l'ottomane, les yeux fixés sur sa personne. Ce fut alors que j'entendis distinctement un léger bruit de pas sur le tapis et près du lit et, ; une seconde après, comme Rowena allait porter le vin à ses lèvres, je vis, — verre, comme de quelque source invisible suspendue je puis l'avoir rêvé, dans l'atmosphère de la — tomber dans le je vis chambre, ou quatre trois grosses gouttes d'un fluide brillant et couleur de rubis. Si je le vis, — Rowena ne sans hésitation, et je le vit pas. Elle avala le vin me gardai bien de lui parler d'une circonstance que je devais, après tout, regarder comme la suggestion d'une imaginatidh surexcitée, et dont tout, ~ les terreurs de ma femme, l'opium et l'heure, augmentait l'activité morbide. Cependant, je ne puis pas me dissimuler qu'immé- diatement après la chute des gouttes rouges, un rapide changement — en mal — s'opéra dans la maladie de ma femme; si bien que, la troisième nuit, les mains de ses serviteurs la préparaient pour la tombe, et que j'étais assis seul, son corps enveloppé dans le suaire, dans cette chambre fantastique qui avait reçu la jeune épouse. — D'étranges visions, engendrées par l'opium, voltigeaient autour de moi comme des ombres. Je pro- menais un œil inquiet sur les sarcophages, dans les coins de la chambre, sur les figures mobiles de la ten- LIGRIA. 419 , tare et sur les lueurs vermiculaires et changeantes de la lampe du plafond. Mes yeux tombèrent alors, — comme je cherchais à me rappeler les circonstances d'une nuit précédente, — sur le même point du cercle lumineux, là ou j'avais vu les traces légères d'une ombre. Mais elle n'y était plus; et, respirant avec plus de liberté, je tournai mes regards vers la fjgure allongée sur le lit. Alors, je moi mille souvenirs de Ligeia, — pâle et rigide sentis fondre sur je sentis refluer vers mon cœur, avec la tumultueuse violence d'une marée, toute cette ineffable douleur que j'avais sentie quand je l'avais vue, c//e aussi, dans son suaire. La nuit avançait, et toujours, — le cœur plein amères dont elle était l'objet, suprême amour, — elle, des pensées les plus mon unique, mon je restais les yeux fixés sur le corps de Rowena. 11 pouvait bien être minuit, peut-être plus tôt, peut- être plus tard, car je n'avais pas pris garde au quand un sanglot, très-bas, tinct, très-léger, temps, mais très-dis- me tira en sursaut de ma rêverie. Je sentis qu'il venait du lit d'ébène, — du lit de mort. Je tendis l'oreille, dans une angoisse de terreur superstitieuse, mais le bruit ne se répéta pas. Je forçai mes yeux à découvrir un mouvement quelconque dans le corps, mais je n'en aperçus pas le moindre. Cependant, il était iuipossible que je me fusse trompé. J'avais entendu le bruit, faible à la vérité, et mon esprit était bien éveillé en moi. Je mon attention maintins résolument et opiniâtrement clouée au cadavre. Quelques minutes s'écoulèrent sans HISTOIRES EXTRAORDINAIRES. 420 aucun incident qui pût jeter un peu de jour sur ce mystère. A la longue, il devint évident qu'une coloration légère, très-faible, à peine sensible, était montée aux joues et avait filtré le long des petites veines dépri- mées des paupières. Sous la pression d'une horreur et d'une terreur inexplicables, pour lesquelles le langage de l'humanité n'a pas d'expression suffisamment éner- mon cœur s'arrêter gique, je sentis les pulsations de et mes membres se roidir sur place. Cependant, le sentiment du devoir me rendit finale- ment mon sang-froid. Je ne pouvais pas douter plus longtemps que nous n'eussions fait prématurément nos apprêts funèbres ; — Rowena vivait encore. 11 était né- cessaire de pratiquer immédiatement quelques tentatives ; mais la tour était tout à fait séparée de la partie de l'abbaye habitée par avait domestiques,— les aucun à portée de la voix, il n'y en — je n'avais aucun moyen de les appeler à mon aide, à moins de quitter la chambre pendant quelques minutes, cela, je — et, quant à ne pouvais m'y hasarder. Je m'efforçai donc de rappeler à moi seul et de fixer l'âme encore voltigeante. Mais, au bout d'un laps de temps très-court, il y eut une rechute évidente joue et de la marmoréenne ; la couleur disparut de la paupière, laissant une pâleur plus que ; les lèvres se serrèrent se recroquevillèrent doublement et dans l'expression spectrale de la mort; une froideur et une viscosité répulsives se répandirent rapidement sur toute la surface du corps, et la complète rigidité cadavérique survint immédiate- LIGEIA. 421 ment. Je retombai en frissonnant sur le lit de repos d'où j'avais été arraché si soudainement, et je m'aban- donnai de nouveau à mes rêves, à mes contemplations passionnées de Ligeia. Une heure Dieu ! s'écoula ainsi, possible? quand — j'eus de nouveau — était-ce, grand la perception bruit vague qui partait de la région du lit. d'un J'écoutai, au comble de l'horreur. Le son se fit entendre de nouveau, c'était un soupir. Je me précipitai vers le corps, je vis, — je lèvres. Une minute après, elles se relâchaient, décou- vis distinctement un tremblement sur les vrant une ligne brillante de dénis de nacre. La stupéfaction lutta alors dans mon esprit avec la profonde ter- reur qui jusque-là l'avait dominé. Je sentis que ma vue s'obscurcissait, que ma raison s'enfuyait ; et ce ne fut que par un violent effort que je trouvai à la longue le courage de me roidirà la tâche que le devoir m'imposait de nouveau. 11 y avait maintenant une carnation imparfaite sur le front, la joue et leur sensible pénétrait tout le la gorge; une cha- corps; et même une légère pulsation remuait imperceptiblement la région du coeur. Ma femme vivait ; et, avec un redoublement d'ar- deur, je me mis en devoir de la ressusciter. Je frictionnai et je bassinai les tempes et les mains, et j'usai de tous les procédés que l'expérience et de nombreuses lectures médicales pouvaient me suggérer. Mais ce fut en vain. Soudainement, la couleur disparut, tion cessa, l'expression de mort revint aux la pulsa- lèvres, et. HISTOIRES EXTRAORDINAIRES. 422 un instant après, tout le corps reprenait sa froideur de glace, son ton livide, sa rigidité complète, son contour amorti, et toute la hideuse caractéristique de ce qui a habité la tombe pendant plusieurs jours. — — s'étonnera-t-on que frissonne en vant ces lignes — de nouveau un sanglot étouffe vint Et puis je retombai dans mes rêves de Ligeia, de nouveau et écri- je ? à mon oreille de la région du lit d'ébene. iMais à quoi bon détailler minutieusement les ineffables horreurs de cette nuit? Raconterai-je combien de fois, coup sur coup, presque jusqu'au petit jour, se répéta ce hideux drame de ressuscitation; que chaque effrayante rechute se changeait en une mort plus rigide et plus irrémé- diable ; que chaque nouvelle agonie ressemblait à une lutte contre quelque invisible adversaire, et que cha- que lutte était suivie de je ne sais quelle étrange altération dans la physionomie du corps? Je me hâte d'en finir. La plus grande partie de la terrible nait était passée, et celle qui était morte remua de nouveau, fois-ci, plus énergiquement que jamais veillant d'une — et, cette quoique se ré- mort plus effrayante et plus irréparable. J'avais depuis longtemps cessé tout effort et tout mou- vement, et je restais cloué sur l'ottomane, désespéré- ment englouti dans un tourbillon d'émotions violentes, dont la moins terrible peut-être, la moins dévorante, était un suprême effroi. Le corps, je le repète, remuait, et mamtenant plus activement qu'il n'avait fait jusquelà. Les couleurs de la vie montaient à la face avec une LIGEIA. énergie singulière, — les 421 membres se relâchaient, — et, sauf que les paupières restaient toujours lourdement fermées, et que les bandeaux et les draperies funèbres communiquaient encore à la figure leur caractère sépulcral, j'aurais rêvé que Rowena avait entièrement Mort. Mais si, dès n'acceptai pas entièrement cette idée, je ne pus pas secoué chaînes les de la lors, je — —à manière d'une personne égarée dans un rêve, — Têtre douter plus longtemps, quand, vacillant, — d'un pas faible, — — se levant du lit, les yeux fermés, et la qui était enveloppé du suaire s'avança audacieusement et palpablement dans le milieu de la chambre. Je ne tremblai pas, foule — je ne bougeai pas, — car une de pensées inexprimables, causées par l'air, la stature, l'allure du fantôme, se ruèrent à Fimproviste dans mon cerveau, et me paralysèrent, rent. Je ne bougeais pas, (Tétait dans mes pensées je — me pétrifiè- contemplais l'apparition. un désordre fou, un tumulte inapaisable. Était-ce bien la vivante Rowena que j'avais en face de moi? cela pouvait-il être vraiment Rowena, — lady Rowena Trevanion de Tremaine, à blonde, aux yeux bleus? Pourquoi, doutais-je? la — Le lourd bandeau oppressait chevelure pourquoi en oui, la bouche; — pourquoi donc cela n'eût-il pas été la bouche respirante de la dame de Tremaine? bien là les — Et — — les joues? roses du midi de sa vie; oui, c'étaient oui, ce pou- vaient être les belles joues de la vivante lady de Tre- maine. — Et le menton, avec les fossettes de la santé, ne pouvait-il pas être le sien ? Mais avait-elle donc grandi ÏIISTOIRKS EXT R A O 4-24 P. DINAI H ES. depuis sa maladie? Quel inexprimable délire s'empara de moi à cette idée se retira ! D'un bond, j'étais à ses pieds à mon contact, l'horrible suaire et elle ! Elle dégagea sa tête de qui l'enveloppait; et alors déborda dans l'atmosphère fouettée de la chambre une masse énorme de longs cheveux désordonnés; ils étaient plus noirs que les ailes de minuit, llieure au plumage de cor- beau! Et alors je vis la figure qui se tenait devant moi ouvrir lentement, lentement — Enfin, sante yeux. d'une voix retenlis- pourrais-je jamais m'y tromper? ; les yeux adorablement étranges de LiGEIA les les voilà donc! criai-je ! — Voilà bien fendus, les yeux noirs, les yeux mon amour perdu, — de lady — de lady METZENGERSTEIN Pestis eram vivus, — moriens tua mors ero. Martin Luther. L'horreur et la fatalité se sont donné carrière dans tous les siècles. A quoi bon mettre une date toire que j'ai à raconter? Qu'il à l'his- me suffise de dire qu'à l'époque dont je parle existait dans le centre de la Hongrie une croyance secrète, mais bien établie, aux doctrines de la métempsycose. De ces doctrines elles- — mêmes, de leur fausseté ou de leur probabilité, je ne qu'une bonne partie de dirai rien. J'affirme, toutefois, noire incrédulité vient, attribue tout noire comme dit la Bruyère, qui malheur à cette cause unique, de ne pouvoir être seuls^. Mais il y avait quelques points dans la superstition hongroise qui tendaient fortement à l'absurde. Les Hongrois différaient très-essentiellement de leurs auto1 . Mercier, dans VAn deux mil quatre cent quarante, soutient s(''rieusement les doctrines de la métempsycose, et J. d'Israeli dit qu'il n'y a pas de système aussi simple et qui répugne moins à Le colonel Ethan Allen, le Green Mountain Boa, l'intelligence. passe aussi pour avoir été sérieux métempsycosiste. — E. A. P. 24. HISTOIRES EXTRAORDINAIRES. 426 Par d'Orient. rites croyaient, — je cite — Vâme, exemple, les ce à termes d'un subtil qu'ils et intelli- gent Parisien, — ne demeu7^e qu'une seule fois dans un coîys sensible. Ainsi, un cheval, un chien, un homme même, ne sont que la ressemblance illusoire de ces élres^. Les familles Berlifitzing été en discord Metzengerstein avaient et pendant des siècles. Jamais on ne deux maisons aussi illustres vit réciproquement aigries par une inimitié aussi mortelle. Cette haine pouvait tirer son origine des paroles d'une ancienne prophétie: — Un grand nom tombera d'une chute terrible, quand, comme le cavalier sur son cheval, la mortalité deMetzengerstein triomphera de l'immortalité de Berlifitzing. Certes, les termes n'avaient que peu ou point de sens. Mais des causes plus vulgaires ont sance — et cela, conséquences donné nais- sans remonter bien haut, également grosses — à des d'événements. En outre, les deux maisons, qui étaient voisines, avaient longtemps exercé une influence rivale dans les affaires d'un gouvernement tumultueux. De plus, des voisins aussi rapprochés sont rarement amis ; et, du haut de leurs terrasses massives, les habitants du château Berhfitzing pouvaient fenêtres même 1. plonger leurs regards dans du palais Metzengerstein. Enfin, les le J'ignore quel est l'auteur de ce texte bizarre et obscur; ce- pendant, je me suis permis de le rectifier légèrement, en l'adaptant au sens moral du récit. Poe cite quelquefois de mémoire et incorrectement. Le sens, après tout, me semble se rapprocher de l'opinion attribuée au pè''e Kircher, enfermés. — C. Bj — que les animaux sont des Esprits METZElNGERSTEIN. 427 déploiement d'une magnificence plus que féodale était peu fait pour calmer les sentiments irritables des Berlifitzing, lieu moins anciens et moins riches. Y a-t-il donc de s'étonner que les termes de cette prédiction, bien que tout à fait saugrenus, aient si bien créé et entretenu la discorde entre deux familles déjà prédis- posées aux querelles par toutes les instigations d'une jalousie héréditaire? — si elle La prophétie semblait inpliquer, impliquaitquelque chose, — un triomphe final ducôtédelamaison déjàpluspuissante, etnaturellement vivait dans la mémoire de la plus faible et de la moins influente, et la remplissait d'une aigre animosité. Wilhelm, comte Berlifitzing, bien haute origine, n'était, à fût d'une récit, qu'un qu'il l'époque de ce vieux radoteurinfirme, et n'avait rien de remarquable, si ce n'est une antipathie invétérée et folle famille de son rival, et une passion si contre la vive pour les chevaux et la chasse, que rien, ni ses infirmités physiques, ni son grand âge, ni l'affaiblissement de son esprit, ne pouvait l'empêcher de prendre journellement sa part des dangers de cet exercice. De l'autre côté, Fre- derick, baron M(^tzengerstein, n'était pas encore majeur. Son père, le ministre G , était mort jeune. Sa mère, madame iMarie, le suivit bientôt. Frederick était à cette époque dans sa dix-huitième année. Dans une ville, dix-huit ans ne sont pas une longue période de temps; mais dans une solitude, dans une aussi magnifique solitude que cette vieille seigneurie, le pendule vibre avec une plus profonde et plus significative solennité. HISTOIRES EXTRAORDINAIRES. 428 Par suite de certaines circonstances résultant de l'administration de son père, le jeune baron, aussitôt après la mort de celui-ci, entra en possession de ses vastes domaines. Hongrie Rarement on posséder un tel avait vu un noble de Ses châteaux patrimoine. étaient innombrables. Leplussplendide et le plus vaste était le palais ses Metzengerstein. La ligne frontière de domaines n'avait jamais été clairement définie; mais son parc principal embrassait un circuit de cinquante milles. L'avènement d'un propriétaire si jeune, et d'un caractère si bien connu, à une fortune si incomparable laissait peu de place aux conjectures relativement à sa ligne probable de conduite. Et, en vérité, dans l'espace de trois jours, la conduite de l'héritier nom d'Hérode et pâlir le re- fit dépassa magnifiquement les espé- rances de ses plus enthousiastes admirateurs. De honteuses débauches, de flagrantes perfidies, des atrocités inouïes, firent bientôt comprendre à ses vassaux trem- blants que rien, ni — ni soumission servile de leur part, scrupules de conscience de la sienne, — ne leur garantirait désormais de sécurité contre les griffes sans remords de ce petit Caligula. Vers la nuit du quatrième jour, on s'aperçut que le feu avait pris aux écuries du château Berlifitzing, et l'opinion unaninK* du voisinage ajouta le crime d'incendie à la liste déjà délits et des atrocités horrible des du baron. Quant au jeune gentilhomme, pendant le tumulte occasionné par cet accident, il se tenait, en apparence . M !•; TZ !<: NG K RsTEIN plongé dans une méditation, 4'20 au haut du palais de famille des Metzengerstein, dans un vaste appartement solitaire. La tenture de tapisserie, riche, quoique fanée, qui pendait mélancoliquement aux murs, représentait les figures fantastiques et majestueuses de mille ancê- tres illustres. Ici des prêtres richement vêtus d'hermine, des dignitaires pontificaux, siégeaient familièrement avec l'autocrate et le souverain, opposaient leur veto aux caprices d'un roi temporel, ou contenaient avec le de la toute-puissance papale fiat le sceptre rebelle du Grand Ennemi, prince des ténèbres. Là, les sombres et grandes figures des princes Metzengerstein — leurs musculeux chevaux de guerre piétinant sur les cadavres des ennemis tombés — ébranlaient fermes par leur forte expression ; les nefs les plus et ici, à leur tour, voluptueuses et blanches comme des cygnes, les images des dames des anciens jours flottaient au loin dans les méandres d'une danse fantastique aux accents d'une mélodie imaginaire. Mais, pendant que le baron prêtait l'oreille ou affectait de prêter Toreille au vacarme toujours croissant des écuries de Berlifitzing, — et peut-être méditai! quelque Irait nouveau, quelque trait décidé d'audace, — ses yeux se tournèrent machinalement vers l'image d'un cheval énorme, d'une couleur hors nature, et représenté dans la tapisserie comme appartenant à un ancêtre sarrasin de la famille de son rival. Le cheva! se tenait sur le — immobile — pendant qu'un peu plus premier plan du tableau, comme une statue, loin. HISTOIRES EXTRAORDINAIRES. 430 derrière lui, son cavalier déconfit mourait sous le poi- gnard d'un Metzengerstein. Sur la lèvre de Frederick surgit une expression diabolique, comme s'il s'apercevait de la direction que son regard avait prise involontairement. Cependant, il ne détourna pas les yeux. Bien loin de là, il ne pouvait d'aucune façon avoir raison de l'anxiété accablante qui comme un drap mor- semblait tomber sur ses sens tuaire, 11 conciliait difficilement ses sensations incohé- rentes comme celles des rêves avec la certitude d'être éveillé. Plus il contemplait, plus absorbant charme, — plus il lui paraissait devenait le impossible d'arracher son regard à la fascination de cette tapisserie. Mais le tumulte du dehors devenant soudainement plus violent, il fit enfin un effort, comme à regret, et tourna son attention vers une explosion de lumière rouge, projetée en plein des écuries enflammées sur les fenêtres de l'appartement. L'action toutefois ne fut que momentanée; son regard retourna machinalement au mur. A son grand étonne- ment, du gigantesque coursier — chose horrible! — avait pendant ce lemps changé de Le cou la tête position. de l'animal, d'abord incliné comme par la compassion vers le corps terrassé de son seigneur, était maintenant étendu, roide et dans toute sa longueur, dans la direction du baron. Les yeux, tout à l'heure invisibles, contenaient maintenant une expression énergique et humaine, et ils brillaient d'un rouge ardent et extraordinaire; et les lèvres distendues de ce cheval à la METZENGEHSTEIN. 431 }3hysionomie enragée laissaient pleinement apercevoir ses dents sépulcrales et dégoûtantes. Stupéfié par la terreur, le jeune seigneur gagna la porte en chancelant. Gomme il un l'ouvrait, éclat de lumière rouge jaillit au loin dans la salle, qui dessina nettement son reflet sur la tapisserie frissonnante ; et, comme le baron hésitait un instant sur le seuil, il tressaillit en voyant que ce reflet prenait la position exacte et remplissait précisément le contour de l'implacable et triomphant meurtrier du Berlifitzing sarrasin. Pour alléger ses esprits affaissés, le baron Frederick chercha précipitamment le plein air. A la porte principale du palais, il rencontra trois écuyers. Ceux-ci, avec beaucoup de difficulté et au grand péril de leur vie, comprimaient les bonds convulsifs d'un cheval gigantesque couleur de feu. — A qui ce cheval? Où l'avez-vous trouvé? demanda le jeune homme d'une voix querelleuse et rauque, reconnaissant immédiatement que le mystérieux coursier de la tapisserie était le parfait pendant du furieux animal qu'il avait devant lui. — C'est votre propriété, monseigneur, répliqua l'un des écuyers, du moins il n'est réclamé par aucun autre propriétaire. Nous l'avons pris comme il s'échappait, tout fumant et écumant de rage, des écuries brûlantes du château Berlifitzing. Supposant qu'il appartenait au haras des chevaux étrangers du vieux comte, nous l'avons ramené comme épave. Mais désavouent tout droit sur la bête ; les domestiques ce qui est étrange, HISTOIRES EXTR AORDLN AI KES. 43-2 puisqu'il porte des traces évidentes du feu, qui prou- vent qu'il l'a échappé belle. — Les lettres \V. V. B. sont également marquées au son front, interrompit un fer très-distinctement sur second écuyer; je supposais donc qu'elles étaient les initiales de Wilhelm von Beiiilitzing, mais tout le monde au château atïirme positivement n'avoir aucune connaissance du cheval. — Extrêmement singulier! dit le jeune baron, avec un air rêveur et comme n'ayant aucune conscience du comme vous dites, un re- sens de ses paroles. C'est, marquable cheval, soit, — un prodigieux cheval bien ! comme vous le remarquez caractère ombrageux et intraitable; à moi, je le veux bien, qu'il avec justesse, d'un ajouta-t-il allons! qu'il soit après une pause ; peut-être un cavalier tel que Frederick de Metzengerstein pourra-t-il dompter le diable même des écuries de Berlifitzing. — Vous vous trompez, monseigneur; le cheval, comme nous vous l'avons dit, je crois, n'appartient pas aux écuries du comte. Si tel eût été le cas, nous con- naissons trop bien notre devoir pour l'amener en pré- sence d'une noble personne de votre famille. — C'est vrai! observa le baron sèchement. Et, à ce moment, un jeune valet de chambre arriva du palais, le teint échauffé et à pas précipités, il chuchota à l'oreille de son maître Thistoire de la disparition sou- daine d'un morceau de la tapisserie, dans une chambre qu'il désigna, entrant ;i!ors d.ins des détails d'un METZENGERSTEIN. 433 caractère minutieux et circonstancié; mais, comme tout cela fut communiqué d'une voix très-basse, pas un mot ne transpira qui pût satisfaire la curiosité excitée des écuyers. Le jeune Frederick, pendant agité d'émotions variées. l'entretien, semblait Néanmoins, il recouvra bien- tôt son calme, et une expression de méchanceté décidée était déjà fixée sur sa physionomie, quand il donna des ordres pérempLoires pour que l'appartement en question fût immédiatement condamné et la clef remise entre ses mains propres. — Avez-vous appris la mort déplorable de Berlifi- tzing, le vieux chasseur? dit au baron un de ses vassaux, après le départ du page, pendant que l'énorme coursier que le gentilhomme venait d'adopter comme sien s'élançait et bondissait avec une furie redoublée à tra- vers la longue avenue qui s'étendait du palais aux écuries de Metzengerstein. — Non, dit le baron se tournant brusquement vers celui qui parlait; mortî dis-tu? — C'est la pure vérité, monseigneur ; et je présume que, pour un seigneur de votre nom, ce n'est pas un renseignement trop désagréable. Un rapide sourire jaillit sur la physionomie du baron. — Comment — Dans est-il mort? ses efforts imprudents pqur sauver la partie préférée de son haras de chasse, il a péri misérable- ment dans les flammes. — En... vé... ri... té...! exclama le baron, comme HISTOIRES EXTRAORDINAIRES. 434 impressionné lentement et graduellement par quelque évidence mystérieuse. — En — Horrible vérité, répéta le vassal. ! dit le jeune homme avec beaucoup de calme. Et il rentra tranquillement dans le palais. A partir de cette époque, une altération marquée eut lieu dans la conduite extérieure du jeune débauché, baron Frederick von Metzengerstein. Véritablement, sa conduite désappointait toutes les espérances et déroutait les intrigues de plus d'une mère. Ses habitudes et ses manières tranchèrent de plus en plus et, moins que jamais, n'offrirent d'analogie sympathique quelconque avec celle de l'aristocratie du voisinage. On ne le voyait jamais au delà des limites de son propre domaine, et, dans le vaste compagnon, monde social, il était absolument sans — à moins que ce grand cheval impétueux, hors nature, couleur de feu, qu'il monta continuelle- ment à partir de cette époque, n'eût en réalité quelque droit mystérieux au titre d'ami. Néanmoins, de nombreuses invitations de la part du voisinage lui arrivaient périodiquement. honorera-t-il notre fête de — « Le baron — sa présence ?» (( Le baron se joindra-t-il à nous pour une chasse au sanglier? )) — (( Metzengerstein ne chasse pas; » zengerstein n'ira pas, » — — «Met- telles étaient ses hautaines et laconiques réponses. Ces insultes répétées ne pouvaient pas être endurées par une noblesse impérieuse. De telles invitations devinrent moins cordiales, — moins fréquentes — avec ; METZENGERSTEIN. temps le elles cessèrent tout à fait. 435 On entendit la veuve de l'infortuné comte Berlifitzing exprimer le vœu (( que le baron fût au logis quand il désirerait n'y pas être, puisqu'il dédaignait la compagnie de ses égaux; et qu'il fût à cheval quand il voudrait n'y pas être, puisqu'il leur préférait la société d'un cheval.» Ceci à coup SLir n'était que l'explosion niaise d'une pique hérédi- taire et prouvait que nos paroles deviennent singuliè- rement absurdes quand nous voulons leur donner une forme extraordinairement énergique. Les gens charitables, néanmoins, attribuaient le changement de manières du jeune gentilhomme au chagrin naturel d'un fils privé prématurément de ses parents, — oubliant toutefois son atroce et insouciante conduite durant les jours qui suivirent immédiatement 11 y en eut quelques-uns qui accusèrent simplement en lui une idée exagérée de son impor- cette perte. tance et de sa dignité. D'autres, à leur tour (et parmi ceux-là peut être cité le médecin de la famille), parlè- rent sans hésiter d'une mélancolie morbide et d'un mal cependant, héréditaire; des insinuations plus téné- breuses, d'une nature plus équivoque, couraient parmi la multitude. En réalité, l'attachement pervers du baron pour sa monture de récente acquisition, — attachement qui semblait prendre une nouvelle force dans chaque nouvel exemple que l'animal donnait de ses féroces et dé- moniaques inclinations, — devint à la longue, aux yeux de tous les gens raisonnables, une tendresse hor- HISTOIRES EXTRAORDINAIRES. 436 rible et contre nature. Dans l'éblouissement du midi, — aux heures profondes de — malade ou bien portant, — dans calme ou dans tempête, — la nuit, le la le jeune Metzengerstein semblait cloué à la selle du cheval colossal dont les intraitables audaces s'accordaient si bien avec son propre caractère. Il y avait de plus des circonstances qui, rapprochées des événements récents, donnaient un caractère surnaturel monstrueux à la manie du cavalier et aux et capacités de la bête. L'espace qu'elle franchissait d'un seul saut avait été soigneusement mesuré, et se trouva dépasser d'une différence stupéfiante les conjectures les plus larges et les plus exagérées. Le baron, en outre, ne se servait pour l'animal d'aucun nom parti- culier, quoique tous distingués par des les chevaux de son haras fussent appellations caractéristiques. Ce cheval-ci avait son écurie à une certaine distance des autres; quant au pansement et atout le service né- et, cessaire, nul, excepté le propriétaire en personne, ne s'était risqué à remplir ces fonctions, ni même à en- trer dans l'enclos où s'élevait son écurie particulière. On observa aussi que, quoique les trois palefreniers qui s'étaient emparés du coursier, quand il fuyait l'in- cendie de Berlifitzing, eussent réussi à arrêter sa course à l'aide d'une chaîne à nœud coulant, cependant aucun des trois ne pouvait affirmer avec certitude que, durant dangereuse cette lors, il lutte, ou à aucun moment depuis eût jamais posé la main sur le corps delà bête. Des preuves d'intelligence particulière dans la conduite METZENGERSTEIN. 437 d'un noble cheval plein d'ardeur ne suffiraient certai- nement pas à exciter une attention déraisonnable; mais il y avait ici certaines circonstances qui eussent vio- lenté les esprits les plus sceptiques et les plus flegma- tiques; et l'on disait que parfois l'animal avait fait re- culer d'horreur la foule curieuse devant la profonde et frappante signification de sa marque, — que parfois le jeune Metzengerstein était devenu pâle et s'était dérobé devant l'expression soudaine de son œil sérieux et quasi humain. Parmi toute la domesticité du baron, il ne se trouva néanmoins personne pour douter de la ferveur extraordinaire d'affection qu'excitaient dans le jeune gentil- homme les qualités brillantes de son cheval; personne, excepté du moins un insignifiant petit page malvenu, dont on rencontrait partout l'offusquante laideur, et dont les opinions avaient aussi peu d'importance qu'il est possible. fois ses 11 avait l'effronterie d'affirmer — si toute- idées valent la peine d'être mentionnées, que son maître ne s'était jamais mis en selle — sans un inexplicable et presque imperceptible frisson, et qu'au retour de chacune de ses longues et habituelles pro- menades une expression de triomphante méchanceté faussait tous les muscles de sa face. Pendant une nuit de tempête, Metzengerstein,- sortant d'un lourd sommeil, descendit comme un ma- niaque de sa chambre, et, montant à cheval en toute hâte, s'élança en bondissant à travers le labyrinthe de la forêt. HISTOIRES EXTUAOriDINAIRES. 438. Un événement aussi commun rer particulièrement l'attention ; ne pouvait pas atti- mais son retour fut attendu avec une intense anxiété par tous ses domestiques, quand, après quelques heures d'absence, les prodigieux et magnifiques bâtiments du palais Metzentrembler jusque gerstein se mirent à craqueter et à dans leurs fondements, sous l'action d'un feu immense et immaîtrisable, — une masse épaisse et livide. Comme les flammes, quand on les aperçut pour la première fois, avaient déjà fait un si terrible progrès que tons les efforts pour sauver une portion quelconque des bâtiments eussent été évidemment inutiles, la population du voisinage se toute tenait paresseusement à Tentour, dans une stupéfaction silencieuse, sinon apathique. Mais un objet terrible et nouveau fixa bientôt l'attention de la multitude, et démontra combien est plus intense l'intérêt excité dans les sentiments d'une foule par la contemplation d'une agonie humaine que celui qui est créé par les plus effrayants spectacles de la matière inanimée. Sur la longue avenue de vieux chênes qui commençait à la forêt et aboutissait à l'entrée principale du palais Metzengerstein, un coursier, portant un cavalier décoiffé et en désordre, se faisait voir bondissant avec une impétuosité qui défiait le démon de la tempête lui-même. Le cavalier n'était évidemment pas le maître de cette course effrénée. L'angoisse efforts convulsifs de sa physionomie de tout son être, , les rendaient témoi- METZENGERSTEIN. gnage d'une lutte surhumaine 439 mais aucun son, excepté ; un cri unique, ne s'échappa de ses lèvres qu'il mordai,t d'outre lacérées, en outre dans l'intensité de sa terreur. En un instant, le choc des sabots retentit avec un bruit aigu et perçant, plus haut que le mugissement des flammes et le glapissement du vent; — un instant encore, et, franchissant d'un seul bond la grande porte et le fossé, le coursier s'élança sur les escaliers branlants du palais et disparut avec son cavalier dans le tourbillon de ce feu chaotique. La furie de la tempête s'apaisa tout à coup et un calme absolu prit solennellement sa place. Une flamme blanche enveloppait toujours suaire, et, le bâtiment comme un ruisselant au loin dans l'atmosphère tran- quille, dardait une lumière d'un éclat surnaturel, pen- dant qu'un nuage de fumée s'abattait pesamment sur les bâtiments sous la forme distincte d'un gigantesque cheval. LE MYSTÈRE DE MARIE ROGET' POUR FAIRE SUITE AU DOUBLE ASSASSINAT DANS LA RUE MORGUE d'événements qui les réelles. Les hommes et les circonstances, en général, modifient le train idéal des événements, en sorte qu'il semble imparfait et leurs conséquences aussi sont également imparfaites. C'est ainsi qu'il en fut de la Réformation au lieu du proIl y a des courent séries idéales parallèlement avec ; ; testantisme est arrivé le luthérianisme. NOV ALIS. même parmi les penseurs les plus calmes, qui n'aient été quelquefois envahies Il 1. y a peu de personnes, Lors de la publication originale de Marie Roget, les notes placées au bas des pages auraient été considérées comme super- Mais plusieurs années se sont écoulées depuis le drame ?<ur lequel ce conte e^st basé, et il nous a paru bon de les ajouter ici, flues. avec quelques mots d'explication relativement au dessein général. Une jeune fille, Mary Cecilia Rogers, fut assassinée dans les environs de New- York et, bien que sa mort eût excité un intérêt in; tense et persistant, le mystère dont elle était enveloppée n'était pas encore résolu à l'époque où ce morceau fut écrit et publié 25. 442 HISTOIRES EXTRAORDINAIRES. par une vague mais saisissante demi - croyance au surnaturel, en face de certaines coïncidences d'un caractère en apparence si merveilleux, que l'esprit se sentait incapable de les admettre comme pures coïnci- dences. De pareils sentiments (car les demi-croyances dont je parle n'ont jamais la parfaite énergie de la pensée), de pareils sentiments ne peuvent être que diffi- cilement comprimés; à moins qu'on n'en réfère à la science de la chance, ou, selon l'appellation technique, au calcul des probabilités. Or, ce calcul essence, purement mathématique ; est, et nous dans son avons ainsi l'anomalie de la science la plus rigoureusement exacte appliquée à l'ombre et à la spiritualité de ce qu'il y a de plus impalpable dans le monde de la spéculation. Les détails extraordinaires que je suis invité à publier forment, comme on le verra, quant à la succes- (novembre 1842). Ici, sous le prétexte de raconter la destinée d'une grisette parisienne, l'auteur a tracé minutieusement les faits essentiels, en même temps que ceux non essentiels et simplement pa- rallèles, du meurtre réel de Mary Rogers. Ainsi tout argument fondé sur la fiction est applicable à la vérité; et la recherche de la vérité est le but. Marie Roget fut composé loin du théâtre du crime, moyens d'investigation que les journaux que l'au- FjB Mystère de et sans autres teur put se procurer. Ainsi fnt-il privé de beaucoup de documents dont il aurait profité s'il avait été dans le pays et s'il avait inspecté les localités. Il n'est pas inutile de rappeler, toutefois, que les aveux de deux personnes (dont l'une est la madame Deluc du roman), faits à différentes époques et longtemps après cette publication, ont pleinement confirmé, rale, mais aussi tous les non-seulement la conclusion géné- principaux détails hypothétiques snr lesquels cette conclusion avait été basée. LE MYSTERE DE MARIE ROGET. sion des époques, la première 443 branche d'une série de coïncidences à peine imaginables, dont tous les lecteurs retrouveront la branche secondaire ou finale dans l'as- Mary Cecilia Rogers, à New-York. sassinat récent de Lorsque, dans un article intitulé Double Assassinat dans la rue Morgue, je m'appliquai, il y a un an à peu près, à dépeindre quelques traits saillants du caractère spirituel de mon ami le chevalier C. Auguste Dupin, il ne me vint pas à l'idée que j'aurais jamais à reprendre le même sujet. Je n'avais pas d'autre but que la pein- ture de ce caractère, et ce but se trouvait parfaitement atteint à travers la série Bizarre de circonstances faites pour mettre en lumière l'idiosyncrasie J'aurais pu ajouter d'autres exemples, de Dupin. mais je n'aurais rien prouvé de plus. Toutefois, des événements récents, ont, dans leur surprenante évolution, éveillé brusque- ment dans ma mémoire quelques détails de surcroît, qui garderont ainsi, je présume, quelque air d'une confession arrachée. Après avoir appris tout ce qui ne m'a été raconté que récemment, étrange que je gardasse le il serait silence sur ce vraiment que j'ai en- tendu et vu il y a déjà longtemps. Après la conclusion de la la tragédie impliquée dans mort de madame L'Espanaye et de sa fille, le cheva- lier Dupin congédia l'affaire de son esprit, et retomba dans ses vieilles habitudes de sombre rêverie. Très- porté, en tout temps, vers l'abstraction, son caractère l'y rejeta bien vite ; et, continuant à occuper notre ap- partement dans le faubourg Saint-Germain, nous aban- HISTOIRES EXTRAORDINAIRES. 444 donnâmes aux vents tout souci de l'avenir, et nous nous assoupîmes tranquillement dans le présent, brodant de nos rêves la trame fastidieuse du monde environnant. Mais ces rêves ne furent pas sans interruption. On devine facilement que le rôle joué par mon ami dans drame de la rue Morgue n'avait pas manqué de faire le impression sur l'esprit de la police parisienne. Parmi ses agents, le lier. il nom de Dupin était devenu un mot fami- Le caractère simple des inductions par lesquelles avait débrouillé le mystère n'ayant jamais été expli- qué au préfet, ni à aucun autre individu, moi excepté, il n'est pas comme surprenant que l'affaire ait été regardée approchant du miracle, ou que les facultés analytiques du chevalier lui aient acquis le crédit merveilleux de l'intuition. Sa franchise l'aurait sans doute poussé à désabuser tout questionneur d'une pareille erreur; mais son indolence fut cause qu'un sujet dont l'intérêt avait cessé pour lui depuis longtemps ne fat pas agité de nouveau. Il arriva ainsi que Dupin devint le fanal vers lequel se tournèrent les yeux de la po- lice, et, en mainte circonstance, des efforts furent faits auprès de lui par la Préfecture pour s'attacher ses talents. L'un des cas les plus remarquables fut l'assassi- nat d'une jeune fille nommée Marie Roget. Cet événement eut lieu deux ans environ après l'hor- reur delà rue Morgue. Marie, dont le nom de baptême et le nom de famille frapperont sans doute l'attention par leur ressemblance avec ceux d'une jeune et Infor- LE MYSTÈRE DE MARIE ROGET. 445 tunée marchande de cigares, était la fille unique de la veuve Estelle Roget. Le père était mort pendant l'en- fance de la fille, et, depuis l'époque de son décès jus- qu'à dix-huit mois avant l'assassinat qui fait le sujet de notre récit, la mère et la fille avaient toujours demeuré ensemble dans la rue Pavée-Saint-Ândré ^ , madame Roget y tenant une pension bourgeoise, avec l'aide de Marie. Les choses allèrent ainsi jusqu'à ce que celle-ci eût atteint sa vingt-deuxième année, quand sa grande beauté attira l'attention d'un parfumeur qui occupait l'une des boutiques du rez-de-chaussée du Palais-Royal, et dont la clientèle était surtout faite des hardis aven- turiers qui infestent le voisinage. M. Le Blanc ^ se doutait bien des avantages qu'il pourrait tirer de la dans son établissement de présence de la belle Marie parfumerie; et ses propositions furent acceptées vive- ment par la jeune fille, bien qu'elles soulevassent chez madame Roget quelque chose de plus que de l'hésitation. Les espérances du boutiquier se réalisèrent, et les charmes de la brillante grisette donnèrent bientôt la vogue à ses salons. Elle tenait son emploi depuis un an environ, quand ses admirateurs furent jetés dans la désolation par sa disparition soudaine de la boutique. M. Le Blanc fut dans l'impossibilité de rendre compte de son absence, et madame Roget devint folle d'inquiétude et de terreur. Les journaux s'emparèrent 4. Nassau-Street. '2. Anderson. immé- HISTOIRES EXTRAORDINAIRES. 446 diatement de la question, et la police était sur le point de faire une investigation sérieuse, quand un beau matin, après l'espace d'une semaine, Marie, en bonne santé, mais avec un comme d'habitude, air légèrement attristé, reparut, comptoir de parfumerie. à son Toute enquête, excepté celle d'un caractère privé, fut immédiatement arrêtée. parfaite ignorance, madame M. Le Blanc professait une comme précédemment. répondirent à Roget toutes les Marie et questions qu'elle avait passé la dernière semaine dans la maison d'un parent, à la campagne. Ainsi l'affaire fut généralement oubliée; car la jeune but ostensible de se soustraire curiosité, fit à tomba, et fille, dans le l'impertinence de la bientôt un adieu définitif au parfumeur, et alla chercher un abri dans la résidence de sa mère, rue Pavée-Saint-André. Il y avait à peu près cinq mois qu'elle était rentrée à la maison, lorsque ses amis furent alarmés par une soudaine et nouvelle disparition. Trois jours s'écoulèrent sans qu'on entendît parler d'elle. Le quatrième jour, on découvrit son corps flottant sur la Seine \ près de la berge qui fait face au quartier de la rue Saint- André, à un endroit peu distant des environs peu fré- quentés de la barrière du Roule ^. L'atrocité du meurtre (car il fut tout d'abord évident qu'un meurtre avait été commis), 1. L'Hudson. 2. Weehawken. la jeunesse et la LE MYSTÈRE DE MARIE ROGET. 447 beauté de la victime, et, par-dessus tout, sa notoriété antérieure, tout conspirait pour produire une intense excitation dans les esprits des sensibles Parisiens. Je ne me souviens pas d'un cas semblable ayant produit un effet aussi vif et aussi général. Pendant quelques semaines, les graves questions politiques du jour furent elles-mêmes noyées dans la discussion de cet unique et absorbant sujet. Le préfet fit des efforts inaccoutumés et toutes les forces ; de la police parisienne furent, jus- qu'à leur maximum, mises en réquisition. Quand le cadavre fut découvert, on était bien loin de supposer que le meurtrier pût échapper, plus d'un temps très-bref, aux recherches qui furent immédiate- ment ordonnées. Ce ne fut qu'à l'expiration d'une semaine qu'on jugea nécessaire d'offrir une récompense; et même cette récompense fut limitée alors à la somme de mille francs. Toutefois, l'investigation continuait avec vigueur, sinon avec discernement, et de nom- breux individus furent interrogés, mais sans résultat; cependant, l'absence totale de fil conducteur dans ce mystère ne faisait qu'accroître l'excitation populaire. A la fin du dixième jour, on pensa qu'il était opportun de doubler la somme primitivement proposée ; et peu à peu, la seconde semaine s'étant écoulée sans amener aucune découverte, et les préventions que Paris a toujours nourries contre la police s'étant exhalées en plusieurs émeutes sérieuses, le préfet prit sur lui d'offrir la somme de vingt mille francs a pour la dénonciation de l'assassin », ou, si plusieurs personnes se trouvaient HISTOIRES EXTRAORDINAIRES. 448 impliquées dans l'affaire, chacun des assassins^ nonçait cette ». « pour la dénonciation de Dans la proclamation qui an- récompense, une pleine amnistie était promise à tout complice qui déposerait spontanément contre son complice; et à la déclaration oflicielle, partout où elle était affichée, s'ajoutait un placard privé, émanant d'un comité de citoyens, qui offrait dix mille francs, en plus de la somme proposée par la préfecture. La récompense entière ne montait pas à moins de trente mille francs ; comme ce qui peut être regardé une somme extraordinaire, si Ton considère l'humble condition de la petite et la fréquence, dans les grandes villes, des atrocités telles que celles en question. Personne ne doutait maintenant que le mystère de cet assassinat ne fut immédiatement élucidé. Mais, quoique, dans un ou deux cas, des arrestations eussent eu lieu qui semblaient promettre un éclaircissement, on ne put rien découvrir qui incriminât les personnes suspectées, et elles furent aussitôt relâchées. Si bizarre que cela puisse paraître, trois semaines écoulées depuis la découverte du cadavre, trois semaines 's'étaient déjà écoulées sans jeter aucune lumière sur la question, et cependant la plus faible rumeur des événements qui agi1. Aux amateurs de la stricte vérité locale, je ferai observer, relativement à ce passage et à d'autres qui suivent, ainsi qu'à plusieurs de Double Assassinat dans la rue Morgue, que l'auteur raconte les choses à l'américaine, et que l'aventure n'est que trèssuperficiellement déguisée; mais que des mœurs parisiennes imaginaires n'infirment pas la valeur de l'analyse, pas plus qu'un plan de Paris imaginaire. — C. B. LE MYSTÈRE DE MARIE ROGET. 449 taient si violemment l'esprit public n'était pas encore arrivée à nos oreilles. Dupin et moi, voués à des recher- ches qui avaient absorbé toute notre attention depuis près d'un mois, nous n'avions, ni l'un ni l'autre, mis le pied dehors; nous n'avions reçu aucune visite,età peine avions-nous jeté un coup d'œil sur les principaux articles politiques d'un des journaux quotidiens. La pre- mière nouvelle du meurtre nous fut apportée par G..., en personnel II vint nous voir le 13 juillet commencement de resta avec nous l'après-midi, et 18.., au assez tard après la nuit tombée. Il était vivement blessé de l'insuccès de ses efforts pour dépister les assassins. Sa réputation, disait -il avec un parisien, était en jeu ; air essentiellement son honneur même, engagé dans la partie. L'œil du public, d'ailleurs, était fixé et il n'était pas sur lui, de sacrifice qu'il ne fût vraiment dis- posé à faire pour l'éclaircissement de ce mystère. 11 termina son discours, passablement drôle, par un compliment relatif à ce qu'il lui plut d'appeler le tact de Dupin, et fit à celui-ci une proposition directe, certaine- ment fort généreuse, dont je n'ai pas le droit de révéler ici la valeur précise, mais qui n'a pas de rapports avec l'objet propre de mon récit. Mon ami repoussa le compliment du mieux qu'il put, mais il accepta tout de suite la proposition, bien que les avantages en fussent absolument conditionnels. Ce 1. Voir Double Assassinat dans la rue Morgue et la Lettre volée. Il est évident que Poe a pensé à M. Gisquet, qui d'ailleurs ne se serait guère reconnu dans le personnage G. — G. B. HISTOIRES EXTRAORDINAIRES. 450 point étant établi, le préfet se répandit tout d'abord en explications de ses propres idées, les entremêlant de longs commentaires sur les dépositions, nous n'étions pas encore en possession. desquelles 11 discourait longuement, et même, sans aucun doute, doctement, lorsque je hasardai à l'aventure une observation sur la nuit qui s'avançait et amenait fermement assis dans son le fauteuil sommeil. Dupin, accoutumé, l'incarnation de l'attention respectueuse. Il était avait gardé ses lunettes durant toute l'entrevue; et, en jetant de temps à autre un coup d'oeil sous leurs vitres vertes, je m'étais convaincu que, pour silencieux qu'il eût été, son sommeil n'en avait pas été moins profond pendant les sept ou huit dernières lourdes heures qui précédè- rent le départ du préfet. Dans la matinée suivante, je me procurai, à la Préfecture, un rapport complet de toutes les dépositions obtenues jusqu'alors, et, à différents bureaux de journaux, un exen>plaire de chacun des numéros lesquels, dans depuis l'origine jusqu'au dernier moment, avait paru un document quelconque, intéressant, relatif à cette triste affaire. Débarrassée de ce qui était positivement marqué de fausseté, cette masse de ren- seignements se réduisait à ceci : Marie Roget avait quitté la maison de sa mère, rue Pavée-Saint-André, le dimanche 22 juin 18.., à neuf heures du matin environ. En sortant, elle avait fait part à M. Jacques Saint-Eustache \ et à lui seul, de son 1. Payne. LE MYSTÈRE DE MARIE ROGET. 451 intention de passer la journée chez une tante, à elle, qui demeurait rue des Drômes. La rue des Drômes est un passage court n'est pas loin à une distance mais très-populeux, qui et étroit, des bords de la rivière et qui est situé de deux milles directe, de la pension sée , dans la ligne suppo- bourgeoise de madame Roget. Saint-Eustache était le prétendant avoué de Maet logeait dans ladite pension, rie, où il prenait éga- lement ses repas. Il devait aller chercher sa fiancée à la brune et la ramener à la maison. Mais, dans l'après- midi, il survint une grosse pluie; et, supposant qu'elle resterait toute la nuit chez sa tante fait dans (comme elle avait des circonstances semblables), il ne jugea pas nécessaire de tenir sa promesse. Comme la nuit s'avançait, on entendit madame Roget (qui infirme) exprimer la crainte Marie » ; « était vieille et de ne plus jamais revoir mais dans le moment on attacha peu d'atten- tion à ce propos. n'était pas quand le jour se fut Le lundi, il fut vérifié que la jeune allée à la rue des Drômes ; et, fille écoulé sans apporter de ses nouvelles, une recherche tardive fut organisée sur différents points de la ville et des environs. Ce ne fut cependant que le quatrième jour depuis l'époque de sa disparition qu'on apprit enfin quelque chose d'important la concernant. Ce jour-là (mercredi 25 juin), un M. Beauvais^ qui avec un ami cherchait les traces de Marie près de la barrière du 1. Crommelin. HISTOIRES EXTRAORDINAIRES. 452 Roule, sur la rive de la Seine opposée à la rue Pavée- Saint-André, fut informé qu'un corps venait d'être ra- mené au rivage par quelques pêcheurs, qui l'avaient trouvé flottant sur le fleuve. En voyant le corps, Beauvais, après quelque hésitation, certifia que c'était celui de la jeune parfumeuse. Son ami le reconnut plus promptement. Le visage était arrosé de sang noir, qui jaillissait en partie de la bouche. on en voit dans Il le n'y avait pas d'écume, cas comme des personnes simplement noyées. Pas de décoloration dans le tissu cellulaire. Autour de la gorge se montraient des meurtrissures et des impressions de doigts. Les bras étaient repliés sur la poitrine et roidis. à moitié ouverte. La main droite crispée, la gauche Le poignet gauche était marqué de deux excoriations circulaires, provenant apparemment de cordes ou d'une corde ayant fait plus d'un tour. Une partie ainsi que le dos dans toute son étendue, mais particu- du poignet droit était aussi très-éraillée, lièrement aux omoplates. Pour amener le corps sur le rivage, les pêcheurs l'avaient attaché à une corde; mais ce n'était pas là ce qui avait produit les excoriations en question. La chair du cou était très-enflée. Il n'y avait pas de coupures apparentes ni de meurtrissures semblant le résultat de coups. On découvrit un mor- ceau de lacet si étroitement serré autour du cou qu'on ne pouvait d'abord l'apercevoir-, enfoui dans il était complètement la chair, et assujetti par un nœud caché juste sous l'oreille gauche. Cela seul aurait suffi pour LE MYSTERE DE MARIE ROGET. produire sait la 453 mort. Le rapport des médecins garantis- fermement le vertueux de caractère la défunte. Elle avait été vaincue, disaient-ils, par la force brutale. Le cadavre de Marie, qnand il fut trouvé, était dans une condition telle, qu'il ne pouvait y avoir, de la part de ses amis, aucune difiiculté à le reconnaître. La toilette était déchirée et d'ailleurs en grand désDans le vêtement extérieur, une bande, large ordre. d'environ un pied, avait été déchirée de bas en haut, depuis l'ourlet jusqu'à la taille, mais non pas arrachée. Elle était roulée trois fois autour jettie dans le ment fait. de la dos par une sorte de taille et assu- nœud très-solide- Le vêtement, immédiatement au-dessous de la robe, était de mousseline fine; et on en avait arraché une bande large de dix-huit pouces, arraché complètement, mais très-régulièrement et avec une grande netteté. On trouva cette bande autour du cou, adapté d'une manière lâche et assujettie avec un nœud serré. Par-dessus cette bande de mousseline et le mor- ceau de lacet, étaient attachées les brides d'un cha- peau, avec le chapeau pendant. Le nœud qui liait les brides n'était pas un nœud comme le font les femmes, mais un nœud coulant, à la manière des matelots. Le corps, après qu'il fut reconnu, ne fut pas, comme c'est l'usage, transporté à la Morgue (cette formalité étant maintenant superflue), mais enterré à la hâte non loin de Grâce l'endroit du rivage où aux efforts de Beauvais, avait été recueilli. l'affaire fut soigneuse- il ment assoupie, autant du moins qu'il fut possible; et HISTOIRES EXTRAOKDlNAIRES. 454 quelques jours s'écoulèrent avant en qu'il résultât aucune émotion publique. A la fin, cependant, un journal hebdomadaire ^ ramassa la question fut exhumé, et une enquête nouvelle ordonnée il ; le cadavre ; mais n'en résulta rien de plus que ce qui avait déjà été observé. Toutefois, les vêtements furent alors présentés à la mère et aux amis de la défunte, qui les reconnurent parfaitement pour ceux portés par la jeune fille quand elle avait quitté la maison. Cependant, l'excitation publique croissait d'heure en heure. Plusieurs individus furent arrêtés et relâchés. Saint-Eustache en particulier parut suspect , et il ne compte rendu intelligible sut pas d'abord donner un de l'emploi qu'il avait fait du dimanche, dans la mati- née duquel Marie avait quitté la maison. Plus tard cependant, il présenta à M. G des affidavit qui expli- quaient d'une manière satisfaisante l'usage qu'il avait fait de chaque le heure de la journée en question. Comme temps s'écoulait sans amener aucune découverte, mille rumeurs contradictoires furent mises en circulation, et les journalistes inspirations. purent lâcher la bride à leurs Parmi toutes ces hypothèses, une particulièrement l'attention ; attira ce fut celle qui admettait que Marie Roget était encore vivante, et que le cadavre découvert dans la Seine était celui de quelque autre infortunée. 11 me paraît utile de soumettre au lecteur quelques-uns des passages relatifs à cette insinuation. 1. The New- York Mercury » LE MYSTÈRE DE MARIE ROGET. 455 Ces passages sont tirés textuellement de Tj^^oi/e S journal dirigé généralement avec une grande habileté. (( Mademoiselle Roget est sortie de la maison de sa mère dimanche matin, 22 juin 18.., avec exprimée d'aller voir sa tante ou quelque autre pa- Depuis cette heure-là, on ne rent, rue des Drômes. trouve personne qui , l'intention vue. l'ait On n'a d'elle aucune trace, aucunes nouvelles Aucune personne quelconque ne s'est présentée, déclarant l'avoir vue ce jour-là, après qu'elle eut quitté le seuil de la maison de sa mère Or, quoique nous n'ayons aucune preuve indiquant que Marie Roget était encore de ce monde, dimanche 22 juin, après neuf heures, nous avons la preuve que jusqu'à cette heure elle était vivante. Mercredi, à midi, un corps de femme a été découvert flottant sur la rive de la barrière du Roule. Même en supposant que Marie Roget ait été jetée dans qu'elle est sortie de la la rivière trois ferait que trois jours écoulés départ, — trois depuis l'instant de son jours tout juste. d'imaginer que le meurtre, time d'un meurtre, ait heures après maison de sa mère, cela ne Mais il est si -toutefois elle absurde a été vic- pu être consommé assez rapi- dement pour permettre aux meurtriers de jeter le corps à la rivière avant le milieu de la nuit. Ceux qui se rendent coupables de si horribles crimes préfèrent les 1. The New- York Brother Jonathan^ édité par H. Hastings Weld» Esquire. HISTOIRES EXTRAORDliNAlRES. 456 ténèbres à la lumière Ainsi nous voyons que, si le corps trouvé dans la rivière de Marie Roget, était celui il dans l'eau plus de deux jours pu n'aurait pas et demi, ou rester au trois maximum. L'expérience prouve que les corps noyés, ou jetés à l'eau immédiatement après une mort violente, ont besoin d'un temps comme de six à dix jours pour qu'une décomposition suffisante ramène à la les surface des eaux. Un cadavre sur lequel on tire le ca- non, et qui s'élève avant que l'immersion ait duré au moins cinq ou six jours, ne manque pas de replonger, on l'abandonne à lui-môme. Maintenant, nous si le demandons, qu'est-ce qui a pu, dans le cas présent, déranger le cours ordinaire de Si le corps, la nature? . . . . dans son état endommagé, avait été gardé sur le rivage jusqu'à mardi soir, on trouverait sur ce rivage quelque trace des meurtriers. douteux que le corps ait pu Il est aussi fort revenir sitôt à la surface, même en admettant qu'il ait été jeté à l'eau deux jours après la mort. Et enfin, il est excessivement improbable que les malfaiteurs qui ont tel commis un meurtre que celui qui est supposé, aient jeté le corps à l'eau sans un poids pour l'entraîner, quand il était si facile de prendre cette précaution. » L'éditeur du journal s'applique ensuite à démontrer que le corps doit être resté dans l'eau non pas simplement trois jours, mais au moins cinq fois trois jours, parce qu'il était si décomposé, que Beauvais a eu beau- coup de peine à le reconnaître. Ce dernier point, toute- LE MYSTERE DE MARIE ROGET. fois, était (( 457 complètement faux. Je continue la citation : Quels sont donc les faits sur lesquels M. Beauvais s'appuie pour dire qu'il ne doute pas que le corps soit celui de Marie Roget? Il a déchiré la manche de la robe et a trouvé, dit-il, des marques qui lui ont prouvé l'identité. Le public a supposé généralement que ces marques devaient consister en une espèce de cicatrice. 11 a passé sa main sur le bras, et y a trouvé du poil, — quelque chose, ce nous semble, d'aussi peu particulier qu'on puisse se cluant que de trouver un bras dans une manche. le figurer, d'aussi peu con- M. Beauvais n'est pas rentré à la maison cette nuit-là, mais il a envoyé un mot à madame Roget, à sept heures, mercredi soir, pour lui dire que l'enquête, tive à sa fille, marchait toujours. rela- Même en admettant que madame Roget, à cause de son âge et de sa douleur, fût incapable de se rendre sur les lieux (ce qui, en vérité, est accorder beaucoup), à coup sûr, il se serait trouvé quelqu'un qui aurait jugé que cela valait bien la peine d'y aller et de suivre l'investigation, si toutefois ils avaient pensé que c'était bien le corps de Marie. Personne n'est venu. On n'a rien entendu dire de la chose, dans dré, qui soit parvenu la dit ni rien rue Pavée-Saint-An- même aux locataires de ladite maison. M. Saint-Eustache, l'amoureux et le futur de Marie, qui avait pris pension chez sa qu'il n'a mère, dépose entendu parler de la découverte du corps de sa promise que le matin suivant, quand M. Beauvais lui-même est entré dans sa chambre et lui en a parlé. ***** 26 458 Qu'une HISTOIRES EXTRAORDINAIRES. nouvelle aussi que celle-là capitale ait été reçue si tranquillement, il y a de quoi nous étonner. » Le journal s'efforce de suggérer ainsi l'idée d'une certaine apathie dans les parents et les amis de Marie, laquelle apathie serait absurde si l'on suppose qu'ils crussent que le corps trouvé était vraiment le sien. L'Étoile cherche, en la somme, à insinuer que Marie, avec connivence de ses amis, s'est absentée de la ville pour des raisons qui compromettent sa vertu; et que ces mêmes amis, ayant découvert sur la Seine un corps ressemblant un peu à celui de la jeune tille, ont profité de l'occasion pour répandre dans le public la nouvelle de sa mort. Mais l'Étoile y a mis beaucoup trop de précipitation. Il a été clairement prouvé qu'aucune apathie de ce genre n'a existé; que la vieille dame était exces- sivement faible, et si agitée, qu'il lui eût été impossible de s'occuper de quoi que ce soit; que Saint-Eustache, bien loin de recevoir la nouvelle froidement, était de- venu fou de douleur et avait donné de tels signes de frénésie, que M. Beauvais avait cru devoir charger un de ses amis et parents de le surveiller et de l'empêcher d'assister à l'examen qui devait suivre l'exhumation, En outre, bien que l'Étoile affirme que le corps a été réenterré aux frais de l'État, — qu'une offre avantageuse de sépulture particulière a été absolument repoussée par la famille n'assistait à la — et qu'aucun membre de cérémonie, — bien que la famille l'Étoile, dis-je, affirme tout cela pour corroborer l'impression qu'elle cherche à produire, — tout cela a été victorieusement LE MYSTÈRE DE MARIE ROGET. réfuté. 459 Dans un des numéros suivants du même jour- on fit un effort pour jeter des soupçons sur Beau- nal, vais lui-même. L'éditeur dit : Un changement vient de s'opérer dans la question. ({ On nous raconte que, dans une certaine occasion, pendant qu'une dame B. était chez madame Roget, M. Beauvais, qui sortait, lui dit qu'un gendarme allait venir, et qu'elle, madame B., eût soin de ne rien dire au gendarme jusqu'à ce qu'il fût de retour et qu'elle lui laissât, à lui, tout le soin de l'affaire Dans la situation présente, il semble que M. Beauvais porte tout le secret de la question, enfermé dans sa est impossible d'avancer d'un pas sans M. Beau- tête. Il vais; de quelque côté que vous tourniez, vous vous heurtez à lui Pour une raison quelconque, il a décidé que personne, excepté lui, ne pourrait se mêler de l'enquête, et il a jeté les parents à l'écart d'une manière fort incongrue, s'il faut en croire leurs récriminations. 11 a paru très- préoccupé de l'idée d'empêcher les parents de voir le cadavre. » Le fait qui suit sembla donner quelque couleur de vraisemblance aux soupçons portés ainsi sur Beauvais. Quelqu'un qui était venu lui rendre visite à son bureau, quelques jours avant la disparition de la jeune fille et pendant l'absence dudit Beauvais, avait observé une rose plantée dans le trou de la serrure, et le mot Marie écrit sur une ardoise fixée à la portée de la main. L'impression générale, autant du moins qu'il nous HISTOIRES EXTRAORDINAIRES. 460 fut possible de l'extraire des papiers publics, était que Marie avait été la victime d'une bande de misérables furieux, qui Pavaient transportée sur mal- rivière, la traitée et assassinée. Cependant, une feuille d'une vaste influence, le Commercial^, combattit très-vivement cette idée populaire. J'extrais un ou deux passages de ses colonnes « : Nous sommes persuadés que l'enquête a jusqu'à présent suivi une fausse piste, tant du moins qu'elle a été dirigée vers la barrière du Roule. Il est impossible qu'une jeune femme, connue, comme était Marie, de plusieurs milliers de personnes, ait pu passer trois bor- nes sans rencontrer quelqu'un à qui son visage fût familier; et quiconque l'aurait vue s'en serait souvenu, car elle inspirait de l'intérêt à tous ceux qui la connaissaient. Elle est sortie juste au moment où les rues sont pleines de monde Il est impossible qu'elle soit barrière du allée à la Roule ou à la rue des Drômes sans avoir été reconnue par une douzaine de personnes; aucune déposition cependant n'affirme qu'on l'ait vue ailleurs que sur le seuil de la maison de sa mère, et il n'y a même aucune preuve qu'elle en soit sortie du tout, excepté le témoi- gnage concernant Vintention exprimée par elle. morceau de sa robe était déchiré, serré autour Un d'elle et noué; c'est ainsi que le corps a pu être porté comme un paquet. Si le meurtre avait été commis à la barrière 1. New-York, Journal of Commerce. LE MYSTÈRE DE MARIE ROGET. 401 du Roule, il n'aurait pas été nécessaire de prendre de telles dispositions. Ce fait, que le corps a été trouvé flottant près de la barrière, n'est pas une preuve rela- tivement au lieu d'où il a été jeté dans l'eau. . . . Un morceau d'un des jupons de l'infortunée jeune fille, long de deux pieds et large d'un pied, avait été arraché, serré autour de son cou et noué derrière sa tête, probablement pour empêcher ses par des drôles qui n'avaient de poche. cris. Cela a été fait même pas un mouchoir » Un jour ou deux avant que le préfet vînt nous rendre visite, la police avait un renseignement obtenu assez important qui semblait détruire l'argumentation du Commercial, au moins dans sa Deux petits garçons, fils d'une partie principale. dame Deluc, vagabon- dant dans les bois, près de la barrière du Roule, avaient pénétré par hasard dans un épais fourré, où se trouvaient trois ou quatre grosses pierres, formant une espèce de siège, avec dossier et tabouret. Sur la pierre supérieure gisait un jupon blanc ; sur la seconde une écharpe de soie. On y trouva aussi une ombrelle, des gants et un mouchoir de poche. Le mouchoir portait le nom (( Marie Roget ». Des lambeaux de vêtements fu- rent découverts sur les ronces environnantes. Le sol était piétiné, les buissons étaient enfoncés; il y avait là toutes les traces d'une lutte. Entre le fourré et la rivière, on découvrit que les palissades étaient abattues, et la terre gardait la trace d'un lourd fardeau qu'on y avait traîné. 2<). HISTOIRES EXTRAORDINAIRES. 4C2 Une feuille hebdomadaire, cette découverte les le Soleil^, donnait sur commentaires suivants, commen- taires qui n'étaient que l'écho des sentiments la presse parisienne (( de toute : Les objets sont évidemment restés moins trois ou quatre semaines ; ils là pendant au étaient complète- ment moisis par Faction de la pluie,et collés ensemble par la moisissure. Tout autour, le gazon avait poussé et même les dominaient partiellement. La soie de l'ombrelle était solide; mais les branches étaient fer- mées, et la partie supérieure, là où Tétoffe était dou- ble et rempliée, étant toute pénétrée de moisissure et pourrie, se déchira aussitôt qu'on l'ouvrit Les fragments de vêtements accrochés aux buissons étaient larges de trois pouces environ et longs de six. L'un était un morceau de l'ourlet de la robe, qui avait été raccommodé, l'autre, non pas l'ourlet. Ils un morceau du jupon, mais ressemblaient à des bandes arra- chées et étaient suspendus au buisson d'épines, à un pied de terre environ Il n'y a donc pas lieu de douter que le théâtre de cet abominable outrage n'ait été enfin découvert. » Aussitôt après cette découverte, parut. Madame Deluc un nouveau témoin raconta qu'elle tenait une au- berge au bord de la route, non loin de la berge de la rivière opposée à la barrière du Roule. Les environs 1. Philadelphie, Saturday Evening Post, édité par C. I. Esquire* Peterson, LE MYSTÈRE DE MARIE ROGET. 4G3 sont solitaires, — le rendez-vous ordinaire des mauvais sujets de la ville, très-solitaires. C'est là, le dimanche, qui traversent la rivière en canot. environ, dans l'après-midi du Vers trois heures dimanche en question» une jeune fille était arrivée à l'auberge, accompagnée par un jeune homme au teint brun. Ils y étaient restés tous deux pendant quelque temps. Après leur départ, ils firent route vers quelque bois épais'du voisinage. L'attention de madame Deluc fut attirée par la toilette que portait la jeune fille, à cause de sa ressemblance avec celle d'une de ses parentes défunte'. Elle remarqua particulièrement une écharpe. Aussitôt après le départ du couple, une bande de mécréants parut, qui firent un tapage affreux, burent et mangèrent sans payer, suivirent la même route que le jeune homme et la jeune fille, revinrent à l'auberge à la brune, puis repassèrent la rivière en grande hâte. Ce fut peu après la tombée de la nuit, dans la même soirée, que madame Deluc, ainsi que son fils aîné, en- tendit des cris de femme dans le voisinage de l'auberge. Les cris furent violents, mais ne durèrent pas longtemps. Madame très- Deluc reconnut non -seulement r écharpe trouvée dans le fourré, mais aussi la robe qui habillait lence \ le cadavre. Un conducteur d'omnibus. déposa également alors qu'il avait Va- vu Marie Roget traverser la Seine en bateau, dans ce dimanche en question, en compagnie d'un jeune 1, Adam, homme d'une HISTOIRES EXTRAORDINAIRES. 464 figure brune. Lui, Valence, connaissait Marie et ne pouvait pas se tromper sur son identité. Les objets trouvés dans le bosquet furent parfaitement reconnus par les parents de Marie. Cette masse de dépositions et d'informations que je récoltais ainsi dans les journaux, à demande de la Dupin, comprenait encore un point, — mais point de la plus haute importance. 11 c'était un paraît qu'immé- diatement après la découverte des objets ci-dessus indiqués on trouva, dans le voisinage du lieu que l'on croyait maintenant avoir été le théâtre du crime, le corps inanimé ou presque inanimé de Saint-Eustache, le fiancé « de Marie. Une fiole vide portant l'étiquette laudanum » était auprès de lui. Son haleine accusait le poison. trouva sur Il mourut sans prononcer une lui une On parole. brièvement son racontant lettre amour pour Marie et son dessein arrêté de suicide. — Je ne crois pas avoir besoin de vous dire, — dit Dupin, comme il achevait la lecture de mes notes, — que c'est là un cas beaucoup plus compliqué que celui de la rue Morgue, duquel il diffère en un point très- important. C'est là un exemple de crime atroce, mais ordinaire. Nous n'y trouvons rien de particulièrement outre. Observez, je vous prie, que c'est la raison pour laquelle le mystère a paru simple ; quoique ce soit justement la même raison qui aurait dû le faire considérer comme plus difficile à résoudre. C'est pourquoi on a d'abord jugé superflu d'offrir une récompense. Les mirmidons de G étaient assez forts pour com- LE MYSTÈRE DE MARIE ROGE prendre comment et pourquoi une vait avoir été telle 405 . atrocité pou- commise. Leur imagination pouvait se — plusieurs modes, — un motif, — plu- figurer un mode, sieurs motifs; et parce qu'il n'était pas impossible que l'un de ces nombreux modes et motifs fût Tunique réel, ils ont considéré comme démontré que être un de le réel devait ceux-là. Mais l'aisance aNeç^ laquelle ils avaient conçu ces idées diverses, et même le caractère plausible dont chacune était revêtue, auraient facilité dû être que de la pris pour des indices de la difficulté plutôt attachée à l'explication de l'énigme. Je vous ai déjà fait observer que c'est par des saillies au-dessus du plan ordinaire des choses que 1^ raison doit trouver sa voie, ou jamais, dans sa recherche de la vérité, et que, dans des cas tels que celui-là, l'important n'est pas tant de se dire tent? » : « Quels sont les que de se dire : « faits Quels sont présentent, qui ne se sont jamais vant? » Dans les qui se présenles faits qui se présentés aupara- investigations faites chez madame L'Espanaye\ les agents de G furent découragés et confondus par cette étrangeté même qui eût été, pour une intelligence bien faite, le plus sûr présage de succès; et cette même intelligence eût été plongée dans le désespoir par le caractère ordinaire de tous les faits qui s'offrent à l'examen dans le cas de la jeune parfu- meuse et qui n'ont encore rien révélé de positif, si ce n'est la présomption des fonctionnaires de la Préfecture. 1. Voir Double Assassinat dans la rue Morgue. HISTOIRES EXTRAORDINAIRES. 4G6 1) Dans le cas de madame L'Espanaye et de sa fille, dès le commencement de notre investigation, il n'y avait pour nous aucun doute qu'un meurtre avait été commis. L'idée de suicide se trouvait tout d'abord exclue. Dans le cas présent, nous avons également à éli- miner toute idée de suicide. Le corps trouvé à la barrière du Roule a été trouvé dans des circonstances qui ne nous permettent aucune hésitation sur ce point important. Mais on a insinué que le cadavre trouvé n'est pas celui de la Marie Roget dont l'assassin ou les assassins sont à découvrir, pour la découverte desquels une récompense est offerte, et qui sont l'unique objet de notre traité avec le préfet. Vous et moi, nous connaissons assez bien ce gentleman. Nous ne devons pas trop nous fier à lui. Soit que, prenant le corps trouvé pour point de départ, et suivant la piste d'un assassin, nous découvrions que ce corps est celui d'une autre personne que Marie; soit que, prenant pour point de départ la Marie encore vivante, nous la retrouvions non assassinée, — dans les deux cas, nous perdons notre peine, puisque c'est avec M. affaire. G que nous avons Donc, pour notre propre but, si ce n'est pour le but de la justice, il est indispensable que notre pre- mier pas soit la constatation de Tidentité du cadavre avec la Marie Roget disparue. » Les arguments de l' Étoile ont trouvé crédit dans le public; et le journal lui-même est convaincu de leur importance, ainsi qu'il résulte de la manière dont il commence un de ses articles sur le sujet en question : LE MYSTÈRE DE MARIE ROGET. (( — Quelques-uns des journaux du matin, 467 dit-il, de lundi. » Pour moi, cet article ne — parnuméro lent de l'article concluant de l'Étoile dans son me paraît guère concluant que relativement au zèle du rédacteur. Nous devons ne pas oublier qu'en général feuilles but de nos le publiques est de créer une sensation, de faire du piquant plutôt que de favoriser la cause de la vérité. Ce dernier but n'est poursuivi que quand il semble coïncider avec le premier. Le journal qui s'accorde avec l'opinion ordinaire (quelque bien fondée que soit d'ailleurs cette opinion) n'obtient pas de crédit parmi la foule. La masse du peuple considère comme profond celui-là seul qui émet des contradictions piquantes de l'idée générale. En logique aussi bien qu'en littérature, c'est ment répigramme qui et le plus est le genre le plus immédiate- Dans les plus bas selon l'ordre du universellement apprécié. deux cas, c'est le genre le mérite. Je » veux dire que c'est le caractère mêlé d'épi- gramme et de mélodrame de cette idée, Roget est encore vivante, — qui l'a — que Marie suggérée à V Étoile, plutôt qu'aucun véritable caractère plausible, lui a et qui assuré un accueil favorable auprès du public. Examinons les points principaux de l'argumentation de ce journal, et prenons bien garde à l'incohérence avec laquelle elle se produit dès le principe. » L'écrivain vise d'abord à nous prouver, par la brièveté de l'intervalle compris entre la disparition de Marie et la découverte du corps flottant, que, ce corps HISTOIRES EXTRAORDINAIRES. '468 ne peut pas être celai de Marie. Réduire cet intervalle à la dimension plus petite possible la devient tout d'abord chose capitale pour l'argumentateur. Dans la recherche inconsidérée de ce but, il se précipite tout d'abord dans la pure supposition, dit-il, — de supposer que a C'est une folie, — meurtre, si un meurtre a le été commis sur cette personne, ait pu être consommé assez vite pour permettre aux meurtriers de jeter le corps dans la rivière avant minuit. » Nous demandons tout de suite, et très-naturellement, pourquoi. Pour- quoi est-ce une folie de supposer que le meurtre a été commis cinq minutes après que la jeune fille a quitté le domicile de sa mère? Pourquoi est-ce une folie de moment supposer que le meurtre a été commis à un quelconque de la journée? 11 s'est commis des assassi- nats à toutes les heures. Mais, que le meurtre ait eu lieu à un moment quelconque entre neuf heures du matin, dimanche, et minuit moins un quart, toujours resté bien assez de il serait temps pour jeter le cada- vre dans la rivière avant minuit. Cette supposition se réduit donc à cela mis le dimanche : ; que le meurtre n'a pas et, si été com- nous permettons à VÈtolle de supposer cela, nous pouvons lui accorder toutes les libertés possibles. On peut imaginer que le paragraphe commençant par meurtre, etc., » « C'est quoiqu'il une folie de supposer que le ait été imprimé sous cette forme par l'Étoile, avait été réellement conçu dans le « C'est cerveau du rédacteur sous cette autre forme : une folie de supposer que le meurtre, si un meurtre a LE MYSTÈRE DE MARIE ROGET. ëlé 469 commis sur cette personne, ait pu être consommé assez vite pour permettre aux meurtriers de jeter le corps dans la rivière avant minuit; disons-nous, de supposer cela, et en c'est une folie, même temps de supposer (comme nous voulons bien le supposer) que le corps n'a été jeté à Peau que passé minuit; passablement mal déduite » opinion mais qui n'est pas aussi , complètement déraisonnable que celle imprimée. )) eu simplement pour but — continua — de réfuter ce passage de l'argumentation de Si j'avais Dupin l'Étoile, j'aurais pu tout aussi bien le laisser oi^i il est. Mais ce n'est pas de VÈtoile que nous avons affaire, mais bien de la vérité. La phrase en question, dans le cas actuel, n'a qu'un sens, et ce sens, je l'ai nettement établi; mais il est essentiel que nous pénétrions der- rière les mots pour chercher une idée que ces mots donnent évidemment à entendre, sans l'exprimer positivement. Le dessein du journaliste était de dire qu'il était improbable, à quelque moment de la journée ou de la nuit de dimanche que le meurtre eût été commis, que les assassins se fussent hasardés à porter le corps à la rivière avant minuit. C'est justement là que gît la supposition dont je me plains. meurtre a été commis à un telles circonstances, porter le corps à la qu'il est rivière. tel On suppose que le endroit et dans de devenu nécessaire de Or, l'assassinat pourrait avoir eu lieu sur le bord de la rivière, ou sur la rivière même; et ainsi le langage du corps à l'eau, auquel on a eu recours à n'importe quel moment du jour ou de m HISTOIRES EXTRAORDINAIRES. la nuit, se serait présenté comme le mode d'action le plus immédiat, le plus sous la main. Vous comprenez que je ne suggère ici rien qui me paraisse plus probable ou qui coïncide avec ma propre opinion. Jusqu'à mêmes de la présent, je n'ai pas en vue les éléments cause. Je désire simplement vous mettre en garde contre le ton général des suggestions de l'Étoile et appeler votre attention sur le caractère de parti pris qui s'y manifeste tout d'abord. » Ayant ainsi prescrit une limite accommodée à ses idées préconçues, ayant supposé que, celui de Marie, il n'aurait pu si pendant un laps de temps très-court, vient à dire « ce corps était que rester dans l'eau le journal en : L'expérience prouve que les corps noyés, ou jetés à l'eau immédiatement après une mort violente, ont besoin d'un temps comme six de qu'une décomposition suffisante face des eaux. à les dix jours ramène pour à la sur- Un cadavre sur lequel on tire le canon, et qui s'élève avant que l'immersion ait duré au moins cinq ou six jours, ne manque pas de replonger, si on l'abandonne à lui-même. » » Ces assertions ont été acceptées tacitement par tous les journaux de Paris, à l'exception du Moniteur^. Cette dernière feuille s'efforce de combattre la partie du paragraphe qui a trait seulement aux corps des noyés, en citant cinq ou six cas dans lesquels 1. les corps de The New-York Commercial Advertiser, édité par Col. Stone. LE MYSTÈRE DE MARIE ROGET. 471 personnes notoirement noyées ont été trouvés flottants après un laps de temps moindre l'Étoile. que celui fixé par Mais il y a quelque chose d'excessivement anti- philosophique dans cette tentative que fait le Moniteur, de repousser Taffirmation générale de VÉtoile par une citation de cas particuliers militant contre cette affir- mation. Quand même il eût été possible d'alléguer cinquante cas, au lieu de cinq, de cadavres trouvés à des eaux au bout de deux ou trois jours, ces la surface auraient pu cinquante exemples légitimement être considérés comme de pures exceptions à la règle de r Étoile, jusqu'à ce que la règle elle-même fût définiti- vement réfutée. Cette règle admise la nie pas, il gumentation de sa force ; (et le Moniteur ne insiste seulement sur les exceptions), l'arl'Étoile reste en possession de toute car cette argumentation ne prétend pas im- pliquer plus qu'une question de probabilité relative- ment à un corps pouvant s'élever à la surface en moins de trois jours; et cette probabilité sera en faveur de l'Étoile jusqu'à ce que les exemples, allégués, soient en si puérilement nombre suffisant pour constituer une règle contraire. » Vous comprenez tout de suite que toute argumen- tation de ce genre doit être dirigée contre la règle elle- même, et, dans ce but, nous devons raisonnée de la règle. Or, général, ni beaucoup le plus lourd que l'eau de la Seine corps léger, faire l'analyse humain n'est, en ni beaucoup plus c'est-à-dire que la pesan- teur spécifique du corps humain, dans sa condition ; HISTOIRES EXTRAORDINAIRES. 472 naturelle, est à peu près égale au vokune d'eau douce qu'il déplace. Les corps des individus gras et charnus, avec de petits os, et généralement des femmes, sont plus légers que ceux des individus maigres, à gros os, et généralement des hommes; et la pesanteur spécifi- que de l'eau d'une rivière est quelque peu influencée par la présence du flux de la mer. Mais, en faisant abstraction de la marée, on peut affirmer que très-peu de corps humains seront submergés, même dans l'eau douce, spontanément, par leur propre nature. Presque tous, tombant dans une rivière, seront aptes à flotter, s'ils laissent s'établir un équilibre convenable entre la pesanteur spécifique de l'eau et leur pesanteur propre, c'est-à-dire s'ils se laissent submerger tout entiers, en exceptant le moins de parties possible. La meilleure position pour celui qui ne sait pas nager est la position verticale de l'homme qui marche sur la terre, tète complètement renversée et submergée, la et les marines restant seules au-dessus l'eau. Dans de telles conditions, la bouche du niveau de nous pourrons tous flotter sans difficulté et sans effort. Il est évident, toutefois, que les pesanteurs du corps et du volume d'eau déplacé sont alors très-rigoureusement balancées, et qu'un rien suffira pour donner à l'un ou à l'autre la prépondérance. Un bras, par exemple, élevé au-dessus de l'eau, et conséquemment privé de son support, est un poids additionnel suffisant pour faire plonger toute la tête, tandis que le secours accidentel du plus petit morceau de bois nous permettra de lever suffi- LE MYSTÈRE DE MARIE ROGET. 473 samment la tête pour regarder autour de nous. Or, dans les efforts d'une personne qui n'a pas la pratique de la natation, les Tair, et il y a en bras se jettent invariablement en même temps obstination à conserver Le résultat est à la tête sa position verticale ordinaire. l'immersion de la bouche et des narines, et, par suite des efforts pour respirer sous l'eau, l'introduction de dans les poumons. L'estomac en absorbe aussi l'eau une grande quantité, et tout le corps s'appesantit de toute la différence de pesanteur entre l'air qui primi- tivement distendait ces cavités et le liquide qui les remplit maintenant. C'est une règle générale, que cette différence suffît pour faire plonger le corps mais ; elle ne suffît pas dans le cas des individus qui ont de petits os et une quantité anormale de matière flasque et graisseuse. Ceux-là flottent » même après qu'ils sont noyés. Le cadavre, que nous supposerons au fond de la rivière, y restera jusqu'à ce que, d'une manière quel- conque, sa pesanteur spécifique devienne de nouveau moindre que celle du volume d'eau qu'il déplace. Cet effet est amené soit par la décomposition, soit autre- ment. La décomposition a pour résultat la génération du gaz qui distend tous les tissus cellulaires et donne aux cadavres cet aspect bouffi qui est si horrible à voir. Quand cette distension est arrivée à volume du corps est sensiblement ce point que le accru sans un accroissement correspondant de matière solide ou de poids, celle sa pesanteur spécifique de l'eau déplacée, et il devient moindre que fait immédiatement son HISTOIRES EXTRAORDINAIRES. 474 apparition à la surface. Mais la décomposition peut être modifiée par d'innombrables elle circonstances; peut être hâtée ou retardée par d'innombrables agents; par la chaleur ou le froid de la saison, par exemple; par l'imprégnation minérale ou la pureté de l'eau sa plus ou moins grande profondeur ; par le : par courant ou la stagnation plus ou moins marqués; et puis par le tempérament originel du corps, selon qu'il était déjà infecté ou pur de maladie avant la mort. Ainsi il est évident que nous ne pouvons, avec exactitude, fixer une époque où le corps devra s'élever par suite de la décomposition. Dans de certaines conditions, ce résultat peut être amené en une heure; dans d'autres, il peut ne pas avoir lieu du tout. Il y a des infusions chi- miques qui peuvent préserver à tout jamais de corruption tout le système animal, par exemple le bichlorure de mercure. Mais, à part la décomposition, avoir et il y a ordinairement dans l'estomac, par la il peut y une génération de gaz fermentation acétique de la matière végétale (ou par d'autres causes dans d'autres cavités), suffisante pour créer une distension qui ra- mène le corps à la surface de l'eau. L'effet produit par le coup de canon est un effet de simple vibration. Il peut dégager le corps du limon ou de la vase molle où il est enseveli, lui permettant ainsi de s'élever, quand d'autres agents l'y ont déjà préparé; ou bien il peut vaincre f adhérence de quelques parties putréfiées du système cellulaire, et faciliter la distension des cavités sous l'influence du gaz. LE MYSTÈRE DE MARIE ROGET. )) Ayant ainsi devant nous toute sujet, nous (( pouvons vérifier — L'expérience prouve dit la 475 philosophie du les assertions de VÈiolle. cette feuille — que les corps noyés, ou jetés à l'eau immédiatement après une mort violente, ont besoin d'un temps comme de six à dix jours, pour qu'une décomposition suffisante les ramène à la surface des eaux. Un cadavre sur lequel on tire le canon, et qui s'élève avant que l'immersion duré au moins cinq ou six jours, ne manque pas de ait replonger si on l'abandonne à lui-même. » )) Tout le paragraphe nous apparaît maintenant comme un tissu d'inconséquences et d'incohérences. L'expérience ne montre jpas toujours que les corps des noyés ont besoin de cinq ou six jours pour qu'une décomposition suffisante leur permette de revenir à surface. La science et la l'expérience réunies prouvent que l'époque de leur réapparition est et doit être nécessairement indéterminée. En outre, si un corps est ramené à la surface de l'eau par un coup de canon, il ne replongera pas de nouveau, lui-même, toutes les fois que la même abandonné à décomposition sera arrivée au degré nécessaire pour permettre le dégage- ment des gaz engendrés. Mais je désire appeler votre attention sur la distinction faite entre les corps des noyés et les corps des personnes jetées à l'eau immé- diatement après une mort violente. Quoique le rédacteur admette cette distinction, cependant il enferme les deux cas dans la même catégorie. J'ai montré comment le corps d'un homme qui se noie acquiert HISTOIRES EXTRAORDINAIRES. 476 une pesanteur spécifique plus considérable que le volume d'eau 'déplacé, et j'ai prouvé qu'il ne s'enfoncerait pas du tout, sans les mouvements par lesquels il jette ses bras au-dessus de l'eau, et les efforts de res- piration qu'il fait sous Peau, qui permettent au liquide de prendre la place de l'air dans poumons. Mais les ces mouvements et ces efforts n'auront pas lieu dans un corps jeté à Veau immédiatement après une mort violente. Ainsi, est dans ce dernier cas, la règle que le corps ne doit pas du tout s' enfoncer, évidemment. Quand VÉtoile ignore est arrivée à la générale — fait que décomposition un point très-avancé, quand la chair a, en grande partie, quitté les os, alors seulement, mais pas avant, nous voyons le corps disparaître sous l'eau. )) Et maintenant que penserons-nous de ce raisonne- ment, — que le cadavre trouvé ne peut pas être celui de Marie Roget, parce que ce cadavre a été trouvé flottant après un laps de trois jours seulement? Si elle a été noyée, elle a pu ne pas s'enfoncer, étant une femme; si elle s'est enfoncée, elle a pu reparaître au bout de vingt-quatre heures, ou même moins. Mais personne ne suppose qu'elle a été noyée ; et, étant morte avant d'être jetée à la rivière, elle aurait flotté et aurait pu être retrouvée à n'importe quelle époque postérieure. « Mais, — dit V Étoile, — si le corps est resté sur le rivage dans son état de détérioration jusqu'à la nuit de mardi, on a dû trouver sur ce rivage quelque trace des meurtriers. » LE MYSTÈRE DE MARIE ROGET. » Ici il est difficile de saisir tout d'abord l'intention du raisonneur. 11 cherche à prévenir ce qu'il imagine pouvoir être une objection à sa théorie, le corps, — à savoir que étant resté deux jours sur le rivage, subir une s'il 477 décomposition rapide, avait été plongé dans l'eau. 11 a dû — plus rapide que suppose que, si tel a été le cas, le corps aurait pu reparaître à la surface mercredi, le seulement, pense que, et il aurait pu reparaître. pressé de prouver que rivage ; car, dans ces conditions-là le 11 est donc très- corps n'est pas resté sur le dans ce cas, on aurait trouvé sur ce rivage quelque trace des meurtriers. Je présume que cette con- séquence vous fera sourire. Vous ne pouvez pas comprendre comme le séjour plus ou moins long du corps sur le rivage aurait pu multiplier les traces des assassins. » INi moi non plus. Le journal continue improbable que meurtre tel les que : « Et enfin, il est excessivement malfaiteurs celui qui qui ont commis un est supposé, aient jeté le corps à l'eau sans un poids pour l'entraîner, quand il était si facile » de prendre cette précaution. » Observez ici la risible confusion d'idées! Personne, pas même l'Étoile, ne conteste qu'un meurtre a été commis sur le corps trouvé. Les traces de violence sont trop évidentes. Le but de notre raisonneur est simple- ment de montrer que ce corps n'est pas celui de Marie. Il désire prouver que Marie n'est pas assassinée, — mais non pas que ce cadavre n'est pas celui d'une personne assassinée. Cependant, son observation ne prouve que 27. HISTOIRES EXTRAORDINAIRES. 478 ce dernier point. n'avait été Voilà un corps auquel Des assassins, attaché. le aucun poids jetant à l'eau, manqué d'y attacher un poids. Donc, il n'auraient pas n'a pas été jeté par des assassins. Voilà tout ce qui est prouvé, si quelque peut chose l'être. La question d'identité n'est même pas abordée, et l'Étoile est très en peine pour contredire maintenant ce qu'elle admettout à rheure. tait vaincus — dit-elle Nous sommes parfaitement con- a — que le cadavre trouvé est celui d'une femme assassinée, n » Et ce n'est pas le seul cas, même dans cette partie de son sujet, où notre raisonneur raisonne, sans s'en apercevoir, contre lui-même. Son but évident, je l'ai déjà dit, est de réduire, autant que possible, l'intervalle de temps compris entre la disparition de Marie et ]a du corps. Cependant, nous découverte insister sur ce point, depuis le moment où elle mère, Nous n'avons — a a quitté la dit-il voyons maison de sa — aucune déposition prouvant que Marie Roget fût encore sur vivants passé neuf heures, )) le que personne n'a vu la jeune fille la terre des dimanche 22 juin. » Gomme son raisonnement est évidemment entaché de parti pris, il aurait mieux fait d'abandonner ce côté de la question vu Marie, ; car, si l'on trouvait quelqu'un qui eût soit lundi, soit tion serait très-réduit, e.t, mardi, l'intervalle en quesd'après sa manière de rai- sonner, la probabilité que ce corps puisse être celui de lagrisette se trouverait diminuée d'autant. fois Il est toute- amusant d'observer que l'Étoile insiste là-dessus LE MYSTÈRE DE MARIE ROGET. 47'.> avec la ferme conviction qu'elle va renforcer son argu- mentation générale. )) Maintenant examinez de nouveau cette partie de l'argumentation qui a trait à la reconnaissance du corps par Beauvais. Relativement au poil sur le bras, l'Étoile montre évidemment de la mauvaise foi. M. Beauvais, n'étant pas bras. 11 un idiot, n'aurait jamais, pour constater d'un corps, argué simplement de poil sur le l'identité n'y a pas de bras sans poil. La généralité des expressions de VÈtoile est une simple perversion des phrases du témoin. Il a dû nécessairement parler de quelque particularité dans ce la poil ; particularité dans couleur, la quantité, la longueur ou la place. )) Le journal dit Son pied était petit « : ; » il y a des milliers de petits pieds. Sa jarretière n'est pas du tout une preuve, non plus que son soulier; car les jarrese vendent par ballots. tières et les souliers en dire autant des fleurs de son chapeau. Un lequel M. Beauvais insiste On peut fait sur fortement est que l'agrafe de la jarretière avait été reculée pour rendre plus étroite. Gela ne prouve rien ; celle-ci car la plupart des femmes emportent chez elles une paire de jarretières et les accommodent à la grosseur de leurs jambes plutôt que de les essayer dans la boutique où elles les achètent. )> Ici il est difïicile de supposer le raisonneur dans son bon sens. Si M. Beauvais, à la recherche du corps de Marie, a découvert un cadavre ressemblant, par les proportions générales et l'aspect, à la jeune fille dispa- HISTOIRES EXTRAORDINAIRES. 480 (même en laissant de rue, il a pu légitimement croire côté la question de Thabillement) qu'il avait abouti au but de sa recherche. Si, outre ce point de proportions générales et de contour, il a trouvé sur le bras une apparence velue déjà observée sur le bras de Marie vivante, son opinion a pu être justement renforcée, et a dû l'être en proportion de la particularité ou du caractère insolite de cette marque velue. Si, le pied de Marie étant petit, les pieds du cadavre se trouvent éga- lement petits, la probabilité que ce cadavre est celui de Marie doit croître dans une proportion, non pas simplement arithmétique, mais singulièrement géométrique ou accumulative. Ajoutez à tout cela des souliers tels qu'on lui en avait vu porter le jour de sa dispari- tion, et, bien que les souliers se vendent par ballots, vous sentirez la probabilité s'augmenter jusqu'à confiner à la certitude. Ce qui, par soi-même, ne serait pas un signe d'identité, devient, par sa position corroborative, la fleurs preuve la plus sûre. Accordez-nous, enfin, les du chapeau correspondant à celles que portait la jeune fille perdue, et nous n'avons plus rien à désirer. Une seule de ces désirer; fleurs, et nous n'avons plus rien à — mais que dirons-nous donc, avons deux, ou trois, si nous en ou plus encore? Chaque unité successive est un témoignage multiple, — une preuve non pas ajoutée à la preuve précédente, mais multipliée par cent ou par mille. Nous découvrons maintenant sur la défunte des jarretières semblables à celles usait la personne vivante; en vérité, il dont y a presque LE MYSTÈRE DE MARTE ROGET. folie à continuer l'enquête. Mais il 481 se trouve que ces jarretières sont resserrées par le reculement de l'agrafe, juste comme Marie avait fait pour les siennes, peu de temps avant de quitter la maison. Douter encore, c'est démence ou hyprocrisie. Ce que VÉtoile dit relative- ment à ce raccourcissement qui doit, selon elle, comme un cas journalier, ne considéré être prouve pas autre' chose que son opiniâtreté dans l'erreur. La nature élastique d'une jarretière à agrafe suffît pour démontrer le caractère exceptionnel de ce raccourcissement. Ce qui est fait ne pour bien s'ajuster besoin d'un perfectionnement que dans doit avoir des cas rares. Ce doit avoir été par suite d'un accident, dans le sens le plus strict, que ces jarretières de Marie ont eu besoin du raccourcissement en question. Elles seules auraient largement suffi pour établir son identité. Mais l'important n'est pas que le cadavre ait les jarretières de la jeune fille perdue, ou ses souliers, ou son chapeau, ou les fleurs de son chapeau, ou ses pieds, ou son aspect et ses proportions générales; est que le — l'important cadavre a chacune de ces choses, et les a prouvé que l'Étoile a réellement, dans de pareilles circonstances, conçu un toutes doute, collectivement. il S'il était n'y aurait, pour son cas, aucun besoin d'une commission de lunatico inquirendo. Elle a cru faire preuve de sagacité en se faisant l'écho des bavardages des hommes de loi, qui, pour la plupart, se contentent de se faire eux-mêmes l'écho des préceptes rectangulaires des cours criminelles. Je vous ferai observer. HISTOIRES EXTRAORDINAIRES. 482 en passant, que beaucoup de ce qu'une cour refuse d'admettre comme preuve est pour l'intelligence ce qu'il y a de meilleur en fait de preuves. Car, se gui- dant d'après les principes généraux en matière de preuves, les principes reconnus et inscrits dans les livres, la cour répugne à dévier vers les raisons parti- culières. Et cet attachement opiniâtre au principe, avec ce dédain rigoureux pour l'exception contradictoire, est un moyen sûr d'atteindre, dans une longue suite de temps, le maximum de vérité auquel il est permis d'atteindre ; la pratique, en masse, est donc philosophique; mais il n'est pas moins certain qu'elle engendre de grandes erreurs dans des cas spéciaux ^ » 0"ant aux insinuations dirigées contre Beauvais, vous n'aurez qu'à souffler dessus pour les dissiper. Vous avez déjà pénétré le véritable caractère de ce gentle- man. C'est un officieux, avec un esprit très-tourné au romanesque et peu de jugement. Tout homme ainsi constitué sera facilement porté, dans un cas d'émotion réeUe; à se conduire de manière à se rendre suspect 1. Une théorie basée sur les qualités d'un objet ne peut pas avoir le développement total demandé par tous les objets auxquels elle doit s'appliquer ; et celui qui arrange des faits par rapport à leurs causes perd la faculté de les estimer selon leurs résultats. Ainsi la jurisprudence de toutes les nations montre que la loi, quand elle devient une science ou un système, cesse d'être la justice. Les erreurs, dans lesquelles une dévotion aveugle aux principes de classification a jeté le droit commun, sont faciles à vérifier si l'on veut observer combien de fois la puissance législative a été obligée d'intervenir pour rétablir l'esprit d'équité qui avait disparu de ses formules. — L and or. LE MYSTÈRE DE MARIE ROGET. 483 aux yeux des personnes trop subtiles ou enclines à la malveillance. M. Beauvais, comme résulte de il vos notes, a eu quelques entrevues personnelles avec l'édi- teur de VÈloile, et il l'a choqué en osant exprimer cette opinion, que, nonobstant la théorie de l'éditeur, le ca- davre était — dit le positivement celui de Marie. journal de Marie, mais — à affirmer que il ne peut pas le (( Il persiste corps est celui ajouter une circon- stance à celles que nous avons déjà commentées, pour faire partager aux autres cette croyance. » Or, sans revenir sur ce point, qu'il eût été impossible, pour faire 'partager aux autres celte croyance, de fournir une preuve plus forte que celles déjà connues, observons ceci: de concevoir un homme parfaite- c'est qu'il est facile ment convaincu, dans un cas de cette espèce, et cependant incapable de produire une seule raison pour convaincre une seconde personne. Rien n'est plus va- gue que les impressions relatives à l'identité d'un individu. Chaque homme reconnaît son voisin, et pourtant il y a bien peu de cas où prêt premier venu sera tout le à donner une raison de reconnaissance. cette L'éditeur de l'Étoile n'a donc pas le droit d'être choqué de la croyance non raisonnée de M. Beauvais. » Les circonstances suspectes dont il est enveloppé cadrent bien mieux avec mon hypothèse d'un caractère officieux, tatillon et romanesque, qu'avec l'insinuation du journaliste relative à sa culpabilité. L'interprétation plus charitable étant adoptée, cune peine à expliquer la nous n'avons plus au- rose dans le trou de la ser- H1SÏ0IP.KS EXTRAORDINAIUES. 484 rare; le mot Marie sur l'ardoise; le fait d'écarter les parents mâles; sa répugnance à leur laisser voir le corps ; la recommandation faite à avec le madame B. de ne pas causer gendarme jusqu'à ce qu'il fût de retour, lui, Beauvais; et enfin cette résolution apparente de ne permettre à personne autre que lui-même de l'enquête. 11 se mêler de me semble incontestable que Beauvais était un des adorateurs de Marie; qu'elle a fait la coquette avec lui ; et qu'il aspirait à faire croire qu'il jouissait de sa confiance et de son intimité complète. Je ne dirai rien de plus sur ce point; et comme l'évidence repousse complètement l'assertion de l'Étoile relativement à cette apathie dont il accuse la mère et les autres parents, apathie qui est inconciliable avec cette qu'ils croient à l'identité du corps de la supposition jeune parfu- meuse, nous procéderons maintenant comme si la question d'identité était établie à notre parfaite satisfaction. — Et que pensez-vous — demandai-je alors — des opinions du Commercial? — Que, par leur nature, elles sont beaucoup plus di- gnes d'attention qu'aucune de celles qui ont été lancées sur le même sujet. Les déductions des prémisses sont philosophiques et subtiles; mais ces prémisses, en deux points au moins, sont basées sur une observation imparfaite. Le Commercial veut faire entendre que Marie a été prise par une bande de vils coquins- non loin de la porte de la maison de sa mère, u possible — dit-il II est im- — qu'une jeune femme connue, comme était Marie, de plusieurs milliers de personnes. LE M\ STÈRE DE MARIE ROGET. ait 485 pu passer trois bornes sans rencontrer quelqu'un à qui son visage fCit familier... n C'est là l'idée d'un homme résidant depuis longtemps dans Paris, — d'un homme public, — dont les allées et les venues dans la ville ont été presque toujours administrations publiques. limitées au voisinage des 11 que sait lai, il va rare- ment à une douzaine de bornes au delà de son propre bureau sans être reconnu et accosté. Et, mesurant l'étendue de la connaissance qu'il a des autres et que les autres ont de lui-même, avec celle de la il compare sa notoriété parfumeuse, ne trouve pas grande différence entre les deux, et arrive tout de suite à cette conclusion qu'elle devait être, dans ses courses, aussi exposée à être reconnue que lui dans les siennes. Cette conclusion ne pourrait être légitime que si ses courses, à elle, avaient été de la même nature méthodique, et confinées dans région que ses courses, à lui. tervalles réguliers, Il la invariable et même espèce de va et vient, à des in- dans une périphérie bornée, rem- plie d'individus que leurs occupations, analogues aux siennes, poussent naturellement à s'intéresser à lui et à observer peuvent sa être, personne. Mais les courses de Marie en général, supposées d'une nature va- gabonde. Dans le cas particulier qui nous occupe, on doit considérer une ligne s' comme très-probable qu'elle a suivi écartant plus qu'à l'ordinaire de ses che- mins accoutumés. Le parallèle que nous avons supposé exister dans l'esprit du Commercial ne serait soutena- ble que dans le cas des deux individus traversant toute HISTOIRES EXTRAORDINAIRES. 486 la ville. Dans ce cas, s'il est accordé que les relations per- sonnelles soient égales, les chances aussi seront égales pour qu'ils rencontrent un nombre égal de connaissances. Pour ma part, je tiens qu'il est, non-seulement possible, mais infiniment probable que Marie a suivi, une quelconque des nom- à n'importe quelle heure, breuses routes conduisant de sa résidence à celle de sa tante, sans rencontrer un seul individu qu'elle con- nût ou de qui elle fût connue. Pour bien juger cette question, pour la juger dans son vrai jour, il nous faut bien penser à Pimmense disproportion qui existe entre les connaissances personnelles de Pindividu le plus répandu de Paris et la population de Paris tout entière. » que paraisse garder encore Mais, quelque force l'insinuation du Commercial, elle sera bien diminuée, si nous prenons en considération l'heure à laquelle jeune fille est sortie, — C'est u dit le Commercial la — au moment où les rues sont pleines de monde, qu'elle est sortie de chez elle.-» Mais pas du tout! 11 était neuf heures du matin. Or, à neuf heures du matin, toute la semaine, excepté le ville sont, il est vrai, remplies le s' dimanche, les rues de la de foule. A neuf heures, dimanche, tout le monde est généralement chez soi, apprêtant pour aller à l'église. Il n'est pas d'homme un peu observateur qui n'ait remarqué l'air particuliè- rement désert de la ville de huit heures à dix heures, chaque dimanche matin. Entre dix et onze, les rues sont pleines de foule, mais jamais à une heure auss^ matinale que celle désignée. LE MYSTERE DE MARIE ROGET. » Il y a un autre point où d'observation ait morceau — dit-il fait défaut 487 il semble que au Commercial. l'esprit Un « — d'un des jupons de l'infortunée jeune fille, de deux pieds de long et d'un pied de large, avait été arraché, serré autour de son cou et noué der- pour empêcher ses rière sa tête, probablement cris. même Cela a été fait par des drôles qui n'avaient pas un mouchoir de poche. » Cette idée est fondée ou ne l'est pas, c'est ce que nous essayerons plus tard d'exa- miner; mais, par ces mots : des drôles qui n'ont pas un mouchoir de poche, l'éditeur veut désigner la classe de brigands la plus vile. Cependant, ceux-là sont justement l'espèce de gens qui ont toujours des mouchoirs, même quand ils manquent de chemise. Vous avez eu occasion d'observer combien, depuis ces mouchoir de poche est dernières années, le devenu indispensable pour le parfait coquin. — Et que devons-nous penser — demandai-je — de l'article du Soleil ? — Que soit c'est grand dommage que son rédacteur ne pas né perroquet, illustre auquel cas il eût été perroquet de sa race. Il le plus a simplement répété des fragments des opinions individuelles déjà expri- mées, qu'il a ramassés, avec une louable industrie, dans tel ou tel autre journal. « Les objets — dit-il sont évidemment restés là pendant trois ou quatre se- maines au moins, et l'on ne peut pas douter que le théâtre de cet effroyable crime n'ait été enfin découvert. » Les faits énoncés ici de nouveau par le Soleil ne sufîi- HISTOIRES EXTRAORDINAIRES. 488 sent pas du tout pour écarter mes propres doutes sur ce sujet, et nous aurons à les examiner plus particu- lièrement dans leurs rapports avec une autre partie de la question. A présent, il faut nous occuper d'autres investiga- )) Vous n'avez pas manqué d'observer une extrême tions. négligence dans l'examen du cadavre. A coup sûr, la question d'identité a été facilement résolue, ou devait l'être; mais il y avait d'autres points à vérifier. Le corps avait-il été, de façon que]conque, dépouillé f La défunte avait-elle sur elle quelques articles de bijouterie quand elle a quitté la maison? Si elle en avait, les a-t-on re- trouvés sur le corps? Ce sont des questions importantes, absolument négligées par l'enquête, d'autres d'une valeur égale qui attiré l'attention. n'ont et il y en a aucunement Nous tâcherons de nous satisfaire par une enquête personnelle. La cause de Saint-Eustache a besoin d'être examinée de nouveau. soupçons contre cet individu ; Je n'ai pas de mais procédons métho- diquement. Nous vérifierons scrupuleusement la validité des attestations relatives aux lieux où on l'a vu le dimanche. Ces sortes de témoignages écrits sont souvent des moyens de mystification. Si nous n'y trouvons rien à redire, nous mettrons Saint-Eustache hors de cause. Son suicide, bien qu'il soit propre à corroborer les soupçons, au cas où on trouverait une supercherie dans les cherie, affidavit, n'est pas, s'il n'y a aucune super- une circonstance inexplicable, ou qui doive nous faire dévier de la ligne de l'analyse ordinaire. LE MYSTERE DE MARIE ROGET. )) la marche Dans 489 que je vous propose maintenant, nous écarterons les points intérieurs du drame et nous concentrerons notre attention sur son contour extérieur. Dans des investigations du genre de celle-ci, on commet assez fréquemment cette erreur, de limiter l'enquête aux faits immédiats et de mépriser absolu- ment les faits collatéraux ou accessoires. routine des testable cours criminelles C'est la dé- de confiner l'instruction et la discussion dans le domaine du relatif apparent. Cependant, l'expérience a prouvé, et une vraie philosophie prouvera toujours qu'une vaste partie de la vérité, la plus considérable peut-être, jaillit des élé- ments en apparence éirangers à l'esprit, si principe , la question. C'est par ce n'est précisément par la lettre de ce que moderne est parvenue à calculer sur l'imprévu. Mais peut-être ne me compre- la science nez-vous pas? L'histoire de la science humaine nous montre d'une manière si continue que c'est aux faits collatéraux, fortuits, accidentels, que nous devons nos plus nombreuses et nos plus précieuses découvertes, qu'il est devenu finalement nécessaire, dans tout aperçu des progrès à venir, de faire une part non- seu- lement très-large, mais la plus large possible aux inventions qui naîtront du hasard, et qui sont tout à fait en dehors des prévisions ordinaires. Il n'est plus philosophique désormais de baser sur ce qui a été une vision de ce qui doit être. V accident doit être admis comme partie de la fondation. Nous faisons du hasard la matière d'un calcul rigoureux. Nous soumettons HISTOIRES EXTRAORDINAIRES. 490 rinattendu et l'inconcevable aux formules mathémati- ques des écoles. » C'est, je le repète, un fait positif que la meilleure partie de la vérité est née de l'accessoire, de Tindirect; et c'est simplement en impliqué dans ce fait, me conformant au que je voudrais, principe dans le cas présent, détourner l'instruction du terrain battu et in- fructueux de l'événement même pour la porter vers les circonstances contemporaines dont il est entouré. Pen- dant que vous vérifierez la validité des affulavit, j'examinerai les journaux d'une manière plus générale que vous n'avez fait. Jusqu'ici, nous n'avons fait que reconnaître le champ de l'investigation mais il serait qu'un examen compréhensif des ; vraiment étrange feuilles publiques, tel que je veux le faire, ne nous apportât pas quelques petits renseignements qui serviraient à donner une direction nouvelle à l'instruction. Conformément à l'idée de Dupin, je me mis à vérifier scrupuleusement les affulavit. Le résultat de mon examen fut une ferme conviction de leur validité et conséquemment de l'innocence de Saint-Eustache. En même temps, mon ami s'appliquait, avec une minutie qui me paraissait absolument superflue, à examiner les collections des divers journaux. Au bout d'une se- maine, il mit sous mes yeux les extraits suivants (( Il : y a trois ans et demi environ, une émotion sem- blable fut causée par la disparition de la même Marie Roget, de la parfumerie de M. Le Blanc, au PalaisRoyal. Cependant, au bout d'une semaine, elle reparut LE MYSTÈRE DE MARIE ROGET. 491 que à son comptoir ordinaire, l'air aussi bien portant possible, sauf une légère pâleur qui ne habituelle. Sa mère et M. était allée la lui était pas Le Blanc déclarèrent qu'elle simplement rendre visite à quelque ami à campagne, et l'affaire fut promptement assoupie. Nous présumons que son absence actuelle frasque de une est même nature, et qu'à l'expiration d'une semaine ou d'un mois nous la verrons revenir parmi nous. » Journal du soir. — Lundi, 23 juin*. Un journal du soir, dans son numéro d'hier, rap- (( pelle une première disparition mystérieuse de made- moiselle Roget. C'est chose connue que, pendant son absence d'une semaine de la parfumerie Le Blanc, elle était en compagnie d'un jeune officier de marine, noté pour ses goûts de débauche. Une brouille, à ce qu'on suppose, la poussa providentiellement à revenir chez Nous savons le nom du Lothario en question, qui elle. est actuellement en congé à Paris; mais, pour des rai- sons qui sautent aux yeux, nous nous abstenons de publier. » le — Le Mercure. — Mardi matin, 2k juin^. « Un attentat du caractère le plus odieux a été commis aux environs de cette ville dans la journée d'avant-hier. Un gentleman, avec sa femme et sa fille, à la tombée de la nuit, a loué, pour traverser la ri- vière, les services de six jeunes gens qui manœuvraient un bateau çàet là, près de la berge de la Seine. Arrivés à la rive opposée, les trois passagers mirent pied à 1. 2. New-York Express. New-York Herald. . HlSTOJRt:S EXTRAORDIIS AI R KS. 492 s'étaient éloignés déjà du bateau jusqu'à terre, et ils le perdre de vue, quand la jeune fille s'aperçut qu'elle y avait laissé son ombrelle. Elle revint pour la cher- cher, fut saisie par cette bande d'hommes, transportée sur le fleuve, bâillonnée, affreusement maltraitée, et finalement déposée sur un point de la rive peu distant de celui où elle était primitivement montée dans le bateau avec ses parents. Les pour le moment à la police ont échappé misérables ; mais elle est sur leur piste, et quelques-uns d'entre eux seront prochaine- ment arrêtés. (( )) — Journal du matin. — 25 juin * Nous avons reçu une ou deux communications qui ont pour objet d'imputer à Mennais^ le crime odieux commis récemment; mais, comme ce gentleman a été pleinement disculpé par une enquête judiciaire, et comme les arguments de nos correspondants semblent marqués de plus de zèle que de sagacité, nous ne jugeons pas convenable de les publier. » — Journal du matin. 28 juin ^. (( Nous avons reçu plusieurs communications assez énergiquement écrites, qui semblent venir de sources diverses et qui poussent à accepter, comme chose certaine, que l'infortunée Marie Roget a été victime d'une de ces nombreuses bandes de coquins qui infestent, le 1. New-York Courier and Inquirer. Mennais était un des individus primitivement soupçonnés et arrêtés; plus tard, il avait été relaclié par suite du manque total 2. de preuves. 3. New-York Courier and Inquirer. LE MYSTERE DE MARIE ROGET. dimanche, les environs de la ville. 493 Notre propre opi- nion est décidément en faveur de cette hypothèse. Nous tâcherons prochainement d'exposer ici quelquesuns de ces argumenis. » — Journal du soir. — Mardi, 31 juin *. Lundi, un des bateliers attachés au service du lise (( a vu sur la Seine un bateau vide s'en allant avec le courant. Les voiles étaient déposées au fond du bateau. Le batelier le remorqua jusqu'au bureau de la navigation. Le matin suivant, ce bateau avait été détaché et avait disparu sans qu'aucun des employés s'en fût aperçu. Le gouvernail est resté au bureau de la navigation. En )) — La Diligence. — Jeudi, 26 juin lisant ces différents extraits, ^. non-seulement il me sembla qu'ils étaient étrangers à la question, mais ne pouvais concevoir aucun moyen de les y ratta- je cher. J'attendais une explication quelconque deDupin. — — n'entre pas actuellement dans Il dit-il — de m'appesantir sur de ces extraits. Je mon intention le premier et le second les ai copiés principalement pour vous montrer l'extrême négligence des agents de la police, qui, si j'en dois croire le préfet, ne se sont pas inquiétés le moins du monde de l'officier de marine auquel il est fait allusion. Cependant, il y aurait de la folie à affirmer que nous n'avons pas le droit de sup- poser une connexion entre la première disparition de Marie. 1. 2. et la seconde Admettons que la première fuite New-York Evening Post. New- York Standar. 28 HISTOIRES EXTRAORDINAIRES. 494 ait eu pour résultat une brouille entre les deux amants et le retour de la jeune fille trahie. Nous pouvons con- sidérer un second enlèvement (si nous savons qu'un second enlèvement a eu lieu) comme indice de nouvelles tentatives de part la du traître, plutôt que comme résultat de nouvelles propositions de la part d'un second individu nous pouvons regarder cette ; deuxième fuite plutôt comme le raccommodage du vieil amour que comme le commencement d'un nouveau. Ou celui qui s'est déjà enfui une fois avec Marie lui aura proposé une évasion nouvelle, ou Marie, à qui des propositions d'enlèvement ont été faites par un individu, en aura agréé de la part d'un autre ; mais il y a dix chances contre une pour suppositions permettez-moi d'attirer votre ! Et, ici, la première de ces attention sur ce fait, que le temps écoulé entre le pre- mier enlèvement connu et le second supposé ne dé- passe que de peu de mois la durée ordinaire des croisières de nos vaisseaux de guerre. L'amant a-t-il été interrompu dans sa première infamie par la nécessité de reprendre la mer, et a-t-il saisi le premier moment de son retour pour renouveler les viles tentatives non absolument accomplies jusque-là, ou du moins non absolument accomplies par lui? Sur toutes ces choses, nous ne savons rien. )) Vous direz peut-être que, dans le second cas, l'en- lèvement que nous imaginons n'a pas eu lieu. Certai- nement non mais pouvons-nous afiirmer qu'il n'y a pas ; eu une tentative manquée? En dehors de Saint-Eus- LE MYSTERE DE MARIE ROGET- 495 tache et peut-être de Beauvais, nous ne trouvons pas d'amants de Marie, reconnus, déclarés, honorables. Il été parlé n'a d'aucun autre. Quel est donc l'amant secret dont les parents (au moins pour la plupart) n'ont jamais entendu parler, mais que Marie rencontre dimanche matin, et qui est entré si profondément le dans sa confiance, qu'elle n'hésite pas à rester avec lui, jusqu'à ce que les ombres du soir descendent, dans les bosquets solitaires de la barrière du Roule ? Quel est, dis-je, cet amant secret dont la plupart, au moins, des n'ont jamais parents, sipfnifient ces singulières entendu parler? Et que paroles de madame Roget, le matin du départ de Marie : « Je crains de ne plus jamais revoir Marie » ? » Mais, nous ne pouvons pas supposer que ma- si dame Roget ait eu connaissance du projet de fuite, ne pouvons-nous pas au moins imaginer que ce projet ait été conçu par la fille? En quittant la maison, elle a donné à entendre qu'elle allait rendre visite à sa tante, rue des Drômes, et Saint-Eustache a été chargé de venir la chercher à la tombée de la nuit. Or, au premier coupd'œil, ce fait milite fortement contre ma suggestion; mais réfléchissons un peu. Qu'elle ait posi- tivement rencontré quelque compagnon, qu'elle traversé avec ait lui la rivière et qu'elle soit arrivée à la barrière du Roule à une heure assez avancée, appro- chant trois heures de faprès-midi, cela est connu. Mais, en consentant à accompagner ainsi cet individu {dans un dessein quelconque, connu ou inconnu de sa HISTOIRES EXTRAORDINAIRES. 496 mhre), elle a dû penser à l'intention qu'elle avait ex- primée en quittant la maison, ainsi qu'à la surprise et aux soupçons qui s'élèveraient dans fiancé, Saint-Eustache, cœur de son le quand, venant chercher à la l'heure marquée, rue des Drômes, il apprendrait qu'elle n'y était pas venue, et quand, de plus, retournant à la pension avec ce renseignement alarmant, il s'aperce- de son absence prolongée de vrait dCi, dis-je, penser à la maison. Elle a tout cela. Elle a dû prévoir le cha- grin de Saint-Eustache, les soupçons de tous ses amis. Il se peut qu'elle n'ait pas eu le courage de revenir pour braver les soupçons; mais les soupçons n'étaient plus qu'une question d'une importance insignifiante pour elle, si nous supposons qu'elle avait l'intention de ne pas revenir. » Nous pouvons imaginer qu'elle (( J'ai rendez-vous avec une certaine personne dans a raisonné ainsi : un but de fuite, ou pour certains autres projets connus de moi seule. pris; il faut Il faut écarter toute chance d'être sur- que nous ayons suffisamment de temps pour déjouer toute poursuite; je donnerai à entendre que je vais rendre visite à ma tante et passer la journée chez elle, rue des Drômes. Je dirai tache de ne venir me chercher qu'à façon, mon absence de la maison, la à Saint-Eus- nuit; de cette prolongée autant que possible, sans exciter de soupçons ni d'inquiétude, pourra s'expliquer, et je gagnerai plus de temps que par tout autre moyen. Si je prie Saint-Eustache de venir me chercher à la brune, il ne viendra certaine- LE MYSTÈRE DE MARIE ROGET. 497 ment pas auparavant; mais, si je néglige tout à fait de le prier de venir, le temps consacré à ma fuite sera diminué, puisque l'on s'attendra à me voir revenir de bonne heure, et que mon absence excitera plus tôt mon dessein l'inquiétude. Or, s'il pouvait entrer dans de reventi% si je n'avais en vue qu'une simple prome- nade avec la personne en question, il ne serait pas de bonne politique de prier Saint-Eustache de venir me chercher; car, en arrivant, il s'apercevrait à coup sûr que je me suis jouée de lui, chose que je pourrais lui cacher à jamais en quittant la maison sans lui notifier mon intention, en revenant avant la nuit et en racontant alors que je suis allée visiter ma tante, rue des Drômes. Mais, comme mon projet est de ne jamais revenir, — du moins avant quelques semaines ou avant — né- que j'aie réussi à cacher certaines choses, la cessité de gagner du temps est le seul point dont j'aie à m'inquiéter. » Vous avez observé, dans vos notes, que l'opinion )) générale, relativement à cette triste affaire, est et a dès été, le principe, que la jeune fille a été victime d'une bande de brigands. Or, l'opinion populaire, dans de certaines conditions, n'est pas faite pour être dé- daignée. Quand elle se lève d'elle-même, quand elle se manifeste d'une manière strictement spontanée, nous devons la considérer comme un phénomène analogue à cette intuition qui est l'idiosyncrase de l'homme de génie. Dans quatre-vingt dix-neuf cas sur cent, je m'en tiendrais à ses décisions. Mais il est très-impor28. HISTOIRES EXTRAORDINAIRES. 498 tant que nous ne découvrions pas de traces palpables d'une suggestion extérieure. L'opinion doit être rigou- reusement la pensée personnelle du public; et il est souvent très-difficile de saisir cette distinction et de la maintenir. Dans cas présent, il me semble, à moi, le que cette opinion publique, relative à une baitde, a été inspirée par l'événement parallèle et accessoire raconté dans le troisième de mes extraits. Tout Paris est excité par la découverte du cadavre de Marie, une fille jeune, belle et célèbre. Ce cadavre est trouvé portant des marques de violence et flottant sur la rivière. Mais il est maintenant avéré qu'à l'époque même ou vers l'é- poque où l'on suppose que la jeune fille a été assassinée, un attentat analogue à celui enduré par la défunte, quoique moins énorme, a été consommé, par une bande de jeunes drôles, sur une autre jeune lille. Kst-il surprenant que le premier attentat connu ait influencé le jugement populaire relativement à l'autre encore obscur? Ce jugement attendait une direction, et fattentat connu semblait l'indiquer avec tant d'op- portunité! Marie, elle aussi, rivière; et c'est sur cette a été trouvée dans la même rivière que l'attentat connu a été consommé. La connexion des deux événements avait en elle quelque chose de si palpable, que c'eût été un miracle que le populaire oubliât de l'ap- précier et de la saisir. Mais, en fait, l'un des deux attentats, connu pour avoir été accompli de telle façon, est un indice, s'il en fut jamais, que l'autre attentat, commis à une époque presque coïncidente, n'a pas été LE MYSTÈRE DE MARIE ROGET. 499 accompli de la même façon. En vérité, on pourrait regarder comme une merveille que, pendant qu'une bande de scélérats consommait, en un lieu donné, un attentat inouï, il se soit trouvé blable, dans la une autre bande sem- même localité, dans la même ville, dans les mêmes circonstances, occupée, avec les mêmes moyens et les mêmes procédés, à commettre un crime d'an caractère exactement semblable et précisément à la même époque ! Et à quoi, je vous prie, l'opinion accidentellement suggérée du populaire nous pousseraitelle à croire, si ce n'est à cette merveilleuse série de coïncidences ? )) Avant d'aller plus loin, considérons le théâtre sup- posé de l'assassinat dans le fourré de la barrière du Roule. Ce bosquet, très-épais, il est vrai, se trouve dans l'extrême voisinage d'une route publique. Dedans, nous dit-on, se trouvent trois ou quatre larges pierres, formant une espèce de siège, avec dossier et tabouret. Sur la pierre supérieure on a découvert un jupon blanc; sur la seconde, une écharpe de soie. Une ombrelle, des gants et un mouchoir de poche ont été également trouvés. Le mouchoir portait le nom : Marie Roget. Des fragfuents de robe étaient attachés aux ronces envi- ronnantes. La terre était piétinée, les buissons enfoncés, et il y avait là toutes les traces d'une lutte vio- lente. )) Malgré l'acclamation dont la presse a salué la dé- couverte de ce fourré, et l'unanimité avec laquelle on a supposé qu'il représentait le théâtre précis du crime, HISTOIRES EXTRAORDINAIRES. 500 il y avait plus d'une bonne raison faut admettre qu'il pour en douter. Si le véritable théâtre avait été, comme l'insinue le Commercial, dans le voisinage de la rue du crime, que nous Pavée-Saint-André, les auteurs supposerons demeurant encore à Paris, auraient naturellement été frappés de terreur par l'attention publi- que, si vivement poussée dans la vraie voie; et tout esprit d'une certaine classe aurait senti tout de suite la nécessité de faire une tentative quelconque pour distraire cette attention. Ainsi, le fourré de la barrière du Roule ayant déjà attiré les soupçons, l'idée de placer les objets en question là où ils ont été trouvés a pu être inspirée très-naturellement. 11 n'y a pas de preuve réelle, quoi qu'en dise le Soleil, que les objets retrouvés soient restés dans petit nombre le fourré plus d'un très- de jours; pendant qu'il est plus que pré- su niable qu'ils n'auraient pas pu rester là, sans attirer durant l'attention, les vingt jours écoulés entre le dimanche fatal et l'après-midi dans laquelle ils ont été découverts par les petits garçons. « Ils tements moisis par l'action de la pluie, tirant cette opinion lui, — étaient complé- — dit le Soleil, de journaux qui ont pailé avant et collés ensemble par la moisissure. avait poussé tout autour et Le gazon même les recouvrait par- tiellement. La soie de l'ombrelle était solide; mais les branches en rieure, là avaient été refermées 011 l'étoffe était la partie supé- double et rempliée, étant ; toute moisie et pourrie par l'humidité, se déchira aussitôt qu'on l'ouvrit. » Relativement au gazon, ayant LE MYSTERE DE MARIE ROGET. poussé tout autour et tiellement, il 501 même recouvrant les objets par- est évident que le fait ne peut avoir été constaté que d'après les dires résultant eux-mêmes des souvenirs des deux petits garçons; car ces enfants enlevèrent les objets et les portèrent à la maison avant qu'ils eussent été vus par une troisième personne. Mais le gazon croît, particulièrement dans une température chaude et humide (comme celle qui régnait à l'époque du meurtre), d'une hauteur de deux ou trois pouces en un jour. Une ombrelle posée sur un terrain récemment gazonné peut, en une seule semaine être complètement cachée par l'herbe soudainement grandie. Et quant à cette moisissure, du Soleil insiste le si sur laquelle l'éditeur opiniâtrement, qu'il n'emploie pas mot moins de trois fois dans le très-court paragraphe cité, ignore-t-il réellement la nature de cette moisis- sure? Faut-il lui apprendre que c'est une de ces nom- breuses classes de fungus, dont le caractère le plus ordinaire est de croître et de mourir en vingt-quatre heures? )) Ainsi nous voyons, au premier coup d'œil, que ce qui avait été si pompeusement allégué pour soutenir cette idée, que les objets étaient restés dans le bosquet pendant trois ou quatre semaines au moins, est abso- lument nul, en tant que preuve quelconque de ce fait. D'autre part, il est excessivement difficile de croire que ces objets aient pu rester dans le fourré en question pendant plus d'une semaine, pendant un intervalle plus long que celui d'un dimanche à l'autre. Ceux qui HISTOIRES EXTRAORDINAIRES. 502 connaissent un peu les alentours de Paris savent l'ex- trême difficulté d'y trouver la retraite, excepté à une grande distance des faubourgs. Un recoin inexploré ou même rarement visité, dans ces bois et ces bosquets» est une chose Qn'un véritable amant insupposable. quelconque de la nature, condamné par son devoir à la poussière et à la chaleur de cette grande métropole, essaye, même pendant les jours ouvrables, d'étancher sa soif de solitude parmi ces décors de beauté natu- relle et champêtre qui nous entourent. Avant qu'il ait pu faire deux pas, sentira l'enchantement naissant il rompu par la voix ou l'irruption personnelle de quelque goujat ou d'une bande de drôles en ribote. 11 cherchera le silence sous les ombrages les plus épais, mais toujours en vain. C'est précisément dans ces coins-là qu'abonde la crapule les plus profanés. ; ce sont là les temples Le cœur navré de dégoût, le prome- neur retournera en hâte vers Paris, comme vers un cloaque d'impureté moins grossière et conséquemment moins odieuse. Mais, si les environs de la ville sont ainsi infestés pendant les jours de la semaine, combien plus encore le sont-ils le dimanche! C'est surtout alors que, délivré des liens du travail ou privé des occasions ordinaires favorables au crime, le goujat de la ville se répand vers les environs, non par amour de la nature champêtre, qu'il méprise de tout son cœur, mais pour échapper aux gênes n'est pas l'air et. aux frais et les conventions sociales. Ce arbres verts qu'il désire, mais l'absolue licence de la campagne. Là, dans l'au- LE MYSTÈRE DE MARIE ROGET. berge, au bord de la route, ou sous bois, n'étant plus 503 l'ombrage des contenu par d'autres regards que ceux de ses dignes compagnons, furieux d'une gaieté mensongère, il se livre aux excès fille de la liberté et du rhum. Je n'avance rien de plus que ce qui sautera aux yeux de tout observateur impartial, quand je répète que le fait de ces objets restant non découverts pendant une période plus longue que d'un dimanche à l'autre, dans un bosquet quelconque des environs de Paris, doit être considéré presque comme un miracle. » Mais les motifs ne nous manquent pas qui nous font soupçonner que les objets ont été placés dans ce fourré dans le but de détourner l'attention du véritable théâtre du crime. Et d'abord, permettez-moi de vous faire remarquer la date de cette découverte. Rapprochez-la de date du cinquième la dans la revue des journaux que j'ai mes extraits, de faite moi-même. Vous verrez que la découverte a suivi, presque immédiatement, les communications urgentes envoyées au journal du soir. Ces communications, quoique variées, et provenant en apparence de sources diverses, ten- même but, — lequel daient toutes au l'attention sur une bande de malfaiteurs était d'attirer comme au- teurs de l'attentat, et sur les alentours de la barrière du Roule comme théâtre du fait. Or, ce qui peut nous étonner, ce n'est pas, naturellement, que les objets aient été trouvés par les petits garçons, à la suite de ces communications et après que l'attention publique a été dirigée de ce côté; mais on pourrait légitimement HISTOIRES EXTRAORDINAIRES. 504 supposer que, si les enfants n'ont pas trouvé les objets pkis tôt, c'est parce que lesdits objets n'étaient pas encore dans le fourré; parce qu'ils y ont été déposés à une époque tardive, — celle de ou une de très- la date, peu antérieure à la date de ces communications, les coupables eux-mêmes, auteurs — par de ces communica- tions. » Ce bosquet était un singulier bosquet, ment singulier. Il était d'une rare l'enceinte de ses murailles naturelles, — excessive- épaisseur. il Dans y avait trois pierres extraordinaires, formant un siège avec dossier et Et ce bosquet, où la nature imitait si bien tabouret. l'art, était dans l'extrême voisinage, à quelques verges, de l'habitation de madame Deluc, de qui les enfants avaient coutume de fouiller soigneusement les fourrés pour récolter de l'écorce de sassafras. raire de parier Serait-il témé- — mille contre un — qu'il ne s'écoulait pas une journée sans qu'un, au moins, de ces petits garçons vînt se cacher dans cette salle de verdure et trôner sur ce trône naturel? Ceux qui hésiteraient à parier, ou n'ont jamais été enfants, ou ont oublié la nature enfantine. Je difficile le de comprendre repète, il est excessivement comment les objets auraient pu, sans être découverts, rester dans ce bosquet plus d'un ou deux jours; et il y a ainsi de bonnes raisons de soupçonner, en dépit de du Soleil, qu'ils ont la dogmatique ignorance été déposés, à une date relative- ment tardive, là où on les a trouvés. » iMais, pour croire que la chose s'est passée ainsi, il LE MYSTERE DE MARIE ROGET. y a encore d'autres raisons, plus fortes celles que je vous ai présentées. 505 qu'aucune de Laissez-moi mainte- nant attirer votre attention sur l'arrangement remar- quablement artificiel des objets. Sur la pierre supé- rieure se trouvait un jupon blanc; sur la seconde, une écharpe de soie; éparpillés alentour, une ombrelle, des gants et un mouchoir de poche xMarie. C'est justement là marqué du nom de un arrangement qu'a dû tel naturellement l'imaginer un esprit peu subtil, visant à trouver un arrangement naturel. Mais ce n'est pas du tout un arrangement réellement naturel. J'aurais mieux aimé voir les choses gisant toutes à terre, et foulées sous les pieds. Dans l'étroite enceinte de ce bosquet, il eût été presque impossible que le jupon et l'écharpe gardassent leur position sur les pierres, exposés aux secousses résultant d'une lutte entre plusieurs personnes. « Il y avait était — dit-on — trace d'une lutte ; la terre piétinée; les buissons étaient enfoncés; » le jupon et l'écharpe des planches. « mais sont trouvés reposant comme sur Les fragments de vêtements accrochés aux buissons étaient larges de trois pouces environ, et longs de six. L'un était un morceau de l'ourlet de la robe, qui avait été raccommodé... Ils ressemblaient à des bandes arrachées...)) Ici, sans s'en apercevoir, le So- employé une phrase excessivement suspecte. Les fragments, tels qu'il nous les décrit, ressemblent à des leil a bandes arrachées, mais à dessein et par la main. C'est un accident des plus rares, qu'un morceau d'un vêtement tel que celui en question puisse être arraché 29 HISTOIRES EXTRAOUDIN AI HES. 506 entièrement ^3iT Faction cVune épine. Par la nature même du tissu, une épine ou un clou qui s'y accroche le déchire rectangulairement, — le divise par deux fentes longitudinales, faisant angle droit, et se rencontrant au sommet par où l'épine est entrée ; — mais que impossible de comprendre que il est pres- morceau soit le complètement arraché. Je n'ai jamais vu cela, ni vous non plus. Pour arracher un morceau d'un tissu, il faut, dans presque tous les cas, deux forces distinctes, agissant en sens différents. Si l'étoffe présente deux bords, — — et par exemple, c'est un mouchoir, si, sire si Ton dé- en arracher une bande, alors, seulement une force unique suffira. Mais, dans est question d'une robe, le alors, cas actuel, il qui ne présente qu'un seul Quant à arracher un morceau du milieu, lequel côté. n'offre aucun côté, ce serait miracle que plusieurs épi- nes le pussent faire, et une seule ne le pourrait. Mais, même quand le tissu présente un côté, il faudra deux épines, agissant, l'une dans les tinctes, et l'autre supposer que la le deux directions dans une seule. Et encore bord n'est pas ourlé. dis- faut-il est ourlé, S'il chose devient presque impossible. Nous avons vu quels grands et nombreux obstacles empêchent que des morceaux soient arrachés par la simple action des épines; cependant, on vous invite à croire que non- seulement un morceau, mais plusieurs morceaux ont été arrachés de cette manière ! Et Vun de ces était L' ourlet de la robe ! Un morceaux autre morceau était une partie de la jupe, mais non pas l'ourlet, — c'est-à-dire LE MYSTÈRE DE MARIE ROGET. qu'il avait été complètement arraché, épines, du milieu dis-je, et 507 par l'action des non du bord de la jupe! des choses auxquelles il Voilà, est bien pardonnable de ne pas croire; cependant, prises collectivement, elles forment un motif moins plausible de suspicion que cette unique circonstance si surprenante, à savoir que les objets aient pu être laissés dans ce bosquet par des meurtriers qui avaient eu la précaution d'emporter le cadavre. Toutefois, vous n'avez pas saisi exactement ma pensée, si vous croyez que mon dessein soit de nier que ce bosquet soit arrivé là ait été le théâtre de l'attentat. Qu'il quelque chose de grave, plus vraisemblablement un Deluc. Mais, en c'est possible; malheur, chez madame somme, c'est un point d'importance secondaire. Nous avons promis de tâcher de découvrir, non pas le lieu, mais les auteurs du meurtre. Tous les arguments que j'ai allégués, malgré toute la minutie que j'y ai mise, n'avaient pour but que de vous prouver, d'abord, la sottise des assertions si positives et si impétueuses du Soleil, ensuite et principalement, de vous amener, par la route la plus naturelle, à une autre idée de doute, — à examiner si cet assassinat a été ou n'a pas pas été l'œuvre d'une bande. » J'attaquerai cette question par une simple allusion aux détails révoltants donnés par le chirurgien interrogé dans l'enquête. Il me suffira de dire que ses conclusions publiées, relativement au nombre des préten- dus goujats, ont été justement ridiculisées, comme fausses et complètement dénuées de base, par tous les HISTOIRES EXTRAORDINAIRES. 508 anatomistes honorables de Paris. Je ne dis pas que la chose n'ait pas pu, le dit ; matériellement, arriver comme il mais je ne vois pas de raisons suffisantes pour sa conclusion; — n'y en pas beaucoup pour avait-il une autre? )) Réfléchissons maintenant sur les traces d'une lutte, et demandons ce qu'on prétend nous prouver par ces traces. La présence d'une bande ? Mais ne prouvent-elles pas plutôt l'absence d'une bande? Quelle espèce de lutte, — quelle lutte assez violente laisser des traces et assez dans tous les sens, longue pour — pouvons-nous imaginer entre une faible fille sans défense et la bande de brigands qu'on suppose? Quelques rudes bras l'empoignant silencieusement, c'en était fait d'elle. La vic- time aurait été absolument passive et à leur discrétion. Vous observerez ici que nos arguments contre le bosquet, adopté comme théâtre de l'attentat, ne s'y appli- quent principalement que comme au théâtre attentat commis par plus d'un seul individu. Si d'un nous ne supposons qu'un seul homme acharné au viol, alors, et seulement ainsi, lutte d'une nature nous pourrons comprendre une assez violente et assez opiniâtre pour laisser des traces aussi visibles. » Autre chose encore. naissant de ce fait, que — J'ai déjà noté les soupçons les objets en question aient pu même demeurer dans le bosquet où on les a découverts. Il semble presque impossible que ces preuves de crime aient été laissées accidentellement là où on les a trouvées. On a eu assez de présence d'esprit (cela est LE MYSTÈRE DE MARIE ROGET. supposé) pour emporter le cadavre ; 5U9 et cependant une preuve plus concluante que ce cadavre même (dont les rapidement altérés par la cor- traits auraient pu être ruption) reste, impudemment étalée sur le théâtre de au mouchoir de poche, por- l'attentat. Je fais allusion tant le nom de la défunte. Si c'est là n'est pas un accident du fait un accident, ce d'une bande. Nous ne pouvons nous l'expliquer que de la part d'un individu. Examinons. C'est un individu qui a commis le meurtre. Le voilà seul avec le spectre de la défunte. Il est épouvanté par ce qui gît immobile devant lui. La fureur de sa passion a disparu, et il y a maintenant dans son cœur une large place pour l'horreur naturelle de la chose faite. Son cœur n'a rien de cette assurance qu'inspire inévitablement la présence de plusieurs. est seul avec la morte. dant, il part. Il Il tremble, il Il est effaré. Cepen- y a nécessité de mettre ce cadavre quelque le porte à la rivière, les autres traces mais il laisse derrière lui du crime; car il lui est difiicile, pour ne pas dire impossible, d'emporter tout cela en une seule fois, et il lui sera loisible de revenir pour re- prendre ce qu'il a laissé. Mais, dans son laborieux voyage vers la rivière, les craintes redoublent en lui. Les bruits de la vie environnent son chemin. Une douzaine de fois il entend ou croit entendre le pas d'un espion. Les lumières mêmes de la ville l'effrayent. A la fin cependant, après de longues et fréquentes pauses pleines d'une profonde angoisse, il atteint les bords de la rivière, et se débarrasse de son sinistre fardeau, au HISTOIRES EXTRAORDINAIRES. 510 moyen d'un bateau peut-être. Mais, maintenant, quel trésor au monde, quelle menace de châtiment, auraient puissance pour contraindre ce meurtrier solitaire à revenir par sa fatigante et périlleuse route, vers le ter- rible bosquet plein de souvenirs glaçants? Il ne revient pas, il laisse les conséquences suivre leur cours. revenir qu'il ne voudrait le Il pourrait pas! Sa seule pensée, c'est de fuir immédiatement. Il tourne le dos pour toujours à ces bosquets pleins d'épouvante, et se sauve comme menacé par le courroux du ciel. » Mais si nous supposions une bande d'individus ? — Leur nombre leur aurait inspiré de l'audace, vérité, l'audace fieffé gredin ; a jamais si, en pu manquer au cœur d'un et c'est de fieffés gredins seulement qu'on suppose une bande composée. Leur nombre, dis-je, les aurait préservés de cette terreur irraisonnée et de cet effarement qui, selon mon hypothèse, ont paralysé l'individu isolé. Admettons, si vous voulez, la possibilité d'une étourderie chez un, deux ou trois d'entre eux; le quatrième aurait réparé cette négligence. Ils n'auraient rien laissé derrière eux ; car leur nombre leur aurait permis de tout emporter à la fois. Ils n'auraient pas eu besoin de revenir. » dans Examinez maintenant le cette circonstance, que, vêtement du dessus du cadavre trouvé, une bande, large environ d'un pied, avait été déchirée de bas en haut, depuis r ourlet jusqu'à la taille, mais non pas arrachée. Elle était roulée trois fois autour de la et assujettie dans le dos par une sorte de taille nœud. Cela a LE MYSTERE DE MARIE ROGET. 511 été fait dans le but évident de fournir une prise pour porter le corps. Or, une troupe d'hommes aurait-elle jamais songé à recourir à un pareil expédient? A trois hommes les membres du cadavre auraient ou quatre fourni une prise non-seulement suffisante, mais la plus commode possible. C'est bien l'invention d'un seul individu, et cela nous ramène à ce fait et la rivière, abattues, et on a découvert que les : Entre le fourré palissades étaient la terre gardait la trace d'un lourd fardeau qu'on y avait tramé ! Mais une troupe d'hommes aurait-elle pris la peine superflue d'abattre une palissade pour tramer un cadavre à travers, puisqu'ils auraient pu, en le soulevant, le faire passer facilement par- dessus? Une troupe d'hommes se avisée de traîner serait-elle même un cadavre, à moins que ce ne fût pour laisser des traces évidentes de cette tramée? )) Et ici il nous faut revenir à une observation du Commercial, sur laquelle je me suis déjà un peu arrêté. Ce journal dit: « Un morceau d'un des jupons de l'infortunée jeune fille avait été arraché, serré autour de son cou, et noué derrière la tête, probablement pour empêcher ses cris. Cela a été fait par des drôles qui n'avaient même pas un mouchoir de poche. )) J'ai » déjà suggéré qu'un parfait coquin n'était ja- mais sans un mouchoir de poche. Mais ce n'est pas sur ce fait que je veux spécialement attirer l'attention. Ce n'est pas faute d'un mouchoir, ni pour le but supposé par le Commercial que cette bande a été employée qui le ; ce prouve, c'est le mouchoir de poche laissé dans HISTOIRES EXTRAORDINAIRES. 512 le bosquet; et ce qui montre que d'empêcher cris, les c'est le but n'était pas que cette bande a été em- ployée de préférence à ce qui aurait beaucoup mieux satisfait au but supposé. la Mais l'instruction, parlant de bande en question, dit qu'elle a ètè trouvée autour du cou, adaptée d'un maiiière assez lâche et assujettie par un nœud serré. vagues, mais Ces termes sont passablement matériellement de diffèrent ceux du Commercial. La bande était large de dix-huit pouces, et devait, repliée et roulée longitudinalement, former une espèce de cordage assez fait fort, quoique de mousseline. Voici ma conclusion. Le meurtrier solitaire ayant porté cadavre jusqu'à une certaine distance le (du bosquet ou d'un autre lieu) au moyen de la bande nouée autour de la taille, a trouvé que le poids, en se servant de ce procédé, excédait ses forces. Il s'est résolu à traîner le fardeau que le fardeau a ; il y a des traces qui prouvent été traîné. Pour ce dessein, il deve- nait nécessaire d'attacher quelque chose comme une corde à l'une des extrémités. C'était autour du cou qu'il était préférable de l'attacher, la tête devant servir à l'empêcher de glisser. Et alors le meurtrier a évidemment pensé à se servir de la bande roulée autour des reins. Il l'aurait sans doute employée, si ce n'eût été l'enroulement de cette bande autour du corps, le nœud gênant par lequel elle était assujettie, et la réflexion qu'il fit qu'elle n'avait pas été complètement arrachée du vêtement. Il était plus facile de détacher une nouvelle bande du jupon. Il l'a arrachée, l'a nouée LE MYSTÈRE DE MARIE ROGET. 513 autour du cou, et a ainsi traîné sa victime jusqu'au bord de la rivière. Que cette bande, dont le mérite était d'être immédiatement à portée de sa main, mais qui ne répondait qu'imparfaitement à son dessein, été ait employée telle quelle, cela démontre que la né- cessité de s'en servir est survenue dans des circon- stances où il n'y avait plus moyen de ravoir le mouchoir, — c'est-à-dire, comme nous l'avons supposé, après avoir quitté le bosquet (si toutefois c'était le bosquet), et sur le )) chemin entre le bosquet et la rivière. madame Deluc Mais, direz-vous, la déposition de désigne spécialement une troupe de drôles, dans voisinage du le bosquet, à l'heure ou vers l'heure du meurtre. Je l'accorde. Je croirais même qu'il y avait bien une douzaine de ces troupes, telle que celle décrite par madame Deluc, à l'heure ou vers l'heure de cette tragédie. Mais la troupe qui a attiré sur elle l'a- nimadversion marquée de madame Deluc, encore que la déposition de celle-ci ait été passablement tardive et soit très-suspecte, est la seule troupe désignée par cette honnête et scrupuleuse vieille dame comme ayant mangé ses gâteaux et avalé son eau-de-vie sans se donner la peine de payer. Et hinc illse wœ? )) Mais quels sont les termes précis de la déposition de madame Deluc? « Une bande de mécréants parut, qui firent un tapage affreux, burent et mangèrent sans même route que le jeune homme payer, suivirent la et la jeune revinrent à l'auberge à la brune, puis fille, repassèrent la rivière en grande hâte. » 29. HISTOIRES EXTRAORDINAIRES. 514 )) Or, cette grande hâte a pu paraître beaucoup plus grande aux yeux de madame Deluc, qui rêvait, avec gâteaux douleur et inquiétude, à sa bière et à ses violés, — bière et gâteaux pour lesquels elle a pu nourrir, jusqu'au dernier moment, une faible espérance faisait tard, de compensation. Autrement, puisqu'il se pourquoi aurait-elle attaché de l'importance à cette hâte? Il n'y a certes pas lieu bande, même de coquins, veuille s'en retourner en quand hâte, de s'étonner de ce qu'une une large elle a rivière à traverser dans de petits bateaux, quand l'orage menace et quand la nuit approche. )) Je dis approche; car la nuit n'était pas encore ar- rivée. Ce ne fut quà la brune que la précipitation indécente de ces mécréants offensa les chastes yeux de madame Deluc. Mais on nous dit que c'est le même soir que madame Deluc, ainsi que son fils aîné, entendit des cris de femme dans le voisinage de l'au- berge. Et par quels termes madame Deluc désigne-t-elle le moment de la soirée où elle a entendu ces cris? Ce fut, dit-elle, peu après la tombée de la nuit. Mais, peu après la tombée de la nuit, c'est au moins la nuit, et le mot à la brune représente encore le jour. Ainsi il est suffisamment clair que la bande a quitté la barrière du Roule avant les cris entendus par hasard par ma- (?) dame Deluc. Et quoique, dans les nombreux comptes rendus de l'instruction, ces deux expressions distinctes soient invariablement citées même dans cette comme je les cite moi- conversation avec vous , aucune MYSTERE DE MARIE ROGET. LE feuille publique, 515 non plus qu'aucun des mirmidons de la police n'a, jusqu'à présent, remarqué l'énorme con- tradiction qu'elles impliquent. » Je n'ai la plus qu'un seul argument à ajouter contre fameuse bande; mais c'est un argument dont le poids est, pour mon intelligence du moins, absolument irrésistible. Dans le cas d'une belle récompense et d'une grâce plénière offertes à tout témoin dénonciateur de ses complices, on ne peut pas supposer un instant qu'un membre quelconque d'une bande de vils coquins, ou d'une association d'hommes quelconque, n'aurait pas, depuis longtemps déjà, trahi ses complices. Chaque individu dans une pareille bande n'est pas encore si avide de la récompense, ni si désireux d'échapper, que terrifié par ridée d'une trahison vivement et tout de même. Que possible. suite, pour n'être le secret n'ait Il trahit pas trahi lui- pas été divulgué, c'est la meilleure des preuves, en somme, que c'est un secret. Les horreurs de cette ténébreuse affaires ne sont con- nues que d'wn ou deux êtres humains et de Dieu. » Ramassons maintenant les vrai , mais positifs , faits, mesquins, il est de notre longue analyse. Nous sommes arrivés à la conviction, soit d'un fatal accident sous le toit de madame Deluc, soit d'un meurtre ac- compli, dans bosquet de la barrière du Roule, par le un amant, ou au moins par un camarade intime et secret de la défunte. Ce camarade a le teint basané. Ce teint, le nœud savant de la ceinture et le nœud coulant des brides du chapeau, désignent un homme HISTOIRES EXTRAORDINAIRES. 51() de mer. Sa camaraderie avec la défunte, jeune fille un peu légère, il est vrai, mais non pas abjecte, le dénonce comme un homme supérieur par le grade à un simple matelot. Or, les communications urgentes, fort bien écrites, envoyées aux journaux, servent à fortifier grandement notre hypothèse. Le fait d'une escapade antérieure, révélé par le Mercure, nous pousse à fondre en un même individu ce marin et cet officier de l'ar- mée de mer, déjà connu pour avoir induit en faute la malheureuse. » Et ici, très-opportunément, se présente une autre considération, celle relative à l'absence prolongée de au teint sombre. Insistons sur ce teint cet individu d'homme, sombre et ba&ané; ce n'est pas un teint légèrement basané que celui qui a pu constituer le seul point de souvenir commun à Valence et à madame Deluc. Mais pourquoi cet homme est-il absent? A-t-il été assassiné par la bande? S'il en est ainsi, pourquoi ne trouve-t-on que les traces de théâtre des deux assassinats doit tique. Et lui, 011 est son la jeune fille? Le être supposé iden- cadavre? Les assassins au- raient très-probablement fait disparaître les deux de la même manière. Non, on peut affirmer que l'homme est vivant, et que ce qui l'empêche de se faire con- naître, c'est la crainte d'être accusé du meurtre. Ce n'est que maintenant, à cette époque tardive, que nous pouvons supposer cette considération agissant avec Miirie; forte- — puisqu'un témoin affirme vu — mais cette crainte n'aurait eu aucune ment sur lui, l'avoir LE MYSTERE DE MARIE ROGET. 517 influence à l'époque du meurtre. Le premier mouvement d'un homme innocent eût été d'annoncer l'attentat et d'aider à retrouver les malfaiteurs. L'intérêt bien entendu conseillait cela. Il a été vu avec la jeune fille; il a traversé la rivière avec elle dans un bac dé- couvert. La dénonciation des assassins aurait apparu, même à un idiot, comme le plus sûr, comme le seul moyen d'échapper lui-même aux soupçons. Nous ne pouvons pas le supposer, dans cette nuit fatale du di- manche, à la fois innocent et non instruit de l'attentat commis. Cependant, ce ne serait que dans ces circonstances impossibles que nous pourrions comprendre qu'il eût manqué, lui vivant, au devoir de dénoncer les assassins. Et quels moyens possédons-nous d'arriver à la vérité? Nous verrons ces moyens se multiplier et de- )) venir plus distincts à mesure que nous avancerons. Passons au crible cette vieille histoire d'une première fuite. Prenons connaissance de l'histoire entière de cet ofïicier, ainsi que des circonstances actuelles où il est placé et des lieux où il se trouvait à l'époque précise du meurtre. Comparons soigneusement entre elles les diverses communications envoyées au journal du soir, ayant pour but d'incriminer une bande. Ceci fait, com- parons ces communications, pour le style et l'écriture, avec celles envoyées au journal du matin, à une épo» que précédente, et insistant si fortement sur la culpabilité de Mennais. Tout cela fini, comparons encore ces communications avec l'écriture connue de l'officier. HISTOIRES EXTRAORDINAIRES. 518 Essayons d'obtenir, par un interrogatoire plus minutieux de madame Deluc et de ses enfants, ainsi que de Valence, le conducteur d'omnibus, quelque chose de plus précis sur l'apparence physique et les allures de l'homme au teint sombre. Des questions, habilement dirigées, tireront, à coup sûr, de quelqu'un de ces témoins des renseignements sur ce point particulier (ou sur — renseignements que les témoins eux- d'autres), mêmes possèdent peut-être sans alors, suivons la trace telier dans la le savoir. Et puis de ce bateau recueilli par le ba- matinée du lundi 23 juin, et qui a dis- paru du bureau de navigation, à l'insu de l'officier de service, et sa7is son gouvernail, à une époque précédant la découverte du cadavre. Avec du soin, avec une per- sévérance convenable, nous suivrons infailliblement ce bateau ; car non-seulement le batelier qui l'a arrêté peut en constater l'identité, mais on a le gouvernail sous la main. Il n'est pas possible que qui que ce soit de gaieté de cœur et sans aucune recherche, abandonné le gouvernail d'un bateau à voiles. 11 n'y a ait, pas eu d'avertissement public relativement à la découa été silencieusement amené au bureau de navigation, et silencieusement il est parti. verte de ce bateau. Mais comment se taire Il fait-il que le propriétaire ou le loca- de ce bateau ait pu, sans annonce publique, à une époque aussi rapprochée que mardi matin, être informé du lieu où était amarré le bateau saisi lundi, à moins que nous ne le avec la Marine, supposions en rapports quelconques — rapports personnels et permanents. LE MYSTÈRE DE MARIE ROGET. 519 impliquant la connaissance des plus petits intérêts et des petites nouvelles locales? » En parlant de l'assassin solitaire traînant son far- deau vers le rivage, j'ai déjà insinué qu'il avait dû se procurer un bateau. Nous comprenons maintenant que Marie Roget a dû être jetée d'un bateau. La chose, trèsnaturellement, s'est passée ainsi. Le cadavre n'a pas dû être confié aux eaux basses de la Les marques rive. particulières, trouvées sur le dos et les épaules de la victime, dénoncent les teau. membrures d'un fond de ba- Que ce corps ait été trouvé sans un poids, cela ne fait que corroborer notre idée. S'il avait été jeté de la rive, on y aurait évidemment attaché un poids. Seu- lement nous pouvons expliquer l'absence de ce poids, en supposant que le meurtrier n'a pas pris la précaution de s'en procurer un avant de pousser au large. Quand il a été au moment de confier rivière, il a le cadavre à la dû, incontestablement, s'apercevoir de son étourderie ; mais il n'avait pas sous la main de quoi y remédier. Il a mieux aimé tout risquer que de retourner à la rive maudite. Une fois délivré de son funèbre chargement, le meurtrier a dû se hâter de retourner vers la ville. Alors, sur quelque quai obscur, il aura sauté à terre. Mais le bateau, l'aura-t-il mis en sûreté? 11 était bien trop pressé pour songer à une pareille niaiserie ! Et même, en l'amarrant au quai, aurait cru attacher une preuve contre lui-même ; il sa pensée la plus naturelle a dû être de chasser loin de lui, aussi loin que possible, tout ce qui avait quelque HISTOIRES EXTRAORDINAIRES. 520 rapport avec son crime. Non-seulement il aura fui loin du quai, mais il n'aura pas permis au bateau d'y rester. Assurément, il l'aura lancé » à la dérive. Poursuivons notre pensée. — Le matin, ble est frappé d'une indicible horreur en le miséra- voyant que son bateau a été ramassé et est retenu dans un lieu où son devoir, peut-être, l'appelle fréquemment. La nuit suivante, sans oser réclamer le gouvernail, il le fait disparaître. Maintenant, où est ce bateau sans gouver- que ce soit là une de nos nail? Allons à la découverte; premières recherches. Avec le premier éclaircissement que nous en pourrons avoir commencera l'aurore de notre succès. Ce bateau nous conduira, avec une rapidité qui nous étonnera nous-mêmes, vers l'homme qui s'en est servi dans la nuit du fatal dimanche. firmation s'augmentera de La con- confirmation, et nous la suivrons le meurtrier à la piste. Pour des raisons que nous ne spécifierons pas, mais qui sautent aux yeux de nos nombreux lecteurs, nous nous sommes permis de supprimer ici, dans le manuscrit remis entre nos mains, la partie où se trouve dé- taillée l'investigation faite à la suite de l'indice, en apparence si léger, découvert parDupin. Nous jugeons seulement convenable de faire savoir que le résultat désiré fut obtenu, et que le préfet remplit ponctuelle- ment, mais non sans répugnance, les termes de son contrat avec le chevalier. LE MYSTÈRE DE MARIE ROGET. L'article de 521 M. Poe conclut en ces termes ^ : On comprendra que je parle de simples coïncidences de rien de plus. Ce que j'ai déjà dit sur ce sujet doit et n'y a dans suffire. Il naturel. Que la mon cœur aucune foi au sur- Nature et Dieu fassent deux, aucun homme, capable de penser, ne le niera. Que ce dernier, ayant créé la première, puisse, à sa volonté, la gouverner ou la modifier, cela est également incontestable. Je dis : à sa volonté; car c'est une question de volonté, et non pas de puissance, comme l'ont supposé d'absurdes logiciens. Ce n'est pas que puisse pas modifier ses lois, la Divinité ne mais nous l'insultons en imaginant une nécessité possible de modification. Ces lois ont été faites, dès l'origine, pour embrasser toutes les contingences qui peuvent être enfouies dans le futur. Car pour Dieu tout est présent. Je répète donc que je parle de ces choses simplement comme de coïncidences. Quelques mots encore. On trouvera dans ma narration de quoi établir un parallèle entre la destinée de la malheureuse Mary Cecilia Rogers, autant du moins que sa destinée est connue, et la destinée d'une nommée Marie Roget jusqu'à une certaine époque de son histoire, — parallèle dont la minutieuse et surprenante exactitude est faite pour embarrasser la raison. Oui, on sera frappé de tout cela. Mais qu'on ne suppose pas un seul instant que, en 1 . Note des éditeurs du Magazine dans lequel fut primitivement publié le Mystère de Marie Roget. HISTOIRES EXTRAORDIIS AIRES. 522 continuant la de Marie depuis triste histoire le point en question et en poursuivant jusqu'à son dénoûment le mystère qui l'enveloppait, j'aie eu le dessein secret de suggérer une extension du parallèle, ou même d'insinuer que les mesures adoptées à Paris pour découvrir l'assassin d'une grisette, ou des mesures fondées sur une méthode de raisonnement analogue, produiraient un résultat analogue. Car, relativement à la dernière partie de la supposition, on doit considérer que la plus légère variation dans les éléments des deux problèmes pourrait engendrer les plus graves erreurs de calcul, en faisant di- verger absolument les deux courants d'événements; à peu près de la même manière qu'en arithmétique une erreur qui, prise individuellement, peut être inappré- par la force accumulative ciable, produit à la longue, de la multiplication, un résultat effroyablement distant de la vérité. Et, relativement à la première partie, nous ne de- vons pas oublier que ce même calcul des probabilités, que j'ai invoqué, parallèle, — interdit toute idée d'extension l'interdit avec du une rigeur d'autant plus impérieuse que ce parallèle a déjà été plus étendu et plus exact. C'est là une proposition anormale qui, bien qu'elle paraisse ressortir du domaine de la pensée gé- nérale, de la pensée étrangère aux mathématiques, n'a, jusqu'à présent, été bien comprise que par les mathématiciens. Rien, par exemple, n'est plus difficile que de convaincre le lecteur non spécialiste que, si un joueur LE MYSTÈRE DE MARIE ROGET. 523 de dés a amené les six deux fois coup sur coup, ce fait est une raison suffisante de parier gros que le troisième coup ne ramènera pas les six. Une opinion de ce genre est généralement rejetée tout d'abord par l'intelligence. On ne comprend pas comment les deux coups joués, et qui sont maintenant déjà complètement enfouis dans le passé, peuvent avoir de l'influence sur le coup qui n'existe que dans le futur. La chance pour amener les six semble être précisément ce qu'elle était à n'im- porte quel moment, c'est-à-dire soumise seulement à l'influence de tous les coups divers que peuvent ame- ner les dés. Et c'est là une réflexion qui semble si parfaitement évidente, que tout effort pour la contre verser est plus souvent accueilli par un sourire moqueur que par une condescendance attentive. L'erreur en question, grosse erreur, grosse souvent de dommages, ne peut pas être critiquée dans les limites qui assignées ici ; soin de l'être. et pour les philosophes Il sufiit me sont elle n'a pas be- de dire qu'elle fait partie d'une infinie série de méprises auxquelles la Raison s'achoppe dans sa route, par sa propension malheureuse à chercher la vérité dans le détaiL FIN DES HISTOIRES EXTRAORDINAIRES ET DU TORIK CINQUIÈME. : TABLE ' 1 Pages. Edgar Poe, sa vie et ses.^csdvres 3 Rouble assassinat dans la rue Morgue La Lettre volée —Le Scarabée d'or. . . Le Canard au ballon. 1 , / .'-. 125 .'' 187 Aventure sans pareille ifuN certain Hans Pfaall 209 Manuscrit trouvé dans une bouteille 287 Une descente dans le M^ïlstrom 307 La Vérité sur le cas de IV^j^Valoemau 337 magnétique. 353 Les souvenirs de M. Auguste' Bedloe 371 Révélation Morella 389 Ligeia 399 Metzengerstein 425 Le Mystère de Marie Roget 441 FIN DE la table. J. CLAYE, IMPRIMEUR, 1, HUE SAINT - BENOIT. |649" ETURN CIRCULATION DEPARTMENT ^^2 j^^.^ Library___^____ .„,..™„.b.R.n.w.<iby"»"'9 Books moy b. M2M0S. DÛÉ^SÂ^iiÔMp^ "^ FORM NO. DD6, BERKELEY, CA 94720 in£„lÇf^'<E'-EY LIBRARIES coaassioET
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GENETIC VARIANTS OF FUNCTIONING OF THE KEY PATHOGENETIC MECHANISMS IN PATIENTS WITH MYOCARDIAL INFARCTION AND ACUTE CARDIORENAL SYNDROME
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Трансляционная медицина / Translyatsionnaya meditsina / Translational Medicine Трансляционная медицина / Translyatsionnaya meditsina / Translational Medicine ISSN 2311-4495 ISSN 2410-5155 (Online) УДК 616.127-005.8:575 ISSN 2311-4495 ISSN 2410-5155 (Online) УДК 616.127-005.8:575 ISSN 2311-4495 ISSN 2410-5155 (Online) УДК 616.127-005.8:575 ГЕНЕТИЧЕСКИЕ ВАРИАНТЫ ФУНКЦИОНИРОВАНИЯ КЛЮЧЕВЫХ ПАТОГЕНЕТИЧЕСКИХ МЕХАНИЗМОВ У ПАЦИЕНТОВ С ИНФАРКТОМ МИОКАРДА И ОСТРЫМ КАРДИОРЕНАЛЬНЫМ СИНДРОМОМ Сиверина А.В., Скородумова Е.А., Костенко В.А., Пивоварова Л.П., Арискина О.Б., Фёдоров А.Н., Скородумова Е.Г. Контактная информация: Сиверина Анна Викторовна «СПб НИИ скорой помощи им. И. И. Джанелидзе» Будапештская ул., д. 3А, Санкт-Петербург, Россия, 192242 E-mail: Gudkovanna_09@mail.ru Государственное бюджетное учреждение «Санкт-Петербургский научно-исследовательский институт скорой помощи им И. И. Джанелидзе», Санкт-Петербург, Россия «Национальный медицинский исследовательский центр им. В.А. Алмазова» Минздрава России, Санкт-Петербург, Россия Статья поступила в редакцию 23.10.2017 и принята к печати 07.02.2017 Статья поступила в редакцию 23.10.2017 и принята к печати 07.02.2017 Abstract Abstract The aim of this study was to evaluate, depending upon presence, the impact of genes polymorphism Leu- 28Pro APOE, G681A CYP2C19, Trp212Тer CYP2C19, Val174Ala SLCO1B1, C786T NOS3 on the clinical course of myocardial infarction (MI ) in patients with associated acute kidney injury (AKI) in hospital. The study included 86 patients treated in SPb Research Institute of Emergency Care. I. I. Dzhanelidze in 2016 about IM. All patients were divided into 2 groups. The fi rst (I) included 34 patients with MI and AKI. The second one — 52 patients with MI without AKI. All patients were taken venous blood, to detect polymorphic genes variants. The method was based upon the analysis of genomic human DNA, isolated from blood leukocytes by polymerase chain reaction. We compared the data of gene analysis and clinical presentation between groups. The represen- tativeness and signifi cance of differences between parameters in the samples evaluated using the nonparametric angular Fischer criteria, t — criteria of Student, single-factor analysis. It has been proven that patients with MI and AKI signifi cantly higher rate of detected mutations in genetic variants such as G681A CYP2C19, C786T NOS3, Val174Ala SLCO1B1, associated with a worsening of the course of myocardial infarction in the hospital period obtained. Key words: gene polymorphism, myocardial infarction, acute kidney injury. GENETIC VARIANTS OF FUNCTIONING OF THE KEY PATHOGENETIC MECHANISMS IN PATIENTS WITH MYOCARDIAL INFARCTION AND ACUTE CARDIORENAL SYNDROME Siverina V. A., Skorodumova E. A., Kostenko V. A., Pivovarova L. P., Ariskina O. B., Fedorov A. N., Skorodumova E. G. Siverina V. A., Skorodumova E. A., Kostenko V. A., Pivovarova L. P., Ariskina O. B., Fedorov A. N., Skorodumova E. G. Corresponding author: Anna V. Siverina St. Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine Budapeshtskaya, str. 3, Saint Petersburg, Russia, 192242 E-mail: Gudkovanna_09@mail.ru Corresponding author: Anna V. Siverina St. Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine Budapeshtskaya, str. 3, Saint Petersburg, Russia, 192242 E-mail: Gudkovanna_09@mail.ru Corresponding author: Anna V. Siverina St. Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine Budapeshtskaya, str. 3, Saint Petersburg, Russia, 192242 E-mail: Gudkovanna_09@mail.ru Received 23 October 2017; accepted 07 February 2018. Saint Petersburg I.I. Dzhanelidze research institute of emergency medicine, Saint Petersburg, Russia Received 23 October 2017; accepted 07 February 2018. Received 23 October 2017; accepted 07 February 2018. Резюме Целью данного исследования явилось оценить клиническое течение инфаркта миокарда (ИМ) у больных с острым повреждением почек (ОПП) на госпитальном этапе в зависимости от наличия полиморфизма генов: Leu28Pro APOE, G681A CYP2C19, Trp212Ter CYP2C19, Val174Ala SLCO1B1, C786T NOS3. В исследование включены 86 пациентов, лечившихся в ГБУ СПб НИИ скорой помощи им. И.И. Джанелидзе в 2016 году по поводу ИМ. Все пациенты были разделены на 2 группы. В первую (I) вошли 34 больных с ИМ и ОПП. Во вторую (II) — 52 человека с ИМ, но без ОПП. Всем пациентам про- изводился забор венозной крови, для выявления полиморфных вариантов генов. Метод основывался на анализе геномной ДНК человека, выделенной из лейкоцитов крови, методом полимеразной цепной реак- ции. Произведено сопоставление полученных лабораторных данных и клинической картины заболевания между группами. Репрезентативность и достоверность различий между параметрами в выборках оценены с использованием углового преобразования Фишера, t-критерия Cтьюдентa, проведен однофакторный анализ. Было доказано, что у больных с ИМ и ОПП достоверно чаще выявляются мутации в генетических вариантах: G681A CYP2C19, C786T NOS3, Val174Ala SLCO1B1, на фоне чего отмечалось ухудшение течения ИМ в госпитальном периоде и увеличение случаев летальных исходов. Ключевые слова: полиморфизм генов, инфаркт миокарда, острое повреждение почек. Для цитирования: Трансляционная медицина. 2017; 4 (6): .6–12 Для цитирования: Трансляционная медицина. 2017; 4 (6): .6–12 6 6 том 4 №6 / 2017 Сердечно-сосудистые заболевания / Cardiovascular medicine Результаты и их обсуждение Результаты и их обсуждение внимание привлекает к себе персонализированная медицина, на развитие которой повлиял прогресс в фармакогенетике. Персонализированная медицина трактуется как «применение геномных и молеку- лярных данных для лучшего оказания медицин- ской помощи, содействия клиническим испытани- ям новых продуктов (в том числе и лекарственных средств), а также помощь в определении предра- сположенности человека к конкретным заболева- ниям или патологическим состояниям» [6]. Распределение полиморфизмов генов в группах приведено в табл. 1. Мутации полиморфизма Leu28Pro гена APOE приводит к изменению структуры молекулы аполи- попротеина, нарушает механизм липидного обмена и потенцирует гиперлипопротеинемию. По дан- ным литературы, частота встречаемости вариан- та 28Pro < 1% в европейских популяциях, однако, риск вероятности развития ИБС при определении этой однонуклеотидной замены (SNP) повышается в 5,3 раза [11]. При сравнении групп достоверной разницы не получено, тем не менее, важно отме- тить, что клинически ИМ у 3 пациентов с гомозиго- той по аллелю С полиморфизма Leu28Pro протекал значительно тяжелее: у 2 больных был отёк легких, у 1 — фибрилляция желудочков. В настоящее время известно много вариантов полиморфных генов, мутации которых влияют на этиопатогенез заболеваний сердечно-сосудистого континуума, в частности, ИМ. Среди них — гены, регулирующие тромбообразование, липидный об- мен, воспалительные реакции, работу ренин-анги- отензин-альдостероновой и симпатоадреналовой систем, функционирование эндотелия [7, 8, 9, 10]. Однако, вопрос о прогностической роли выявляе- мых мутаций в полиморфных генах при ИМ с ОПП не изучен. у ф р ц уд Ген CYP2C19 «чувствительности к клопидо- грелю» отвечает за выработку активного фермента, который трансформирует пролекарство клопидо- грел в активный тиоловый метаболит [12]. Мутации в полиморфных варианты G681A гена CYP2C19*2 и Trp212Ter гена CYP2C19*3 обусловливают сни- жение или утрату функциональной способности энзима, причем, как в гомозиготном, так и в гете- розиготном состоянии [13]. В российской популя- ции частота носительства полиморфизма G681A гена CYP2C19*2 составляет 11,4 — 13,3% [14]. У больных ИБС частота встречаемости значитель- но выше — 27,3% [15]. Гомозиготы по аллелю «дикого типа» *1*1 достоверно чаще встречались во второй группе, относительно первой, р ˂ 0,05. Гетерозиготы — носители только одного мутант- ного аллеля *1*2 и *1*3, приблизительно с одина- ковой частотой встречались во всех группах, p ˃ 0,05. К группе «слабых метаболизаторов» относи- лись больные, генотип которых состоял из двух аллелей со сниженной функциональной активно- стью, таких как *2*3, *2*2, *3*3. Статистически значимая разница между группами была отмечена только по генотипу *2*2, где в I выборке он встре- чался у 20,6% (7 человек) и у 3,8% (2 больных) вто- рой, р ˂ 0,05. Цель исследования Оценить особенности клинического течения инфаркта миокарда у больных с острым повре- ждением почек на госпитальном этапе с учетом влияния мутантных генотипов полиморфизма основных генов, связанных с ключевыми аспекта- ми патогенеза заболевания. Результаты и их обсуждение Был проведен однофакторный анализ, который также подтвердил достоверность получен- ных результатов. For citation: Translyatsionnaya meditsina= Translational Medicine. 2017; 4 (6): 6–12. исход регистрировался в 21% случаев, против 1% в группе с ИМ без ОПП [4]. Немаловажное влия- ние на прогноз больных с ОКРС оказывает и ста- дия ОПП [5]. При наблюдении за 637 пациентами с ОКРС риск неблагоприятных исходов на 30-й день при I стадии ОПП (KDIGO) составлял 3,9%, при II ст. — 19,4%, а при III ст.- до 50 % [5]. За послед- ние 20 лет были усовершенствованы тактика и об- щие принципы лечения больных с острым коронар- ным синдромом (ОКС). Наряду со значительным расширением спектра эффективных фармаколо- гических средств, получили широкое распростра- нение эндоваскулярные и хирургические методы восстановления кровотока в коронарных артериях. Однако, неутешительно высокие цифры летально- сти стимулируют поиск более эффективных ме- тодов диагностики и лечения ИБС. Всё большее исход регистрировался в 21% случаев, против 1% в группе с ИМ без ОПП [4]. Немаловажное влия- ние на прогноз больных с ОКРС оказывает и ста- дия ОПП [5]. При наблюдении за 637 пациентами с ОКРС риск неблагоприятных исходов на 30-й день при I стадии ОПП (KDIGO) составлял 3,9%, при II ст. — 19,4%, а при III ст.- до 50 % [5]. За послед- ние 20 лет были усовершенствованы тактика и об- щие принципы лечения больных с острым коронар- ным синдромом (ОКС). Наряду со значительным расширением спектра эффективных фармаколо- гических средств, получили широкое распростра- нение эндоваскулярные и хирургические методы восстановления кровотока в коронарных артериях. Однако, неутешительно высокие цифры летально- сти стимулируют поиск более эффективных ме- тодов диагностики и лечения ИБС. Всё большее На сегодняшний день ишемическая болезнь сердца (ИБС) и, особенно такая ее форма как ин- фаркт миокарда (ИМ), занимают лидирующие по- зиции, как по количеству смертей, так и по числу случаев инвалидизации населения. По данным фе- деральной службы государственной статистики, в 2012 году от ИМ умерло 563 000 человек, что со- ставляет чуть больше половины от общего количе- ства летальных исходов в следствие ИБС [1]. Раз- витие острого повреждения почек (ОПП) на фоне протекающего ИМ укладывается в понятие — острого кардиоренального синдрома (ОКРС) [2]. По данным различных источников, ОКРС при ИМ встречается в 40–70 и в 32% случаев ассоциирует- ся с неблагоприятными ближайшими исходами [3]. Так, в исследовании, представленном Маkenzi и соавторами, у пациентов с ИМ и ОПП летальный 7 том 4 № 6 / 2017 Сердечно-сосудистые заболевания / Cardiovascular medicine Материалы и методы р Обследовано 86 пациентов, лечившихся в ГБУ СПб НИИ скорой помощи им. И.И. Джанелидзе в 2016 году по поводу ИМ, из них мужчин — 60,2%, женщин — 39,8%. Средний возраст больных со- ставил 62,1 ± 4,2 лет. Все пациенты были разделе- ны на 2 группы. В первую (I) вошли 34 больных с ИМ и ОПП. Во вторую (II) — 52 человека с ИМ, но без ОПП. Выборки были сопоставимы по полу и возрасту. Выявление полиморфизма генов осно- вывалось на анализе геномной ДНК человека, вы- деленной из лейкоцитов крови, методом полиме- разной цепной реакции «SNP-ЭКСПРЕСС» ООО НПФ «Литех». Cтaтиcтичеcкaя обрaботкa полу- ченных дaнных проводилacь c иcпользовaнием прогрaммы Microsoft offi ce Excel 10.0. Для оценки рaзличий выборочных cовокупноcтей иcпользовa- ли критерий Cтьюдентa (t). Cрaвнение кaчеcтвен- ных признaков проводили нa оcновaнии углового преобразования Фишера, также определяли отно- шения шaнcов (ОШ) c 95% доверительным интер- валом (ДИ). Для вcех видов aнaлизa проводилacь оценкa репрезентaтивноcти полученных резуль- тaтов. Cтaтиcтичеcки знaчимыми cчитaлиcь рaз- личия при р < 0,05. Изменение работы белков-переносчиков под влиянием генетических мутаций (генетическо- го полиморфизма) ассоциировано со снижением функциональной активности транспортера органи- ческих анионов (ОАТР-С) у носителей полиморф- ного варианта Val174Ala гена SLCO1B1. ОАТР-С осуществляет перенос статинов из крови в печень для последующей биотрансформации. Носитель- ство мутантного аллеля сопряжено с замедлением функционирования транспортера, накоплением том 4 № 6/ 2017 8 Сердечно-сосудистые заболевания / Cardiovascular medicine Сердечно-сосудистые заболевания / Cardiovascular medicine Таблица 1. Рисунок 2- Госпитальная летальность Рисунок 2- Госпитальная летальность Следствием более тяжелого течения ИМ у больных с ОПП, явились высокие цифры госпи- тальной летальности у этой категории пациентов: I — 20,6% , II — 7,7% , p < 0,05. Материалы и методы Подавление или снижение активности eNOS приводит к недостатку оксида азота — дисфункции эндотелия, которой, К исследованным полиморфизмам относил- ся также C786T гена NOS3, который отвечает за выработку эндотелиальной оксид азота (NO) — 9 том 4 № 6 / 2017 ердечно-сосудистые заболевания / Cardiovascular medicine Сердечно-сосудистые заболевания / Cardiovascular medicine Сердечно-сосудистые заболевания / Cardiovascular medicine Рисунок 1. Встречаемость сердечной недостаточности в выборках Рисунок 1. Встречаемость сердечной недостаточности в выборках Рисунок 1. Встречаемость сердечной недостаточности в выборках Рисунок 1. Встречаемость сердечной недостаточности в выборках согласно классической теории «ответ на повре- ждение», отводится основная роль в атерогенезе, а также развитии атеротромбоза. SNP C786T досто- верно чаще встречается в I группе, как с генотипом CT — 44,1%, так и с TT — 23,5%, по сравнению со II — 21 и 5,8%, p ˂ 0,05. согласно классической теории «ответ на повре- ждение», отводится основная роль в атерогенезе, а также развитии атеротромбоза. SNP C786T досто- верно чаще встречается в I группе, как с генотипом CT — 44,1%, так и с TT — 23,5%, по сравнению со II — 21 и 5,8%, p ˂ 0,05. пациентов с ИМ и ОПП — 23,3%, относительно группы сравнения — 7,0%, p < 0,05. ХСН II функ- ционального класса (ФК) по Нью-Йоркской клас- сификации (NYHA) чаще встречалась у больных II выборки — 67,4%, против 55,9% первой, p > 0,05. Однако, ХСН III-IV ФК преобладала у пациентов с ИМ и ОПП — 32,4%, II — 15,4%, p < 0,05. Же- лудочковые нарушения сердечного ритма высо- ких градаций значительно чаще регистрировались в группе с ИМ и ОПП — 35,3%, по сравнению с контрольной выборкой — 19,2%, p < 0,05. Рециди- вы острого коронарного синдрома (ОКС) в 3 раза чаще встречались в первой группе (17,6%), относи- тельно группы контроля (5,8%), p < 0,05. Причём, у 14,7% человек с ИМ и ОПП, перенёсших реци- див ОКС, при повторной коронароангиографии выявлены тромбозы стентов, в контрольной груп- пе данное осложнение не определялось. Этот факт ассоциируется с высокой частотой встречаемости генотипа *2*2 гена CYP2C19 у пациентов первой выборки. Клиническая картина в группах была следу- ющей: Q-ИМ в первой группе диагностирован в 52,9%, во второй — 53,8% случаев, p > 0,05. Не Q-ИМ в I — 47,1%, во II — 46,2%, достоверной разницы между выборками не выявлено, p > 0,05. ИМ в прошлом перенесли 32,4% пациентов первой выборки, 15,4% — второй, p < 0,05. Гипертони- чеcкая болезнь диагностирована у 98,9% больных общей группы. Распределение частоты случаев острой и хронической сердечной недостаточности (ХСН) представлено на рис. 1. При анализе диагностики острой сердечной недостаточности (ОСН) по II класса по Killip ста- тистически значимая разница отмечалась между I группой 32,4% и контролем 11,5%, p < 0,01. ОСН Killip III–IV классов в 3 раза чаще наблюдалась у Распределение госпитальной летальности в группах, представлено на рис. 2. Рисунок 2- Госпитальная летальность Рисунок 2- Госпитальная летальность Материалы и методы Распределение полиморфизмов генов в группах I и II Группа Полиморфизм Группа I n = 34 Группа II n = 52 t / р ОШ с 95% ДИ APOE Leu28 Pro n (%) LeuLeu 25 (73,5) 41 (78,8) t = 0,56 p > 0,05 0,75 [0,27; 2,05] LeuPro 7 (20,6) 10 (19,2) t = 0,15 p > 0,05 1,08 [0,37;3,21] ProPro 2 (5,9) 1 (2,0) t = 0,15 p > 0,05 3,19 [0,29;36,60] CYP2C19 G681A и Тrp212Ter n (%) *1*1 12 (35,3) 37 (71,2) t = 3,47 p ˂ 0,05 0,22 [0,09;0,56] *1*2 8 (23,5) 9 (17,4) t = 0,69 p ˃ 0,05 1,47 [0,50;4,28] *1*3 3 (8,8) 2 (3,8) t = 0,90 р ˃ 0,05 2,42 [0,38;15,30] *2*3 2 (5,9) 1 (1,9) t = 0,89 p ˃ 0,05 3,18 [0,28;36,60] *2*2 7 (20,6) 2 (3,8) t = 2,25 p ˂ 0,05 6,48 [1,26;33,40] *3*3 2 (5,9) 1 (1,9) t = 0,89 p ˃ 0,05 3,19 [0,28;36,60] SLCO1B1 Val174Ala n (%) ValVal 14 (41,2) 25 (48,1) t = 0,63 p ˃ 0,05 0,76 [0,32;1,81] ValAla 13 (38,2) 25 (48,1) t = 0,91 p ˃ 0,05 0,67 [0,28;1,61] AlaAla 7 (20,6) 2 (3,8) t = 2,25 p ˂ 0,05 6,74 [1,31;34,71] NOS3 C786T n (%) CC 11 (32,4) 38 (73,1) t = 4,03 p ˂ 0,05 0,18 [0,07;0,45] CT 15 (44,1) 11 (21,2) t = 2,25 p˂0,05 2,94 [1,14;7,69] TT 8 (23,5) 3 (5,8) t = 2,23 p ˂ 0,05 5,03 [1,23;20,58] Таблица 1. Распределение полиморфизмов генов в группах I и II статинов в крови с развитием нежелательных явле- ний в мышечной ткани. Это диктует необходимость индивидуального подхода к выбору максимальной дозы препарата с учетом генотипа [16]. Пациенты с гомозиготой по аллелю Аla варианта Val174Ala гена SLCO1B1 достоверно чаще встречаются в пер- вой выборке 20,6%, против 3,8%, p < 0,05. синтазы (eNOS). Этот фермент участвует в синте- зе NO эндотелием и, следовательно, в регуляции сосудистого тонуса, кровотока и артериального давления. NO играет роль и в патогенезе ИБС, по- скольку угнетает пролиферацию гладкомышечных клеток, а также обладает протекторным эффектом в отношении агрегации тромбоцитов и ингибирует адгезию лейкоцитов к эндотелию. Список литературы / References 12. Brandt JT, Close SL, Iturria SJ, et al. Common polymorphisms of CYP2C19 and CYP2C9 affect the pharmacokinetic and pharmacodynamic response to clopidogrel but not prasugrel. J Thromb Haemost. 2007; 5 (12): 2429-2436. 1. The Demographic Yearbook of Russia — 2014. Web page in Russian. [Демографический ежегодник России — 2014. Электронный ресурс]. URL: http://www.gks.ru/bgd/ regl/B14_16/Main.htm (11 октября 2017). 2. Skorodumova EA, Kostenko VA, Ilyina VA, Siverina AV, Fedorov AN. Interference of background chronic kidney disease and acute tubular damage in patients with myocardial infarction. Translational Medicine = Translyatsionnaya meditsina. 2016; 3 (2): 40–45. In Russian [Скороду- мова Е.А., Костенко В.А., Ильина В.А., Сиверина А.В.,Фёдоров А.Н. Взаимовлияние фоновой хрониче- ской болезни почек и острого канальцевого поврежде- ния у пациентов с инфарктом миокарда. Трансляцион- ная медицина. 2016; 3 (2): 40–45]. 13. Zelenskaya EM, Slepuhina AA, Koch NV, et al. Genetic, pathophysiological and clinical aspects of antiplatelet therapy. Pharmacogenetics and Рharmacogenomics. 2015; 13. Zelenskaya EM, Slepuhina AA, Koch NV, et al. Genetic, pathophysiological and clinical aspects of antiplatelet therapy. Pharmacogenetics and Рharmacogenomics. 2015; 1:12-19. In Russian [Зеленская Е.М., Cлепухина А.А., Кох Н.В. и др.. Генетические, патофизиологические и клинические аспекты антиагрегантной терапии. Фарма- когенетика и Фармакогеномика. 2015; 1: 12–19]. 1. Mirzaev KB, Sychev DA, Karkischenko VN, et al. CYP2C19*2, CYP2C19*3, CYP2C19*17 allele and genotype frequencies in clopidogrel-treated patients with coronary heart disease from the Russian population. Biomedicine = Biomedistina. 2013; 1: 117–128. InRussian [Мирзаев К.Б., Сычёв Д.А., Каркищенко В.Н. и др. Ча- стота полиморфных маркеров CYP2C19*2, CYP2C19*3, CYP2C19*17 среди русской популяции и сравнение рас- пространенности CYP2C19*2 у пациентов с ишемической болезнью сердца, получающих терапию клопидогрелем, и здоровых добровольцев. Биомедицина. 2013;1: 117–128]. 3. Smirnov AV, Dobronravov VA, Rumyantsev ASh, Kayukov IG. Acute kidney injury. Moscow: Medical information Agency, 2015: 228–238. In Russian. [Смирнов А. В., Добронравов В. А., Румянцев А. Ш., Каюков И. Г. Острое повреждение почек. M.: Медицинское информа- ционное агентство, 2015: 228–238]. 4. Marenzi G, Gabiati A, Bertoli SV, et al. Incidence and relevance of acute kidney injury in patients hospitalized with acute coronary syndromes. Am.J.Cardiol. 2013; 20 (1): 816–822. 5. Roy AK, McGorrian C, Treacy C, et al. A Comparison of Traditional and Novel Defi nitions (RIFLE, AKIN, and KDIGO) of Acute Kidney Injury for the Prediction of Outcomes in Acute Decompensated Heart Failure. Cardiorenal Med. 2013; 3 (1): 26–37. 2. Komarov AL, Panchenko EP, Donnikov AE et al. Конфликт интересов / Confl ict of interest Авторы заявили об отсутствии потенциального конфликта интересов. / The authors declare no confl ict of interest. Список литературы / References Factors Determining Clinical Effectiveness of Clopidogrel and Prognosis of Patients With Stable Ischemic Heart Disease. Cardiology = Kardiologiya. 2011; 2: 8–18. In Russian. [Ко- маров А.Л., Панченко E.П., Донников А.Е. др. Факторы, определяющие клиническую эффективность клопидогре- ла и прогноз у больных со стабильной формой ишемиче- ской болезни сердца. Кардиология. 2011; 2: 8–18]. 2. Komarov AL, Panchenko EP, Donnikov AE et al. Factors Determining Clinical Effectiveness of Clopidogrel and Prognosis of Patients With Stable Ischemic Heart Disease. Cardiology = Kardiologiya. 2011; 2: 8–18. In Russian. [Ко- маров А.Л., Панченко E.П., Донников А.Е. др. Факторы, определяющие клиническую эффективность клопидогре- ла и прогноз у больных со стабильной формой ишемиче- ской болезни сердца. Кардиология. 2011; 2: 8–18]. 6. Abrahams E, Ginsburg GS, Silver M. The Personalized Medicine Coalition: goals and strategies. Am. J. Pharmacogenomics. 2005; 5: 345–355. 7. Bochkov NP, PuzyrevVP, Smirnyagina SA. Clinical genetics. Moscow: Geotar-Media, 2011. p. 592. In Russian [Бочков Н.П., Пузырёв В.П., Смирнихина С.А.. Клини- ческая генетика. М.: Гэотар-Медиа, 2011. с. 592]. 3. Sychev DA, Shuev GN, Prokofi ev AB. Applied aspects of SLCO1B1 pharmacogenetic testing for predicting of statin-induced myopathy and personalization of statins therapy. Rational Pharmacotherapy in Cardiology. 2013; 9 (6): 698–700. In Russian. [Сычев Д.А., Шуев Г.Н., Про- кофьев А.Б. Прикладные аспекты применения фармако- генетического тестирования по SLCO1B1 для прогно- зирования развития статин индуцированной миопатии и персонализации применения статинов. Рациональная Фармакотерапия в Кардиологии. 2013; 9 (6): 698–700]. 8. Pchelina SN, Sirotkina OV, Sheidina AM, et al. Genetic Factors of Risk of Development of Myocardial Infarction in Young Men Living in North-West Region of Russia. Cardiolody = Kardiologiya. 2007; 7: 29–34. InRussian. [Пчелина С.Н., Сироткина О.В., Шейдина А.М. и др. Генетические факторы риска инфаркта миокарда у мужчин молодого возраста, проживающих в северо- западном регионе России. Кардиология. 2007; 7: 29–34]. Выводы 1. У больных с ОПП на фоне ИМ статистиче- ски чаще регистрировались носители: генотипа *2*2 гена CYP2C19, ответственного за чувстви- тельность к клопидогрелю; генотипа AlaAla гена SLCO1B1, определяющего чувствительность к ста- тинам; аллеля T как в гетерозиготном так и гомо- зиготном положении гена NOS3, обеспечивающего выработку NO. 2. У пациентов ОПП и ИМ достоверно чаще выявлялись мутантные аллели исследуемых генов, на фоне чего отмечалось ухудшение течения ИМ в госпитальном периоде: увеличивалось количест- во случаев ОСН и ХСН, желудочковых нарушений сердечного ритма, рецидивов ОКС и летальных ис- ходов. 2. У пациентов ОПП и ИМ достоверно чаще выявлялись мутантные аллели исследуемых генов, на фоне чего отмечалось ухудшение течения ИМ в госпитальном периоде: увеличивалось количест- во случаев ОСН и ХСН, желудочковых нарушений сердечного ритма, рецидивов ОКС и летальных ис- ходов. Следствием более тяжелого течения ИМ у больных с ОПП, явились высокие цифры госпи- тальной летальности у этой категории пациентов: I — 20,6% , II — 7,7% , p < 0,05. Следствием более тяжелого течения ИМ у больных с ОПП, явились высокие цифры госпи- тальной летальности у этой категории пациентов: I — 20,6% , II — 7,7% , p < 0,05. том 4 № 6/ 2017 10 Сердечно-сосудистые заболевания / Cardiovascular medicine Конфликт интересов / Confl ict of interest 11. Postgenomic and nanotechnological innovations. ApoE (apolipoprotein E). Web page in Russian. [«ПИННИ» (Постгеномные и нанотехнологические инновации) ApoE (аполипопротеин Е). Электронный ресурс.] URL: http://пинни.рф/information/genes-information/132.html (11 октября 2017). Сердечно-сосудистые заболевания / Cardiovascular medicine St. Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine; St. Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine; Пивоварова Людмила Павловна, д.м.н., руководи- тель отдела лабораторной диагностики, ГБУ «СПб НИИ скорой помощи им. И. И. Джанелидзе»; Viktor A. Kostenko, PhD, head of Department of emergency cardiology and rheumаtology St. Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine; Арискина Ольга Борисовна, к.б.н., научный сотруд- ник, отдел лабораторной диагностики, ГБУ «СПб НИИ скорой помощи им. И. И. Джанелидзе»; I. I. Dzhanelidze Research Institute of Emergency Medicine; Фёдоров Артём Николаевич, врач-кардиолог, отде- ла неотложной кардиологии и ревматологии, ГБУ «СПб НИИ скорой помощи им. И. И. Джанелидзе»; Lyudmila P. Pivovarova, MD, head of Department of laboratory diagnostics, St. Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine; Olga B. Ariskina, PhD, researcher of Department of laboratory diagnostics, St. Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine; Скородумова Елизавета Геннадьевна, м.н.с., отдел не- отложной кардиологии и ревматологии, ГБУ «СПб НИИ скорой помощи им. И. И. Джанелидзе»; Информация об авторах: 9. Shesternya PA, Shulman VA, Nikulina SYu. Genetic aspects of myocardial infarction: problems and perspectives. Russian Journal Of Cardiology. 2012; 1 (93): 4–9. In Russian. [Шестерня П А, Шульман ВА, Никулина С Ю. Генетические аспекты инфаркта миокарда: проблемы и перспективы. Российский кардиологический журнал. 2012; 1 (93): 4–9]. Сиверина Анна Викторовна, врач-кардиолог, от- дел неотложной кардиологии и ревматологии, ГБУ «СПб НИИ скорой помощи им. И. И. Джанелидзе»; Сиверина Анна Викторовна, врач-кардиолог, от- дел неотложной кардиологии и ревматологии, ГБУ «СПб НИИ скорой помощи им. И. И. Джанелидзе»; Скородумова Елена Андреевна, д.м.н., старший науч- ный сотрудник, отдел неотложной кардиологии и ревма- тологии, ГБУ «СПб НИИ скорой помощи им. И. И. Джа- нелидзе»; 10. Schunkert H, Erdmann J, Samani NJ. Genetics of myocardial infarction: a progress report. Eur. HeartJ. 2010; 31 (8): 918–925. 10. Schunkert H, Erdmann J, Samani NJ. Genetics of myocardial infarction: a progress report. Eur. HeartJ. 2010; 31 (8): 918–925. Костенко Виктор Авенирович, к.м.н., руководитель отдела неотложной кардиологии и ревматологии, ГБУ «СПб НИИ скорой помощи им. И. И. Джанелидзе»; 11 том 4 № 6 / 2017 Сердечно-сосудистые заболевания / Cardiovascular medicine Author information: Author information: Artyom N. Fedorov, cardiologist of Department of emergency cardiology and rheumаtology, St. Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine; Anna V. Siverina, cardiologist of Department of emergency cardiology and rheumаtology, St. Petersburg I. I. Dzhanelidze Research Institute of EmergencyMedicine; Elizaveta G. Skorodumova, MSc, of Department of emergency cardiology and rheumаtology, St. Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine. Elena A.,Skorodumova MD., senior researcher at Department of emergency cardiology and rheumаtology, 12 том 4 № 6/ 2017 том 4 № 6/ 2017 том 4 № 6/ 2017
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https://www.frontiersin.org/articles/10.3389/fmicb.2018.02461/pdf
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Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling
Frontiers in microbiology
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ORIGINAL RESEARCH published: 17 October 2018 doi: 10.3389/fmicb.2018.02461 ORIGINAL RESEARCH published: 17 October 2018 doi: 10.3389/fmicb.2018.02461 ORIGINAL RESEARCH published: 17 October 2018 doi: 10.3389/fmicb.2018.02461 Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling Alvaro Mauricio Florez1*, Miguel Orlando Suarez-Barrera2,3, Gloria M. Morales2, Karen Viviana Rivera2, Sergio Orduz4, Rodrigo Ochoa5, Diego Guerra5 and Carlos Muskus5 1 RG Microbial Ecology: Metabolism, Genomics & Evolution, Microbiomas Foundation, Chía, Colombia, 2 Laboratorio de Biología Molecular y Biotecnología, Universidad de Santander, Bucaramanga, Colombia, 3 Escuela de Medicina, Facultad de Salud, Universidad Industrial de Santander, Bucaramanga, Colombia, 4 Grupo Biología Funcional, Laboratorio de Prospección y Diseño de Biomoléculas, Escuela de Biociencias, Universidad Nacional, Sede Medellín, Colombia, 5 Programa de Estudio y Control de Enfermedades Tropicales PECET, Unidad de Biología Molecular y Computacional-UBMC, Universidad de Antioquía, Medellín, Colombia Keywords: Bacillus thuringiensis, Cry11, DNA shuffling, docking, Aedes aegypti, Culex quinquefasciatus Edited by: Marc Strous, University of Calgary, Canada The Cry11 family belongs to a large group of δ-endotoxins that share three distinct structural domains. Among the dipteran-active toxins referred to as three-domain Cry11 toxins, the Cry11Aa protein from Bacillus thuringiensis subsp. israelensis (Bti) has been the most extensively studied. Despite the potential of Bti as an effective biological control agent, the understanding of Cry11 toxins remains incomplete. In this study, five Cry11 variants obtained via DNA shuffling displayed toxic activity against Aedes aegypti and Culex quinquefasciatus. Three of these Cry11 variants (8, 23, and 79) were characterized via 3D modeling and analysis of docking with ALP1. The relevant mutations in these variants, such as deletions, insertions and point mutations, are discussed in relation to their structural domains, toxic activities and toxin-receptor interactions. Importantly, deletion of the N-terminal segment in domain I was not associated with any change in toxic activity, and domain III exhibited higher sequence variability than domains I and II. Variant 8 exhibited up to 3.78- and 6.09-fold higher toxicity to A. aegypti than Cry11Bb and Cry11Aa, respectively. Importantly, variant 79 showed an α-helix conformation at the C-terminus and formed crystals retaining toxic activity. These findings indicate that five Cry11 variants were preferentially reassembled from the cry11Aa gene during DNA shuffling. The mutations described in loop 2 and loop 3 of domain II provide valuable information regarding the activity of Cry11 toxins against A. aegypti and C. quinquefasciatus larvae and reveal new insights into the application of directed evolution strategies to study the genetic variability of specific domains in cry11 family genes. Reviewed by: Christopher L. Hemme, The University of Rhode Island, United States Evgeniya V. Nazarova, Cornell University, United States *Correspondence: Alvaro Mauricio Florez amflorez@microbiomas.org Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Received: 06 April 2018 Accepted: 25 September 2018 Published: 17 October 2018 INTRODUCTION toxicity, it is highly synergistic with Bti Cry toxins and aids to overcome resistance in mosquitoes to Cry toxins (Wirth et al., 1997). Due to the synergistic interactions of Bt subsp. israelensis toxins, this bacterium has been used worldwide to control mosquito larvae of the genera Aedes, Culex and Anopheles, which are involved in the transmission of diseases including malaria, hemorrhagic fever, dengue fever, lymphatic filariasis, yellow fever (Ben-Dov, 2014), Chikungunya and Zika (Chouin- Carneiro et al., 2016; Gardner et al., 2016; Tsetsarkin et al., 2016). Bacillus thuringiensis (Bt), a Gram-positive bacterium characterized by the production of Cry δ-endotoxins capable of killing insects, has been used since the late 1930s as a biological control agent (Melo et al., 2016). A total of 308 holotype toxins are clustered into 75 Cry proteins (Crickmore et al., 2014) (revised February, 2018). The tertiary structures of nine Cry toxins determined via X-ray crystallography to date contain three conserved domains with specific functions and implicated in the structural stability of the protein. The domain I is a bundle of 7–8 α helices involved in pore formation, domain II is a β-prism with exposed loops regions involved in receptor binding and, domain III is a β-sandwich and has influence on receptor binding, ion channel formation and insect specificity (Li et al., 1991; Grochulski et al., 1995; Derbyshire et al., 2001; Galitsky et al., 2001; Morse et al., 2001; Guo et al., 2009; Hui et al., 2012; Figure 1A). ) Cry11Aa from Bti is a 72 kDa protoxin that is activated by gut enzymes via the proteolytic removal of 28 residues from its N-terminus and proteolytic cleavage into two fragments of 38 and 30 kDa that remain associated and retain toxicity (Dai and Gill, 1993; Revina et al., 2004; de Barros Moreira Beltrao and Silva-Filha, 2007). The Cry11Aa toxin has higher activity against Aedes and Culex than against Anopheles (Revina et al., 2004; Otieno-Ayayo et al., 2008). In A. aegypti, this toxin interacts with two midgut brush border membrane receptors; a GPI anchored and alkaline phosphatase (ALP1) (Fernandez et al., 2006) and also binds to Cyt1Aa as a kind of membrane-bound receptor of Cry11Aa increasing the toxic activity (Perez et al., 2005). Other midgut proteins different to the receptor alkaline phosphatase (ALP1) such as ATP binding protein, increases the toxicity of Cry11Aa against C. quinquefasciatus (Zhang L. et al., 2017). INTRODUCTION Other two toxins, Cry11Bb (94 kDa) and Cry11Ba (81 kDa), share a similar insect specificity and are phylogenetically related to Cry11Aa. Cry11Bb and Cry11Ba are produced by Bt subsp. medellin and Bt subsp. jegathesan, respectively (Delecluse et al., 1995; Orduz et al., 1998). g ) The mechanisms by which Bt induces death in insects are controversial and have not been completely elucidated (Vachon et al., 2012). Currently, there are two mechanisms related to Bt- induced toxicity in insects that have been accepted; the sequential binding and signaling pathways (Zhang Q. et al., 2017). The sequential binding mechanism has been extensively studied and is based on the formation of pores in epithelial cells in the midgut of targeted insects, which results in toxin-receptor interactions, osmotic imbalance and cell death (Bravo et al., 2007; Pardo- Lopez et al., 2013). After crystal ingestion, Cry toxins become solubilized, and specific proteases present in the lumen of the midgut activate the toxins, which then bind to specific receptors located in the insect midgut. In some Cry toxins, this event induces the proteolytic removal of helix α1 (Aronson, 2000), triggering Cry toxin oligomerization, insertion of oligomeric structures altering membrane stability, receptor and production of channels or pores, ultimately leading to cell lysis and insect death (Bravo et al., 2004; Figure 1B). The signaling pathways is a recent proposed mechanism in which the activation of signaling cascades, leads to increased cyclic AMP and protein kinase activities, resulting in cell death (Zhang et al., 2006). Both of these mechanisms contain gaps. In the sequential binding mechanism, the presence of several types of resistance and the link between proteolysis and pore formation are not understood (Melo et al., 2016). In the signaling pathway mechanism, how Cry toxin-receptor interactions mediate toxic activity is unclear (Melo et al., 2016). In this context, it has been suggested that Cry toxins can cause death based on their ability to induce both pore formation and ion channel activation (Zhang Q. et al., 2017). Although the tertiary structure of Cry11 toxins have not been determined by X-ray crystallography, Cry11Aa have been the most studied among this group using protein engineering tools. Several mutations haven been introduced into different domains that are implicated in its toxicity. Citation: Florez AM, Suarez-Barrera MO, Morales GM, Rivera KV, Orduz S, Ochoa R, Guerra D and Muskus C (2018) Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling. Front. Microbiol. 9:2461. doi: 10.3389/fmicb.2018.02461 October 2018 | Volume 9 | Article 2461 1 Frontiers in Microbiology | www.frontiersin.org Cry11 Variants Obtained via DNA Shuffling Florez et al. Frontiers in Microbiology | www.frontiersin.org INTRODUCTION Therefore, studies have focused on domain I developing N-terminally truncated forms of Cry11Aa (Pang et al., 1992) or modifications in domains II and III altering the interactions with its receptor in the midgut, confirming the importance of these domains for Cry11Aa-mediated toxicity (Fernandez et al., 2005, 2009). Based on phage display and site-directed mutagenesis, the exposed regions of loop α8, β4 and loop 3 in domain II of Cry11Aa have been shown to be involved in the interaction of Cry11Aa with A. aegypti brush border membrane vesicles (BBMVs). Specifically, two mutations in loop α8, V262E and E266A, reduced the toxic activity of Cry11Aa against A. aegypti (Fernandez et al., 2005). There are also mutations in loop α-8 that are involved in Cry11Aa–ALP1 receptor interaction, that affect the Cyt1Aa and Cry11Aa interaction reducing the synergism between these proteins and decreasing their toxic activity (Perez et al., 2005). Other binding sites have been described for the interaction of Cry11Aa with the receptor ALP1 as an important secondary receptor for Cry11Aa and Cry11Ba (Chen et al., 2017). The involved regions are located in loop 2 of domain II and β18-β19 of domain III of Cry11Aa, which interact with ALP1 regions R59–G102 and N257–I296, respectively (Fernandez et al., 2009). The pBtoxis megaplasmid from Bt subsp. israelensis (Bti) contains four Cry proteins encoded by the cry4Aa, cry4Ba, cry10Aa, and cry11Aa genes and two Cyt proteins encoded by the cyt1Aa and cyt2Ba genes (Berry et al., 2002). The cry genes produce 134, 128, 78, and 72 kDa polypeptides, respectively, all of which possess larvicidal activity higher than of the Cyt’s proteins (Ben-Dov, 2014). However, the high toxic activity of Bti is the result of synergistic interactions between all of them (Ben-Dov, 2014). That is the case of Cyt1Aa that despite the low October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 2 Cry11 Variants Obtained via DNA Shuffling Florez et al. FIGURE 1 | Structure of the Cry toxins, domains, and their mode of action. (A) Ribbon diagram of Cry deduced 3D structure. Three domains are colored in red blue and green, respectively. (B) Sequential binding mechanism. 1. The toxin binds to GPI-anchored APN and ALP receptors in the lipid rafts; 2. Binding to cadherin receptor 3. Proteolytic cleavage of the helix α1 at N-terminal end; 4. N-terminal cleavage induces the formation of pre-pore oligomer 5. INTRODUCTION Increasing of the oligomer binding affinity to GPI-anchored APN and ALP receptors; 6. Oligomer inserts into the membrane, leading to pore-formation and cell lysis; and 7. Cellular death. FIGURE 1 | Structure of the Cry toxins, domains, and their mode of action. (A) Ribbon diagram of Cry deduced 3D structure. Three domains are colored in red blue and green, respectively. (B) Sequential binding mechanism. 1. The toxin binds to GPI-anchored APN and ALP receptors in the lipid rafts; 2. Binding to cadherin receptor 3. Proteolytic cleavage of the helix α1 at N-terminal end; 4. N-terminal cleavage induces the formation of pre-pore oligomer 5. Increasing of the oligomer binding affinity to GPI-anchored APN and ALP receptors; 6. Oligomer inserts into the membrane, leading to pore-formation and cell lysis; and 7. Cellular death. Since the three Cry11 toxins are phylogenetically related and exhibit similar specificity to insect species, it is possible to infer similarities at structural level that can be analyzed after mixing their genes in order to create novel proteins with improved properties. Therefore, considering the lack of studies focused on this approach, we designed a DNA shuffling strategy to obtain variants with increased toxicity to A. aegypti and C. quinquefasciatus. DNA shuffling has been used alone or in combination with phage display and the staggered extension process via homologous recombination to increase the activity of Cry toxins against specific insect pests (Lucena et al., 2014). This technique is a powerful approach based on recombination between parental genes in a single DNA shuffling reaction following random fragmentation (Stemmer, 1994). and toxicity in which are implicated domains II and III preferably. Frontiers in Microbiology | www.frontiersin.org DNA Shuffling g Three micrograms of each PCR product obtained from the TA cloning constructs and from pGEBb with lengths of 2.5 kb (cry11Aa), 1.6 kb (cry11Ba-1), 0.78 kb (cry11Ba-2), and 3.5 kb (cry11Bb) were mixed in 25 µl of 50 mM Tris–HCl, pH 7.4, and 10 mM of MnCl2. In the same tube, 0.0006 U DNase I (Life Technologies) was added to a final volume of 50 µl. The reaction was incubated between 5 and 20 min at room temperature to optimize production of fragments ranging between 25 and 250 bp. The reaction was stopped by adding 25 µl of 25 mM EDTA. The DNase I digestion products were separated via electrophoresis and purified with the QIAEX II Gel Extraction Kit (QIAGEN). Forty microliters of the pooled of purified fragments were used as template for a PCR without primers in 1× Pfx buffer, 0.3 mM dNTPs, and 2.5 U Pfx50 polymerase in a final volume of 50 µl under the following conditions: 94◦C for 3 min, 45 cycles of 94◦C for 30 s, 48◦C for 3 min and 68◦C for 1 min (with a 12-s increase in extension time per cycle), and a final extension step at 68◦C for 7 min. The products for reassembly were validated via agarose gel electrophoresis and purified using the Wizard PCR Clean-Up system. Test Primers for Reassembly via PCR Test Primers for Reassembly via PCR The pTOAa, PTOBa-1 and pTOBa-2 constructs were used as templates in 50-µl PCRs that contained 0.32 µM PCR4 primers as described in Supplementary Table 1, 1× Pfx polymerase buffer, 0.4 mM dNTPs, 2.5 mM MgCl2 and 0.5 U Pfx50 polymerase (Life Technologies) in 1× reaction buffer that contained 1 mM MgSO4. The amplification conditions were denaturation at 94◦C for 4 min followed by 35 cycles of 45 s at 94◦C, 45 s at 59–67◦C, and 4 min 30 s at 68◦C and a final extension step of 10 min at 68◦C. The PCR products were separated via electrophoresis and purified using the Wizard purification system (Promega). To generate the pGE7 construct, 50 ng of pGEBb-1 plasmid DNA was used as a template. The PCR was performed in a final volume of 50 µl that contained 0.32 µM of each primer as described in Supplementary Table 1, 1× Pfx polymerase buffer, 0.3 mM dNTPs, 1 mM MgSO4 and 0.5 U Pfx50 polymerase. The PCR conditions were denaturation for 4 min at 94◦C followed by 35 cycles of 94◦C for 45 s, 68◦C for 45 s, and 68◦C for 4 min 30 s and a final extension step at 68◦C for 10 min. The PCR products were separated via agarose gel electrophoresis and purified using the Wizard R⃝SV Gel and PCR Clean-Up System (Promega). Isolation of cry11 Genes via PCR The cry11 genes were amplified via PCR using specific primers and plasmid DNA from constructs pBTM3, pSV2 and pJEG90.1 as templates. Briefly, reactions were conducted in a final volume of 50 µl that contained 20 ng of plasmid DNA, 0.5 µM primers, 1× Taq polymerase buffer, 0.4 mM dNTPs, 1.5 mM MgCl2 and 0.5 U GoTaq polymerase (Promega). The amplification conditions were denaturation at 94◦C for 5 min followed by 35 cycles of 45 s at 94◦C, 45 s at 55◦C, and 4 min 30 s at 72◦C, and a final extension at 72◦C for 10 min. The PCR products were separated via electrophoresis and purified using the PCR Clean- Up System (Promega). The cry11Ba gene was obtained via two independent PCRs, producing a 1.8-kb product (denoted Ba1) corresponding to cry11Ba with a deletion of 246-bp downstream of the ATG start site and a 0.9-kb fragment (denoted as Ba-2) that contained a 0.75-kb fragment of cry11Ba including the stop codon and a 249-bp segment homologous to the multiple cloning site (MCS) of the pHT315 shuttle vector. Microbial Strains, Clone Selection and Gene Constructs For DNA manipulation, Escherichia coli DH5α, JM109 (Promega) and DH5αTOP10© (Life Technologies) cells were grown at 37◦C in Luria Bertani (LB) culture medium supplemented with ampicillin (50 µg/ml) and X-gal (50 mg/ml). A Bt 4Q2-81 strain carrying the pBTM3 plasmid and expressing the cry11Bb gene from Bt subsp. medellin (Restrepo et al., 1997) and a second Bt 4Q2-81 strain carrying the pJEG90.1 plasmid and expressing the cry11Ba gene from Bt subsp. jegathesan (Delecluse et al., 1995) were cultured as previously described (Restrepo et al., 1997) in M1 medium supplemented with 30 µg/ml tetracycline and 20 µg/ml erythromycin. Crystal production was evaluated via phase contrast microscopy. E. coli DH5α cells harboring the pSV2 plasmid, which carried the p19 gene upstream of cry11Aa from Here, we report five variants that were reassembled from the cry11Aa gene that exhibit from moderate to high toxic activities against A. aegypti and C. quinquefasciatus mosquito larvae. Variant 8 was the most toxic to the mosquito larvae, and variants 23 and 79 displayed important differences in 3D structure, toxin-ALP1 interactions October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 3 Cry11 Variants Obtained via DNA Shuffling Florez et al. Bt subsp. israelensis, were cultured in M1 medium supplemented with 20 µg/ml chloramphenicol; this strain was obtained from Dr. Neil Crickmore from the University of Sussex. The pTOAa, pTOBa-1, pTOBa-2, and pTOBb plasmids carrying the cry11Aa, cry11Ba-1, cry11Ba-2, and cry11Bb genes, respectively, were amplified via PCR and cloned using the TOPO TA Cloning R⃝ system (Life Technologies) (Supplementary Table 1). To obtain pGEBb-1, the pTOBb vector was digested with EcoRI and BamHI, releasing an insert of 3.5 kb that was ligated into pGEM7zf (+) (Promega). The DNA shuffling library was cloned into the TA TOPO cloning system. Selected variants were subcloned into the pSV2 expression vector using HindIII and SacI and transformed into BMB171 cells in LB supplemented with 6 µg/ml chloramphenicol. The acrystaliferous strain BMB171 was used to produce the variants and was donated by Dr. Ziniu Yu from the State Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, Hubei, China. pTOAa containing a 2.5-kb insert, pTOBa-1 containing a 1.6- kb insert, pTOBa-2 containing a 0.78-kb insert and pTOBb containing a 3.5-kb insert were sequenced by Macrogen, Inc. (Seoul, South Korea) using M13/T7 primers and the primer pair pCR4F (5′-GATAACAATTTCACACAGGA-3′) and pCR4R (5′-TTGTAAAACGACGGCCAGTG-3′). Cultures, Solubilization, Cry Protein Quantification, and SDS–PAGE Cultures expressing variants 1, 8, 23, 79, and 81 and recombinant Cry11Aa and Cry11Bb, including the Bt acrystaliferous BMB171 strain, were grown in 10 ml of LB supplemented with chloramphenicol (6 µg/ml) for 7 days at 30◦C and 300 rpm. After 48 h of incubation, the culture purity was confirmed via microscopic observation of spores, crystals, and lysed cells. The final culture was collected via centrifugation at 11,200 × g for 15 min at 4◦C. The supernatant was discarded, and 1 ml of 1 M NaCl was added to the pellet, which was shaken for 1 h at 30◦C and 50 rpm to neutralize protease activity. Then, Larvae Each bioassay consisted of two replicates, each with 30 first instar larvae in 1 ml at 24◦C for each variant at 7 different concentrations under the same environmental conditions. A total of 420 larvae were used for each variant. Larval mortality was determined by counting the number of live larvae after 24 and 48 h, and 50% lethal concentrations were determined statistically via Probit analysis which employ a transformation from sigmoid dose-response curve to a straight linear and then analyzed by a regression on the relationship. The calculation of the average lethal concentration (LC50) was made using the R-Project Software3. Transformation of Bacillus thuringiensis The final cultures were centrifuged at 11,200 × g at 4◦C for 10 min, and the precipitate was washed twice in 1× PBS. The pellet was resuspended in 1/10 of the original volume, and 100 µl of the samples were placed on glass slides and dried overnight at room temperature. The samples were coated with a thin layer of gold on a Denton Desk Vacuum IV and analyzed using a JEOL JSM 5010 LV scanning electron microscope. Transformation of Bacillus thuringiensis The PCR products were cloned using TOPO Zero Blunt PCR cloning kit and then subcloned into the pSV2 expression vector using HindIII and SacI. The resulting vectors were transformed into BMB171 cells via electroporation using a Bio- Rad MicropulserTM. BMB171 cells were grown in LB-glycine 0.12% up to an OD600 of 0.15, corresponding to the early exponential phase, and then transformed with 500 ng of the constructs. The electroporation conditions were 2 kV/cm, 200 , and 25 µF for 4 ms. Transformed cells were revitalized via incubation in 500 µl of LB for 2 h at 30◦C at 50 rpm. Two hundred microliters of transformed cells were plated on 60-mm Petri dishes containing LB agar supplemented with 6 µg/ml chloramphenicol. Colony counts and percent efficiency of transformation were calculated after 48 h. Endospore and crystal formation was evaluated via scanning electron microscopy (SEM). cry11 Gene Cloning, Insert Validation, and Sequencing The cry11 genes obtained via PCR were cloned using the TOPO TA system. Recombinant variants for each product were selected, and plasmid DNA was extracted using the Wizard Plus Minipreps kit (Promega). Verification of each insert was performed via digestion of 50 ng of DNA with EcoRI, followed by separation via agarose gel electrophoresis. The released inserts were also used as templates for PCR to confirm the presence of the cry11 genes in the inserts. Each reaction was conducted in a final volume of 25 µl that contained 0.4 µM each of the forward primer 5′-TTAGAAGATACGCCAGATCAAGC-3′ and the reverse primer 5′-CATTTGTACTTGAAGTTGTAATCCC- 3′ (Bravo, 1997; Bravo et al., 1998) in 1× Taq polymerase buffer, 0.4 mM dNTPs, 2.5 mM MgCl2 and 0.12 U GoTaq polymerase. The amplification conditions were 5 min at 94◦C followed by 35 cycles of 45 s at 94◦C, 45 s at 51◦C, and 1 min at 72◦C and a final extension step of 6 min at 72◦C. The full-length sequences were amplified using two sets of primers, PCR4F/R and PGE7F/R, and the combination of these primers, PCR4F/PGE7R, along with a template of 1 µl of the products of the primerless PCR, to a final volume of 50 µl. The PCR was conducted in 1× Pfx50 buffer, 0.3 µM dNTPs, 0.3 µM primers, and 5 U Pfx50 under the following conditions: 4 min at 94◦C, 25 cycles of 94◦C for 45 s, 55◦C for 1 min, and 68◦C for October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 4 Cry11 Variants Obtained via DNA Shuffling Florez et al. 4 min (with a 20-s increase in extension time per cycle), and a final extension step at 68◦C for 10 min. The second PCR product, corresponding to the DNA shuffling product, was analyzed via agarose gel electrophoresis and purified using the Wizard PCR Prep DNA Purification System. the suspension was washed twice with 1 ml of 1× PBS and centrifuged at 11,200 × g for 5 min at 4◦C. The number of spores was determined via heat shock using 100 µl of each culture and incubated at 72◦C for 20 min. Afterward, the samples were incubated at 4◦C for 10 min and were diluted by 10−1 to 10−5 in a final volume of 100 µl. The dilutions were plated on LB agar supplemented with chloramphenicol (6 µg/ml) and incubated at 30◦C for 24 h. 3https://www.r-project.org/ 4http://zhanglab.ccmb.med.umich.edu/I-TASSER/ Cloning, DNA Sequencing and Homology Analysis To quantify Cry protein production, 200 µl of the final sporulated culture of each variant was solubilized by adding 800 µl of solubilization buffer (50 mM NaOH, 10 mM EDTA, pH 11.7), incubating the culture at 4◦C overnight and centrifuging the culture at 25,200 × g for 1 h at 4◦C. The supernatant was collected, and the volume was adjusted to 1 ml with Tris-base (0.1 M, pH 7.4). The protein concentration was determined using the Bradford protein assay (Bradford, 1976) and was confirmed via SDS–PAGE using bovine serum albumin (BSA) as a standard. The protein samples were electrophoresed on 10% SDS–PAGE gels at 80 V for 90 min using a Bio-Rad mini protein system (Bio- Rad Laboratories). A total of 5 µg of protein was loaded per lane, and the protein bands were visualized by staining with Coomassie Brilliant Blue R-250 solution for 30 min. y The shuffled PCR products were cloned using the TOPO Zero Blunt PCR cloning kit (Life Technologies) according to the manufacturer’s instructions and chemically competent E. coli DH5αTOP10 cells (Life Technologies). Sequencing data were used to select clones according to their open reading frame (ORF) and DNA identity to the parental genes. Plasmid DNA from each clone was isolated using a Wizard Minipreps kit, and the DNA was sent to Macrogen, Inc., South Korea, for sequencing. The forward and reverse primers used for sequencing were as follows: M13 forward (−20): 5′-GTAAACGACGGCCAG-3′; M13 reverse, 5′-CAGGAAACAGCTATGAC-3′. Gene homology analysis was performed using BLASTn and BLASTx, available at1. Sequence alignments were performed using ClustalW (Thompson et al., 1994), available on the web2. 1http://blast.ncbi.nlm.nih.gov/Blast.cgi 2http://www.expasy.org Molecular Docking of Cry11 Domains With ALP1 Parental Cry11Aa and variants 8, 23, and 79 were analyzed to identify their interactions with the receptor ALP1 from A. aegypti (UniProtKB ID; Q16WV8). For this protein, two regions that interact with Cry11Aa were identified by epitope mapping. These regions are located within R59-G102 and N257-I296 in ALP1, which interact with residues in loop α8 of Cry11Aa domain II and residues R561-N570 in Cry11Aa domain III, respectively (Fernandez et al., 2009). The structure of ALP1 was modeled using the I-TASSER server taking into account folding recognition by threading. The model was evaluated according to Z-scores obtained using ProSA-web (see text footnote6), and a Ramachandran plot was generated using Swiss-MODEL (see text footnote5). For docking analysis, the interactions between Cry11Aa and ALP1 regions were analyzed using the Cry11Aa-interacting domains as peptides with rigid conformations based on the predicted 3D structures obtained in the previous step. The ALP1 structure and the peptides obtained from Cry11Aa and each variant were parameterized using AutoDockTools11 via the addition of polar hydrogens to each residue’s side chain to facilitate the formation of hydrogen bonds. The structures were also treated with Gasteiger partial charges to facilitate electrostatic interactions among other molecular entities. Docking analysis was performed using AutoDock Vina12 considering an exhaustiveness set to 80, which is proportional to the length of the ligand. For the simulated interactions with both regions of ALP1, 3D grid cubic boxes with sides of 32 Å in length and a grid space of 1.0Å were located on the defined active site center, covering all the residues of interest and allowing the entrance of the full peptide structures into the protein cavities. Subsequently, the different docking conformations for each variant were illustrated and analyzed using LIGPLOT13. Four PCR products of 3.5, 2.7, 1.8, and 0.9 kb corresponding to cry11 genes were obtained (Supplementary Figure 1A), as confirmed via DNA sequencing. The 3.5-kb PCR product contained a 2.2-kb fragment encoding the Cry11Bb protein. Three segments were also identified downstream of the last stop codon. The first segment consisted of 234-bp and showed 93% identity to cry11Bb2 (accession number HM68615.1). The second segment was a 129-bp fragment that showed 93% identity to the complementary strand of the IS2140 insertion element (accession number M23740.1) and was used to distinguish the reassembled products from the cry11Bb gene. Parental cry11 Genes cry11Aa, cry11Ba, and cry11Bb were used as parental genes to be fragmented in DNA shuffling based on their closely phylogenetical relationship, similarities at structural protein level and toxic specificity to similar insect species. This approach has been used for in vitro recombination of families of homologous genes in order to create novel proteins with improved properties and is useful for those in which the three- dimensional structure is unknown. In contrast to other random mutagenesis protocols, this technique introduces mutations by random DNA fragmentation and PCR reassembly in a cyclic process that alternates gene diversification, screening and selection of functional variants (Stemmer, 1994). 3D Structure Prediction and Validation and Secondary Structure Analysis of Non-conserved Regions The amino acid sequences of parental Cry11Aa and variants 8, 23, and 79 were modeled via threading methodology using the free local server I-TASSER4. From the five models obtained by the October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 5 Cry11 Variants Obtained via DNA Shuffling Florez et al. RESULTS program, the first model of each structure was selected according to the best C and TM scores. These structures were geometrically and energetically validated to assess the quality of the generated 3D model using different servers, such as the Ramachandran SWISS-MODEL5, the Z-score and energy graph in the ProSA- web server6, ERRAT7, and Verify3D8. The structures obtained for variants 8, 23, and 79 were aligned with the structure of Cry11Aa. For variant 79, complementary analysis based on the predicted secondary structure of non-conserved regions was performed using JPred9. Subsequently, the ab initio method was used to predict the 3D structure of variant 79 using Robetta server10. 5http://swissmodel.expasy.org/workspace/?func=tools_structureassessment1 6https://prosa.services.came.sbg.ac.at/prosa.php 7http://services.mbi.ucla.edu/ERRAT/ 8http://services.mbi.ucla.edu/Verify_3D/ 9http://www.compbio.dundee.ac.uk/jpred/ 10http://robetta.bakerlab.org/ 11http://autodock.scripps.edu/resources/adt 12http://vina.scripps.edu/ 13http://www.ebi.ac.uk/thornton-srv/software/LIGPLOT/ Molecular Docking of Cry11 Domains With ALP1 The third segment, a 173-bp fragment, showed 83% identity to the complementary strand of the cry30Aa gene (accession number AJ251978.1). y g The 2.7-kb PCR product contained a 1.9-kb fragment encoding the Cry11Aa protein as well as two additional segments of 580- and 90-bp that were identified upstream of the first ATG and downstream from the stop codon, respectively. The 580-bp fragment was homologous to the p19 accessory protein gene (GenBank: CAD30080.1) and was used to distinguish the reassembled products from cry11Aa gene. The downstream 90- bp segment was homologous to the MCS of pSV2. Sequence analysis showed that the 1.8-kb and 0.9-kb PCR products from cry11Ba, denoted as Ba1 and Ba2, respectively, shared a 407-bp segment. A deletion of 246-bp downstream of ATG start site was used to recognized the reassembled products from cry11Ba gene. No mutations were detected in the DNA sequences of the PCR products of any of the parental genes. Frontiers in Microbiology | www.frontiersin.org Assembly of Full-Length cry11 Genes Assembly of Full-Length cry11 Genes The primers used for DNA shuffling were tested via conventional PCR and random fragmentation, and the results indicated that the parental gene amplifications were successful (Supplementary Figure 1A). The purified PCR products (Supplementary Figure 1B) were mixed and treated with DNase I for 7, 8, or 9 min. However, only the products treated for 8 min (Supplementary Figure 1C) generated fragments between 25 and 200-bp. These products were reassembled, resulting fragments between 1 and 10-kb (Supplementary Figure 1D). After the final assembly using the PCR4F and pGE7R primers, we observed fragments between 0.25 and 2-kb (Supplementary Figure 1E). The assembly reaction products were cloned as described in the 5http://swissmodel.expasy.org/workspace/?func=tools_structureassessment1 6https://prosa.services.came.sbg.ac.at/prosa.php 7http://services.mbi.ucla.edu/ERRAT/ 8http://services.mbi.ucla.edu/Verify_3D/ 9http://www.compbio.dundee.ac.uk/jpred/ 10http://robetta.bakerlab.org/ 11http://autodock.scripps.edu/resources/adt 12http://vina.scripps.edu/ 13http://www.ebi.ac.uk/thornton-srv/software/LIGPLOT/ October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 6 Cry11 Variants Obtained via DNA Shuffling Florez et al. expression, did not show crystal formation (Supplementary Figure 3B). Materials and Methods. A total of 94 variants were obtained, and 10 of these variants did not contain an insert. For the remaining variants, 34 were <1.0-kb, 14 were between 1.1 and 2-kb, 22 were >2.1-kb and, and 14 did not show homology. Characteristics and Sequence Homology of cry11 Variants In accordance with sequence identity, the variants 8, 23, and 79 were reassembled products from the cry11Aa parental gene. The toxic activity against A. aegypti larvae was up to 3.78- and 6.09-fold higher for variant 8 than for Cry11Bb and Cry11Aa, respectively. No significant differences were observed against C. quinquefasciatus. Variants 23 and 79 showed lower and higher mutation rates than Cry11Aa, respectively, although both variants retained toxic activity against C. quinquefasciatus. Additionally, variants 23 and 79 exhibited moderate and high toxicity to A. aegypti, respectively. Surprisingly, variant 79, despite of high variations still retaining toxic activity. Recombinant Cry11Bb exhibited high toxic activity to both A. aegypti and C. quinquefasciatus larvae, while variants 23 and 81 exhibited lower toxic activity than control against A. aegypti. The results of toxicity assays of five variants for the two types of larvae are shown in Figure 2. Based on their mutations and bioassay results, variants 8, 23, and 79 were chosen for analysis of their 3D structure and interaction with ALP1. According to the sequence analysis of the Cry11 variants, 14 of them did not show homology to any known endogenous Cry11 toxin. Among those 14 variants, six variants were >2.1-kb and eight were between 1 and 2-kb. The 22 variants that displayed sequence homology and a similar size to the full-length parental genes (>2.1-kb) were clustered into three groups. The first group consisted of variants 1, 8, 23, 28, 54, 79, and 81; these variants contained the p19 gene located upstream from the ATG start site and showed homology to cry11Aa. A second group of ten variants, including 16, 36, 51, 57, 61, 68, 71, 75, 77, and 85, showed DNA homology to cry11Aa but lacked the p19 gene. The third group of variants, 14, 17, 67, 76, and 86, showed homology to the cry11B genes. Among the 22 variants that were between 1 and 2-kb, 14 showed homology to the cry11B genes, and eight did not have homology to any of the cry11 genes used. All 34 variants that were <1-kb showed homology to the cry11B genes, and their reassembled products contained only domain III. Variants 8 and 23 Are Similar to Their Parental Protein, Whereas Variant 79 Shows Structural Differences Among all variants obtained via DNA shuffling, variants 1, 8, 23, 28, 54, 79, and 81 were selected for characterization. Homology analysis of the deduced amino acid sequences of Cry11Aa and variants 1, 8, 23 and 81 showed a high degree of conservation with few amino acid changes, which were preferentially located in domain III (Supplementary Figure 2A). Variants 28 and 54 exhibited 100% identity to Cry11Aa. Therefore, these mutants were excluded from further analysis. Comparative analysis of Cry11Aa, variant 23 (aa 1-643), variant 8 (aa 1-568), and variant 79 (aa 1-551) showed that the two first variants are highly conserved in the extension of the sequences. However, many variations relative to Cry11Aa were present in variant 79 at the end of the sequence, particularly beginning from aa 286. In addition to polymorphisms, several insertions/deletions were present at the end of this sequence (Supplementary Figures 2B,C). The Z-score, ERRAT and Verify3D results obtained for Cry11Aa and variants 8, 23, and 79 are shown in Supplementary Table 2. Multiple alignment including structural alignment of Cry11Aa and its variants showed that variants 8 and 23 are similar to their parental protein due to high sequence conservation (Supplementary Figures 2A,B). The similarity of these variants with respect to Cry11Aa was 87.4 and 98.9%, respectively. These variants also showed similar structural conformation to Cry11Aa (Figures 3A–D). However, variant 79 exhibited structural differences in the non-conserved region compared with its parental protein (Figures 3E,F). The similarity of variant 79 with respect to Cry11Aa was 55,7%. The predicted secondary structure in the non-conserved region of variant 79 predominantly contains α helices instead of β-sheets, which are found in the corresponding region of Cry11A. This result was also found based on ab initio analysis, thus confirming the high prevalence of α helices in this region (Figure 3G). Finally, secondary structure analysis of the deduced amino acid sequence from variants 8, 23, and 79 predicted the presence of α helix, β-sheets and loops (Supplementary Figure 2C). The accession numbers and particular characteristics of the variants 1, 8, 23, 79, and 81 genes are described in Table 1. Protein Expression and Crystal Formation The Ramachandran analysis of ALP1 showed that 76% of its residues were in favorable zones according to phi and psi angle positions and were involved in interactions with the parental Cry11Aa protein or its variants. None of the residues of ALP1 positioned in unfavorable zones (Supplementary Figure 4A) were involved in interactions with the parental Cry11Aa protein or its variants. The Z-score (−7.27) and the energy obtained from the ProSA-web server were below 0. Both SDS–PAGE revealed that all variants contained a similar pattern of solubilized proteins, and degradation was not observed (Supplementary Figure 3A). According to SEM analysis, variants 1, 8, 23, 79, and 81 as well as the parental proteins Cry11Aa and Cry11Bb form crystals (Supplementary Figure 3B). Strain BMB171, which was used as the plasmid recipient for protein October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 7 Cry11 Variants Obtained via DNA Shuffling Florez et al. TABLE 1 | Molecular characteristics of Cry11 variants obtained via DNA shuffling. DI DII DIII Variants GenBank accession number Identity (%) cry11Aa Mutation rate (%) Del. (nt) Subs. (nt) Ins. (nt) Subs. (nt) Del. (nt) Variant 1 MH068786 84,6 15 219 0 73 6 1 Variant 8 MH068787 87,7 13 219 6 0 13 0 Variant 23 MH068788 98,9 1 9 6 5 2 0 Variant 79 MH068789 80,1 20 326 7 42 21 0 Variant 81 MH068790 90,7 8 153 0 8 2 0 TOTAL 926 19 128 44 1 D, domain; Del, deletions; Subs, substitutions; Ins, insertions. TABLE 1 | Molecular characteristics of Cry11 variants obtained via DNA shuffling. TABLE 1 | Molecular characteristics of Cry11 variants obtained via DNA shuffling. FIGURE 2 | Half lethal concentrations of Cry11 variants obtained via DNA shuffling in Aedes aegypti and Culex quinquefasciatus larvae. The values are expressed as / l f t l i t 95% fid li it (CL) FIGURE 2 | Half lethal concentrations of Cry11 variants obtained via DNA shuffling in Aedes aegypti and Culex quinquefasciatus larvae. The values are expressed as ng/ml of spore-crystal mixtures, 95% confidence limit (CL). FIGURE 2 | Half lethal concentrations of Cry11 variants obtained via DNA shuffling in Aedes aegypti and Culex quinquefasciatus larvae. The values are expressed as ng/ml of spore-crystal mixtures, 95% confidence limit (CL). Protein Expression and Crystal Formation Amino acids F316 and W319 of variant 8 formed three hydrogen bonds with amino acids E98 and Q100 of ALP1 (Figure 4C) in contrast to the two hydrogen bonds found in the interaction between F389 and Q391 of the parental Cry11Aa protein with Y478 and E105 of ALP1 (Figure 4D). For variant 23, a single hydrogen bond between G388 and Q98 of ALP1 was found (Figure 4E), whereas variant 79 formed five hydrophobic interactions with nine amino acids of ALP1 (Figure 4F). analyses matched the score reported in the PDB crystallographic database, and the interactions were found to be energetically stable (Supplementary Figures 4B–D). The interaction of Cry11Aa with ALP1 involves a peptide of 12 amino acids, 389FTQWFQSTLYGW400, within loop 2 in domain II of Cry11Aa that was conserved in variants 8 and 23 (Supplementary Figure 2A). However, in variant 79, the only first five amino acids, 281FTQWF285, were found (Supplementary Figure 2B). The identified interactions of ALP1 with the twelve-amino acid peptide indicated that W319 and F320 of variant 8 form hydrogen bonds with Y478 and S381 of ALP1, respectively (Figure 4A). The protein complex between variant 8 and ALP1 was also stabilized by five and eight hydrophobic interactions, respectively (Figure 4A). For variant 23, seven hydrophobic interactions with eleven amino acids of ALP1 were found (Supplementary Figure 5). For Cry11Aa, nine hydrophobic interactions with five amino acids and one hydrogen bond with Q391 of ALP1 were found (Figure 4B). The identified interactions of ALP1 with the five-amino acid peptide of variant 79 described above were also analyzed for the interactions of ALP1 with Cry11Aa as well as variants 8 and 23. According to the data for Cry11Aa, the peptide 564RVQSQNSGNN573, located in the β18β19 region of domain III, was found in all variants with exception of variant 79 (Supplementary Figure 2C). The LIGPLOT analysis of Cry11Aa showed three hydrophobic interactions of Cry11Aa with surface-exposed amino acids of ALP1 (Figure 5A). However, in variant 23, only one stable interaction through a hydrogen bond between R561 of the variant protein and G261 of ALP1 was observed (Figure 5B). In variant 8, two hydrogen bonds between R491 of the variant protein and N259 of ALP1 as well as three hydrophobic interactions of R491, V492, and Q493 of the variant protein with V258, G257, and G 261 of ALP1 were formed (Figure 5C). Protein Expression and Crystal Formation October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 8 Cry11 Variants Obtained via DNA Shuffling Florez et al. Florez et al. Cry11 Variants Obtained via DNA Shuffling FIGURE 3 | Prediction of the 3D Structures of Cry11Aa and Variants 8, 23, and 79. (A) Conserved region of variant 8 in light blue and Cry11Aa in beige, RMSD: 1,084 with 247 aa. (B) Non-conserved region of variant 8 in light blue and Cry11Aa in beige. (C) Conserved region of variant 23 in light blue and Cry11Aa in beige, RMSD: 1,132 with 488 aa. (D) Non-conserved region of variant 23 in light blue and Cry11Aa in beige. (E) Conserved region of variant 79 in light blue and Cry11Aa in beige, RMSD: 1,084 with 247 aa. (F) Non-conserved region of variant 79 in light blue and Cry11Aa in beige. (G) Ribbon representation of the non-conserved region of variant 79 generated using the Robetta server. FIGURE 3 | Prediction of the 3D Structures of Cry11Aa and Variants 8, 23, and 79. (A) Conserved region of variant 8 in light blue and Cry11Aa in beige, RMSD: 1,084 with 247 aa. (B) Non-conserved region of variant 8 in light blue and Cry11Aa in beige. (C) Conserved region of variant 23 in light blue and Cry11Aa in beige, RMSD: 1,132 with 488 aa. (D) Non-conserved region of variant 23 in light blue and Cry11Aa in beige. (E) Conserved region of variant 79 in light blue and Cry11Aa in beige, RMSD: 1,084 with 247 aa. (F) Non-conserved region of variant 79 in light blue and Cry11Aa in beige. (G) Ribbon representation of the non-conserved region of variant 79 generated using the Robetta server. DISCUSSION to increase the activity of Bt Cry toxins (Lucena et al., 2014). Other approaches based on phage-assisted continuous evolution (PACE) (Badran et al., 2016), in vitro template-change PCR (Shu et al., 2016), site-directed mutagenesis, and error- prone PCR have also been used successfully to identify novel Directed evolution approaches such as phage display, DNA shuffling and staggered extension process shuffling combined with Red/Et homologous recombination have been proposed October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 9 Cry11 Variants Obtained via DNA Shuffling Florez et al. FIGURE 4 | Molecular docking of the interactions of domain II of Cry11Aa and its variants with ALP1. (A,B) Interactions formed by 11-amino acid peptides within domain II of Cry11Aa. (A) Variant 8 (B) Cry11Aa. (C–F) Interactions formed by 5-amino acid peptides within domain II of Cry11Aa (C) variant 8 (D) Cry11Aa (E) Variant 23 (F) Variant 79. FIGURE 4 | Molecular docking of the interactions of domain II of Cry11Aa and its variants with ALP1. (A,B) Interactions formed by 11-amino acid peptides within domain II of Cry11Aa. (A) Variant 8 (B) Cry11Aa. (C–F) Interactions formed by 5-amino acid peptides within domain II of Cry11Aa (C) variant 8 (D) Cry11Aa (E) Variant 23 (F) Variant 79. receptors expressed on the surface of insect midgut cells and to understand the effects of different cry gene mutations on the mechanism of action of Cry toxins (Lucena et al., 2014). In this study, we report five Cry toxin variants produced via reassembly during DNA shuffling of the cry11Aa gene that showed toxic activity against A. aegypti and C. quinquefasciatus larvae. used to recognize those genes reassembled from cry11Ba, and an internal sequence corresponding to the specific primers was used to reassemble the variants from the construct containing the cry11Bb gene. The p19 gene was present upstream of the first ATG codon in variants 1, 8, 23, 28, 54, 79, and 81. According to sequence analysis, all variants displayed some degree of identity to cry11Aa; this observation indicated that all variants were preferentially reassembled from this parental gene during DNA shuffling (Table 1). DNA shuffling was designed using internal sequences in the parental constructs, including the identification of specific sites for priming during reassembly. We used the upstream p19 gene to identify the genes that were reassembled from cry11Aa. Frontiers in Microbiology | www.frontiersin.org DISCUSSION A deletion of 246 bp downstream of the ATG start site was Variants 1 and 79 retained toxic activity against A. aegypti and C. quinquefasciatus despite lacking 8.0 and 11.9 kDa N-terminal regions, respectively. These variants exhibited the October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 10 Cry11 Variants Obtained via DNA Shuffling Florez et al. FIGURE 5 | Interactions visualized in LigPlot of ALP1 against peptides of Cry11Aa and its variants 8 and 23. (A) Cry11Aa, (B) Variant 23, and (C) Variant 8. highest mutation rates, 15 and 20%, respectively. The mutations f d l d f variant 79 did not show mutations in helices α-4 and α-5, h h l d f l d FIGURE 5 | Interactions visualized in LigPlot of ALP1 against peptides of Cry11Aa and its variants 8 and 23. (A) Cry11Aa, (B) Variant 23, and (C) Variant 8. FIGURE 5 | Interactions visualized in LigPlot of ALP1 against peptides of Cry11Aa and its variants 8 and 23. (A) Cry11Aa, (B) Variant 23, and (C) Variant 8. s visualized in LigPlot of ALP1 against peptides of Cry11Aa and its variants 8 and 23. (A) Cry11Aa, (B) Variant 23, and (C) Variant 8 FIGURE 5 | Interactions visualized in LigPlot of ALP1 against peptides of Cry11Aa and its variants 8 and 23. (A) Cry11Aa, (B) Va variant 79 did not show mutations in helices α-4 and α-5, which are implicated in pore formation, or in regions involved in toxin-receptor interactions, such as loop α8 and strand β4 (Supplementary Figure 2C). However, among the 260 amino acids at the C- terminus that were modified in variant 79, we found five amino acids located in loop α-2 (F281, T282, Q283, W284, and F285) that generate nine hydrophobic interactions with ALP1 (Figure 4F). These five amino acids in Cry11Aa and in variants 8 and 23 also form more stable interactions with ALP1 than hydrogen bonds (Figures 4C–E). Additionally, according to 3D and secondary structural analyses, the non- conserved C-terminal region of variant 79 has an unusual highest mutation rates, 15 and 20%, respectively. The mutations found in variant 1 were not in regions involved in pore formation or toxin-receptor interactions, explaining the toxicity of variant 1 to A. aegypti based on bioassays. DISCUSSION In the same region, in variant 23, four out of the nine amino acids of ALP1 that initially interacted with the wild type Cry11Aa toxin were involved in hydrophobic interactions; this evidence suggests that these changes could affect the stability of the toxin-receptor interaction (Supplementary Figure 5). The interaction between the β18-β19 region in domain III of variant 23 and ALP1 also appears to be characterized by a lack of hydrophobic interactions and the formation of a stable hydrogen bond between R561 of variant 23 and Q261 of ALP1 (Figure 5B). The observed differences in the interaction of ALP1 with wild type Cry11Aa and variant 23 based on docking analysis could be explained by the peptide conformations in the original model (Cry11Aa and variant 23 were considered as rigid peptides folded in the original conformation). In this manner, a technological limitation of docking analysis was overcome by producing interactions with the complete protein. Based on these findings, we suggest that the changes found in loop 2 of domain II and the interactions observed in the β18-β19 region of domain III affect the stability of the interaction of variant 23 with ALP1, thereby producing the moderate toxicity of variant 23 to A. aegypti. ( g ) The N-terminal deletions found in variants 1, 8, 23, 79, and 81 were between 3 and 108 amino acids in length (0.33 and 11.8 kDa). However, these deletions are not implicated in the differences in toxic activity against A. aegypti and C. quinquefasciatus larvae between variants. Only one study has reported an N-terminal deletion in the Cry11Aa toxin where a truncated protein lacking 9.6 kDa was non-toxic to A. aegypti (Pang et al., 1992). In other Cry toxins such as Cry2a, deletion of 42 amino acids at the N-terminus increased the toxic activity against Spodoptera littoralis, Helicoverpa armigera, and Agrotis ipsilon (Mandal et al., 2007). Furthermore, in Cry1Ac, a deletion of 56 amino acids at the N-terminus, which included helix α-1, increase the toxic activity against Pectinophora gossypiella by 107-fold (Mandal et al., 2007) and against Plutella xylostella and Ostrinia nubilalis by 350-fold (Tabashnik et al., 2011). However, the toxic activity of Cry4Ba against A. DISCUSSION The formation of two hydrogen bonds in loop 2 of domain II produced by W319 and F320 with two amino acids of ALP1 including one hydrophobic interaction (Q321) in the same cavity (Figure 4A), as well as two hydrogen bonds formed in domain III by R491 with N259 of ALP1 and three hydrophobic interactions, could explain the toxicity toward A. aegypti mediated by ALP1 (Figure 4C). α-helix conformation (Figure 3G). The structural conformation of domains II and III of variant 79 has not previously been observed in Cry toxins; according to BLASTP analysis, this protein region is completely new. Therefore, we cannot discard the possibility that interactions of loop α8 in domain I of variant 79 with loop α-2 of ALP1 could be sufficient to explain the toxicity of this variant. α-helix conformation (Figure 3G). The structural conformation of domains II and III of variant 79 has not previously been observed in Cry toxins; according to BLASTP analysis, this protein region is completely new. Therefore, we cannot discard the possibility that interactions of loop α8 in domain I of variant 79 with loop α-2 of ALP1 could be sufficient to explain the toxicity of this variant. Variants 23 and 81 contain N-terminal deletions of 0.33 and 5.5 kDa, respectively. The mutation rates of variants 23 and 81 were less than those of other variants, and these two variants displayed 6.8- and 11.6-fold lower toxic activity against A. aegypti larvae, respectively, relative to Cry11Aa (Table 1 and Figure 2). However, these variants retained high activity against C. quinquefasciatus. The LC50s of variants 23 and 81 for C. quinquefasciatus are similar with the values that have been reported for Cry11Aa (van Frankenhuyzen, 2009) and were comparable to those of the parental protein in our bioassays. Loop α8, strand β4 and loop 3 in domain II of these variants did not contain mutations, but differences in docking analysis results for variant 23 compared to the other variants could explain the moderate toxicity of variant 23 to A. aegypti. In loop 2, mutation S392F caused loss of the hydrogen bond formed via the interaction between Q391 of the wild type protein and N24 of ALP1, potentially reducing energetic stability and decreasing the specificity of the intermolecular interactions. DISCUSSION The insertion of 22 amino acids at the end of the C-terminus with seven substitutions did not affect the toxic activity of variant 1. The results of the bioassays with C. quinquefasciatus larvae showed that variant 1 has similar toxic activity to Cry11Aa but 7.63-fold lower toxic activity than Cry11Bb. In the other hand, toxic activity of variant 79 was unexpected, despite its numerous mutations, its toxic activity against A. aegypti and C. quinquefasciatus was high (Figure 2). The deduced amino acid sequence of October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 11 Cry11 Variants Obtained via DNA Shuffling Florez et al. using the synthetic peptide 447LTYNRIEYDSPTTEN461 in binding assays in the presence of A. aegypti BBMV (Fernandez et al., 2009). So far, mutations in loop 3 have been reported in Cry4Ba toxin that produces an increase of toxicity of 1.38 and 700 times toward A. aegypti and C. quinquefasciatus, respectively (Abdullah et al., 2003). In our study, the interactions found by docking analysis in loop 2 of domain II and strands β18-β19 in domain III suggest a role in the stability of the interaction with ALP1. The formation of two hydrogen bonds in loop 2 of domain II produced by W319 and F320 with two amino acids of ALP1 including one hydrophobic interaction (Q321) in the same cavity (Figure 4A), as well as two hydrogen bonds formed in domain III by R491 with N259 of ALP1 and three hydrophobic interactions, could explain the toxicity toward A. aegypti mediated by ALP1 (Figure 4C). using the synthetic peptide 447LTYNRIEYDSPTTEN461 in binding assays in the presence of A. aegypti BBMV (Fernandez et al., 2009). So far, mutations in loop 3 have been reported in Cry4Ba toxin that produces an increase of toxicity of 1.38 and 700 times toward A. aegypti and C. quinquefasciatus, respectively (Abdullah et al., 2003). In our study, the interactions found by docking analysis in loop 2 of domain II and strands β18-β19 in domain III suggest a role in the stability of the interaction with ALP1. Frontiers in Microbiology | www.frontiersin.org REFERENCES Bravo, A., Sarabia, S., Lopez, L., Ontiveros, H., Abarca, C., Ortiz, A., et al. (1998). Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl. Environ. Microbiol. 64, 4965–4972. Abdullah, M. A., Alzate, O., Mohammad, M., Mcnall, R. J., Adang, M. J., and Dean, D. H. (2003). Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering. Appl. Environ. Microbiol. 69, 5343–5353. doi: 10.1128/AEM.69.9.5343-5353.2003 Chen, J., Aimanova, K., and Gill, S. S. (2017). Functional characterization of Aedes aegypti alkaline phosphatase ALP1 involved in the toxicity of Cry toxins from Bacillus thuringiensis subsp. israelensis and jegathesan. Peptides 98, 78–85. doi: 10.1016/j.peptides.2017.05.011 Aronson, A. (2000). Incorporation of protease K into larval insect membrane vesicles does not result in disruption of integrity or function of the pore-forming Bacillus thuringiensis delta-endotoxin. Appl. Environ. Microbiol. 66, 4568–4570. doi: 10.1128/AEM.66.10.4568-4570.2000 Chouin-Carneiro, T., Vega-Rua, A., Vazeille, M., Yebakima, A., Girod, R., Goindin, D., et al. (2016). Differential susceptibilities of Aedes aegypti and Aedes albopictus from the Americas to Zika Virus. PLoS Negl. Trop. Dis. 10:e0004543. doi: 10.1371/journal.pntd.0004543 Badran, A. H., Guzov, V. M., Huai, Q., Kemp, M. M., Vishwanath, P., Kain, W., et al. (2016). Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance. Nature 533, 58–63. doi: 10.1038/nature 17938 Crickmore, N., Baum, J., Bravo, A., Lereclus, D., Narva, K., Sampson, K., et al. (2014). Bacillus Thuringiensis Toxin Nomenclature. Available at: http://www. btnomenclature.info/ Ben-Dov, E. (2014). Bacillus thuringiensis subsp. israelensis and Its Dipteran- Specific Toxins. Toxins 6, 1222–1243. doi: 10.3390/toxins6041222 Dai, S. M., and Gill, S. S. (1993). In vitro and in vivo proteolysis of the Bacillus thuringiensis subsp. israelensis CryIVD protein by Culex quinquefasciatus larval midgut proteases. Insect Biochem. Mol. Biol. 23, 273–283. doi: 10.1016/0965- 1748(93)90008-G Berry, C., O’neil, S., Ben-Dov, E., Jones, A. F., Murphy, L., Quail, M. A., et al. (2002). Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbiol. 68, 5082–5095. doi: 10.1128/AEM.68.10.5082-5095.2002 de Barros Moreira Beltrao, H., and Silva-Filha, M. H. (2007). Interaction of Bacillus thuringiensis svar. israelensis Cry toxins with binding sites from Aedes aegypti (Diptera: Culicidae) larvae midgut. FEMS Microbiol. Lett. 266, 163–169. doi: 10.1111/j.1574-6968.2006.00527.x Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding. Anal. Biochem. 72, 248–254. doi: 10.1016/0003-2697(76)9 0527-3 Delecluse, A., Rosso, M. L., and Ragni, A. (1995). FUNDING This work was supported by grants from the Colombian Department of Science, Technology and Innovation, COLCIENCIAS, 1299-12-16813 and 5201-545-31565. ACKNOWLEDGMENTS We thank Andres Castañeda, Laura Natalia Parra, Luz Dary Rodriguez, Liliana Torcoroma, and Juan David Estupiñan for technical assistance. We thank Andres Castañeda, Laura Natalia Parra, Luz Dary Rodriguez, Liliana Torcoroma, and Juan David Estupiñan for technical assistance. AUTHOR CONTRIBUTIONS AF conceived the study, was in charge of overall direction and planning and wrote the manuscript with input from all authors. MS-B carried out the experiments and worked out almost all of the technical details. GM performed directed evolution techniques and sequencing analysis. KR assisted with MS-B measurements and bioassays. SO contributed to electronic SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02461/full#supplementary-material DISCUSSION Variant 8, which contained several substitutions in domains II and III, was the most interesting variant produced in this study due to its high toxicity to the two mosquito species. These findings confirm the importance of these domains in Cry toxin-receptor interactions and in Cry protein toxicity. The substitutions found in variants 8 and 23 provide new information about the role of loops 2 and 3 of domain II in Cry toxin-ALP1 interactions. Importantly, the α helix conformation of the C-terminus of variant 79 based on secondary structure analysis corresponds to a new protein structure with toxic activity. We believe that DNA reassembly via DNA shuffling following random fragmentation could be a good strategy to generate random mutations in specific cry genes to design new and more potent toxins. Overall, the data presented in this report indicate that the N-terminal deletions observed in all variants did not affect their toxicity to A. aegypti or C. quinquefasciatus. Variant 8, which contained several substitutions in domains II and III, was the most interesting variant produced in this study due to its high toxicity to the two mosquito species. These findings confirm the importance of these domains in Cry toxin-receptor interactions and in Cry protein toxicity. The substitutions found in variants 8 and 23 provide new information about the role of loops 2 and 3 of domain II in Cry toxin-ALP1 interactions. Importantly, the α helix conformation of the C-terminus of variant 79 based on secondary structure analysis corresponds to a new protein structure with toxic activity. We believe that DNA reassembly via DNA shuffling following random fragmentation could be a good strategy to generate random mutations in specific cry genes to design new and more potent toxins. microscopy analysis and devised the project. RO carried out the molecular docking analysis. DG contributed to the analysis of 3D structure prediction and validation and secondary structure analysis of non-conserved regions. CM contributed to the interpretation of the 3D structure prediction and docking results. DISCUSSION aegypti was abolished when more than 38 amino acids were removed from the N-terminus (Pao-intara et al., 1988) and, in the case of chimeric proteins formed by a fusion of N-terminus of Cry4Ba and the C-terminus of Cry1Ac, an increase of toxicity against C. pipiens larvae was observed (Zghal et al., 2017). Although these findings confirm the importance of the N-terminal region in the toxicity of Cry proteins, the N-terminal deletions found in our variants did not affect their toxic activity against A. aegypti or C. quinquefasciatus larvae. The difference in toxicity against A. aegypti and C. quinquefasciatus (Figure 2) could be explained by the presence of compounds either in midgut juice or membranes- bond proteases. Although the roles of these compounds and proteases have not been tested in variants obtained by DNA shuffling, there is evidence that the capacity to processing the protoxin depends on specific proteases located in the larval midgut and favored by alkaline conditions (Ben-Dov, 2014). In Cry11Aa toxin, the treatment with proteases generates fragments with different molecular weight and toxic activity against A. aegypti (Revina et al., 2004; de Barros Moreira Beltrao and Silva-Filha, 2007), whereas in C. quinquefasciatus, the processing pattern differ from those that are active to A. aegypti (Dai and Gill, 1993). Therefore, we suggest that the toxicity differences found in A. aegypti and C. quinquefasciatus could be explained by mechanism that dependent on the host. Variant 8 was the most important of this study despite a deletion of 8.0-kDa at the N-terminus and similar 3D structure to Cry11Aa (Figures 3A,B), it showed an increased toxic activity of 6.09 times compared to Cry11Aa toward A. aegypti without significant differences against C. quinquefasciatus (Figure 2). Importantly, this variant did not show substitutions in regions involved in pore formation such as helices α4 and α5 neither in regions involved in toxin-receptor interaction such as loop α8 and strand β4. However, three substitutions T453A, R456G, and P462R in loop 3 located in domain II were found (Supplementary Figure 2A). In Cry11Aa, this loop did not shown any interaction October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 12 Cry11 Variants Obtained via DNA Shuffling Florez et al. Overall, the data presented in this report indicate that the N-terminal deletions observed in all variants did not affect their toxicity to A. aegypti or C. quinquefasciatus. REFERENCES L., et al. (2014). Molecular approaches to improve the insecticidal activity of Bacillus thuringiensis cry toxins. Toxins 6, 2393–2423. doi: 10.3390/toxins6082393 Vachon, V., Laprade, R., and Schwartz, J. L. (2012). 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Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II. FEBS Lett. 579, 3508–3514. doi: 10.1016/j.febslet.2005.05.032 Restrepo, N., Gutierrez, D., Patino, M. M., Thiery, I., Delecluse, A., and Orduz, S. (1997). Cloning, expression and toxicity of a mosquitocidal toxin gene of Bacillus thuringiensis subsp. medellin. Mem. Inst. Oswaldo Cruz 92, 257–262. doi: 10.1590/S0074-02761997000200021 Revina, L. P., Kostina, L. I., Ganushkina, L. A., Mikhailova, A. L., Zalunin, I. A., and Chestukhina, G. G. (2004). Reconstruction of Bacillus thuringiensis ssp. REFERENCES N., Zaritsky, A., Wirth, M. C., Manasherob, R., Khasdan, V., Cahan, R., et al. (2008). Variations in the mosquito larvicidal activities of toxins from Bacillus thuringiensis ssp. israelensis. Environ. Microbiol. 10, 2191–2199. doi: 10.1111/j.1462-2920.2008.01696.x Pang, Y., Frutos, R., and Federici, B. A. (1992). Synthesis and toxicity of full- length and truncated bacterial CryIVD mosquitocidal proteins expressed in lepidopteran cells using a baculovirus vector. J. Gen. Virol. 73( Pt 1), 89–101. doi: 10.1099/0022-1317-73-1-89 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Pao-intara, M., Angsuthanasombat, C., and Panyim, S. (1988). The mosquito larvicidal activity of 130 kDa delta-endotoxin of Bacillus thuringiensis var. israelensis resides in the 72 kDa amino-terminal fragment. Biochem. Biophys. Res. Commun. 153, 294–300. doi: 10.1016/S0006-291X(88)81221-X Copyright © 2018 Florez, Suarez-Barrera, Morales, Rivera, Orduz, Ochoa, Guerra and Muskus. 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. Pardo-Lopez, L., Soberon, M., and Bravo, A. (2013). Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. FEMS Microbiol. Rev. 37, 3–22. doi: 10.1111/ j.1574-6976.2012.00341.x October 2018 | Volume 9 | Article 2461 Frontiers in Microbiology | www.frontiersin.org 14
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Age- and Gender-Related Mean Hearing Threshold in a Highly-Screened Population: The Korean National Health and Nutrition Examination Survey 2010–2012
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Objective This study aimed to identify the mean hearing thresholds in the South Korean population to establish reference data and to identify the age- and gender-related characteristics. Editor: Pietro Cipresso, IRCCS Istituto Auxologico Italiano, ITALY OPEN ACCESS Citation: Park YH, Shin S-H, Byun SW, Kim JY (2016) Age- and Gender-Related Mean Hearing Threshold in a Highly-Screened Population: The Korean National Health and Nutrition Examination Survey 2010–2012. PLoS ONE 11(3): e0150783. doi:10.1371/journal.pone.0150783 Background In evaluating hearing disability in medicolegal work, the apportionment of age- and gender- related sensorineural hearing loss should be considered as a prior factor, especially for the elderly. However, in the literature written in the English language no studies have reported on the age- and gender-related mean hearing threshold for the South Korean population. Age- and Gender-Related Mean Hearing Threshold in a Highly-Screened Population: The Korean National Health and Nutrition Examination Survey 2010–2012 Yun Hwi Park, Seung-Ho Shin*☯, Sung Wan Byun*☯, Ju Yeon Kim ☯These authors contributed equally to this work. * drshinsh@gmail.com (SS); byunsw66@gmail.com (SB) ☯These authors contributed equally to this work. * drshinsh@gmail.com (SS); byunsw66@gmail.com (SB) RESEARCH ARTICLE Methods This study is based on the Korea National Health and Nutrition Examination Survey (KNHANES) 2010–2012, which was conducted by the Korean government, the data of which was disclosed to the public. A total of 15,606 participants (unweighted) representing 33,011,778 Koreans (weighted) with normal tympanic membrane and no history of regular or occupational noise exposure were selected and analyzed in this study. The relationship between the hearing threshold level and frequency, age, and gender was investigated and analyzed in a highly-screened population by considering the sample weights of a complex survey design. Copyright: © 2016 Park et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Conclusions The data from KNHANES 2010–2012 showed gender differences at hearing thresholds of 3 kHz, 4 kHz, and 6 kHz in a highly-screened population. The most significant gender differ- ence in relation to hearing threshold was observed at 4 kHz. The hearing thresholds at all of the tested frequencies worsened with increasing age. The mean hearing thresholds sug- gested in this study will be useful for the formulation of healthcare-related hearing policies and used as reference data for disability ratings for hearing loss due to various causes. Results Funding: The work was supported by the Ewha Womans University Research Grant of 2014. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. A gender ratio difference was found between the unweighted and the weighted designs: male:female, 41.0%: 59.0% (unweighted, participants) vs. 47.2%:52.8% (weighted, repre- senting population). As age increased, the hearing threshold increased for all frequencies. Hearing thresholds of 3 kHz, 4 kHz, and 6 kHz showed a statistical difference between both genders for people older than 30, with the 4 kHz frequency showing the largest difference. This paper presents details about the mean hearing threshold based on age and gender. Competing Interests: The authors have declared that no competing interests exist. 1 / 13 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 Age-Related Gender Difference of Mean Hearing Threshold Level Introduction When court-ordered hearing examination is requested due to car accidents or work-related injuries, the age and gender of the patient are very important when determining the allocation of causation among the multiple factors that caused or significantly contributed to the injury or disease, and the resulting impairment. If a victim has no record of having undergone a hear- ing test prior to the car accident or work-related injury, the hearing threshold should be esti- mated. For example, in medicolegal cases, it might be difficult to consider if the pre-accident hearing threshold of a 70-year-old man is similar to that of a 20-year-old woman. Only a few reports have provided reference data for age- and gender-related hearing threshold levels for Koreans, and the studies that did had limitations in that their study population was uncertain [1] or the study sample suffered from selection bias [2]. For example, the data were collected from the health check-ups of healthy examinees [2] or from patients at a single institution [3, 4]. Recently, some articles have been published about hearing loss based on the Korean National Health and Nutrition Examination Survey (KNHANES) 2010–2012, which was con- ducted by the Korea Centers for Disease Control and Prevention. However, no study has reported on mean hearing thresholds for the Korean population using a complex survey design to analyze the data. When a complex design is ignored during statistical analysis, it results in biased estimates and overstated significance levels [5–7]. The KNHANES 2010–2012 was a complex, stratified, multistage, probability-cluster survey of a representative sample of the non-institutionalized civilian population in South Korea. Therefore, it provides reliable audiological data for stratified samples representing the entire Korean population. This present study aims to extract and objectively analyze the hearing data and to show the age- and gender-related authentic mean hearing threshold. PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 Subjects and Methods Study Design (Complex Survey Design), Population, and Data Collection Selection of Subjects To determine the mean hearing thresholds of the Korean population not affected by factors that are hazardous to hearing, including middle ear disease or noise exposure, better ears were selected and then the following exclusion criteria were established: participants with abnormal tympanic membrane on otoscopy or a history of regular or occupational noise exposure were excluded. To determine which ear (right or left) was better in individual participants, six-tone average thresholds (0.5, 1, 2, 3, 4, and 6 Hz), four-tone averages (0.5, 1, 2, and 3 kHz), and thresholds at 1 kHz, 2 kHz, 0.5 kHz, 3 kHz, 4 kHz, and 6 kHz, in both ears, were compared one-by-one. For example, if the six-tone average for the right ear is smaller than the left ear, the right ear is the better ear regardless of all the other thresholds. If the six-tone averages for both ears are the same, the four-tone averages for both ears are compared. Table 1 shows the detailed exclusion procedures; 83.5% (weighted) of the representative popu- lation were included in this study. Age-Related Gender Difference of Mean Hearing Threshold Level Table 1. Selection of Participants with Normal Tympanic Membrane and No History of Noise Exposure. Participants (Unweighted, n = 18,582) Representative Population (Weighted, n = 39,515,552) Exclusion criteria No Yes No Yes Abnormal ear drum (A) 17,476 (94.0%) 1,106 (6.0%) 37,457,444 (94.8%) 2,058,108 (5.2%) History of noise exposure (B) 16,590 (89.3%) 1,992 (10.7%) 34,805,691 (88.1%) 4,709,861 (11.9%) (A) or (B) 15,606 (84.0%) 2,976 (16.0%) 33,011,778 (83.5%) 6,503,774 (16.5%) doi:10.1371/journal.pone.0150783.t001 Table 1. Selection of Participants with Normal Tympanic Membrane and No History of Noise Exposure. doi:10.1371/journal.pone.0150783.t001 the sampling rate, response rate, and age/sex proportions of the reference population into account in order to provide representative estimates of the Korean civilian population. Previ- ous articles have described the KNHANES methodology in detail [5, 8–11]. the sampling rate, response rate, and age/sex proportions of the reference population into account in order to provide representative estimates of the Korean civilian population. Previ- ous articles have described the KNHANES methodology in detail [5, 8–11]. A total of 23,621 individuals (10,611 males and 13,010 females), representing 47,761,044 individuals (23,884,864 males and 23,876,180 females) in South Korea participated in the sur- vey from July 2010 to December 2012. Among them, pure tone audiometry data were acquired from 8,147 male participants representing 19,826,055 Korean men and 10,435 female partici- pants representing 19,689,497 women, whose ages ranged from 12 to 85. Study Design (Complex Survey Design), Population, and Data Collection Study Design (Complex Survey Design), Population, and Data Collection The KNHANES is an ongoing cross-sectional survey of the civilian non-institutionalized popu- lation of South Korea. Many field survey teams performed interviews and physical and labora- tory examinations. Those teams included otolaryngologists, ophthalmologists, and nurse examiners with mobile examination units. The KNHANES participants were not randomly sampled. Since the survey was designed using a complex, stratified, multistage probability-sampling model based on the National Cen- sus Data, individual participants were not equally representative of the Korean population. The KNHANES website provides survey datasets that include information regarding the survey location, strata by age, sex, and various other factors, and the sample weight for each partici- pant. In all of the analyses, survey sample weights were used, which were calculated by taking 2 / 13 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 Audiometric Measure Pure-tone audiometric testing was performed with a GSI SA-203 audiometer (Entomed; Lena Nodin, Sweden). Testing was conducted in a sound-treated booth inside the mobile examina- tion center reserved for KNHANES. Otolaryngologists, who had been trained to operate the audiometer, provided instructions to participants and obtained the recordings. All audiometric testing was done under the supervision of an otologist. Only air conduction thresholds were measured. Supra-auricular headphones were used in the sound-treated booth. The otolaryngol- ogist provided basic instructions to the participant regarding the automated hearing test. Auto- mated testing was programmed according to a modified Hughson-Westlake procedure with appropriate masking. The lowest level at which the subject responded to 50% of the pure tone was set as the threshold. The automated hearing test involving air-conducted pure-tone stimuli showed good test-retest reliability and validity comparable to the manual pure-tone audio test [12, 13]. Participants responded by pushing a button when they heard a tone, and the results were automatically recorded [9]. The following frequencies were tested: 0.5 kHz, 1 kHz, 2 kHz, 3 kHz, 4 kHz, and 6 kHz. A retest threshold for 1 kHz was obtained to confirm threshold mea- surement consistency. Unless the difference between the two thresholds at 1 kHz was less than 3 / 13 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 Age-Related Gender Difference of Mean Hearing Threshold Level 5 dB, another audiogram was automatically repeated until that difference was less than 5 dB. To compare the hearing thresholds, the four-tone average (0.5 kHz, 1 kHz, 2 kHz, and 3 kHz) was calculated. The four-tone average is recommended by the American Academy of Otolaryn- gology-Head and Neck Surgery (AAO-HNS) [14]. Statistical Analysis Statistical analyses and graphing were performed using R software (version 3.2.1; The R Foun- dation, Vienna, Austria, URL http://www.R-project.org). The statistical values, including mean hearing thresholds and frequency averages, were calculated with the 'survey' package from the R repository sites. The package was developed for analyses of complex survey samples and this made it possible to provide statistics taking sample weights and strata into consideration. Cross-tabulations, the calculations of means, and t-tests were performed using the 'svytables', 'svymean', and 'svyttest' functions, respectively (in contrast to 'mean', 'xtabs', and 't.test' func- tions for simple random sampling), according to gender and age. P values<0.05 were consid- ered statistically significant. Cross-tabulations, the calculations of means, and t-tests were performed using the 'svytables', 'svymean', and 'svyttest' functions, respectively (in contrast to 'mean', 'xtabs', and 't.test' func- tions for simple random sampling), according to gender and age. P values<0.05 were consid- ered statistically significant. To find the distribution difference of the unweighted sample number and the representative population number by age and gender, a plot was made with triple Y axes (Fig 1). Mean hear- ing thresholds by age and gender according to frequency were calculated. Plots were made to discover the relationship between hearing threshold level and frequency, age, and gender (Fig 2). To compare the mean hearing threshold of both genders for each age group (12–19, 20–29, 30–39, 40–49, 50–59, 60–69, 70–79, and 80–85) at the six tested frequencies and the four-tone averages, multiple t-tests, adjusted by Bonferroni correction and based on the complex survey design, were performed. Plots were made to verify the gender difference of the four-tone aver- ages (Fig 3). A mean pure tone audiogram based on age group is shown in Fig 4. Ethics Statement The KNHANES obtained a written informed consent from each participant prior to conduct- ing the survey, and the Institutional Review Board of Ewha Womans University Mokdong Hospital approved this present research study (IRB No. ECT-201509-033). Results In the KNHANES 2010–2012 data, the number of highly-screened participants who underwent audiometric testing was 15,606, representing 33,011,778 Koreans (Table 2). The age of the par- ticipants ranged from 12 to 85. The mean age was 45.8±19.0 (unweighted) and 40.9±17.8 (weighted). Differences in the gender ratio were found between the unweighted and weighted designs: M:F, 41.0%: 59.0% (unweighted, participants) vs. 47.2%:52.8% (weighted, representing population) (Fig 1). In comparing the proportion of the unweighted and weighted groups by age (12–19, 20–29, 30–39, 40–49, and 50–59) in males, the unweighted groups were found to be under-sampled in contrast to the representative population. The unweighted groups by age (60–69, 70–79, and 80–85) in males were over-sampled in contrast to the weighted groups by the representative population. Table 3 and Fig 2 show the mean hearing threshold levels as a function of gender, fre- quency, and age. In addition, it provides the four-tone average threshold. Irrespective of gender and frequency, as the age of the participants increased, their hearing threshold increased. Table 3 shows the statistical difference of the mean hearing threshold level by gender and age at 0.5 kHz, 1 kHz, 2 kHz, 3 kHz, 4 kHz, and 6 kHz. At frequencies of 0.5 kHz, 1 kHz, and 2 kHz, there was little statistical difference in the mean threshold level between males and 4 / 13 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 Age-Related Gender Difference of Mean Hearing Threshold Level Fig 1. Age- and gender-related distribution of participants (unweighted, the left 1st axis) and the corresponding population (weighted, the left 2nd axis). The blue and red columns show the distribution of 15,606 participants, and the cyan and pink columns represent 33,011,778 members of the Korean population, according to gender and age groups. The proportion of the participants is different from the proportion of the study population. This figure shows Fig 1. Age- and gender-related distribution of participants (unweighted, the left 1st axis) and the corresponding population (weighted, the left 2nd axis). The blue and red columns show the distribution of 15,606 participants, and the cyan and pink columns represent 33,011,778 members of the Korean population, according to gender and age groups. The proportion of the participants is different from the proportion of the study population. This figure shows Fig 1. Age- and gender-related distribution of participants (unweighted, the left 1st axis) and the corresponding population (weighted, the left 2nd axis). Age-Related Gender Difference of Mean Hearing Threshold Level the difference between the occupation percentage of the unweighted and the weighted sample numbers, according to age group. If the unweighted design is used for analysis, male participants in their teens, twenties, thirties, forties, and fifties are under-sampled and participants in their sixties, seventies, and eighties are over-sampled. These ratio differences would affect the final outcome. In order to produce unbiased cross-section estimates for the entire Korean population, the specialized statistical technique of complex sampling design, considering survey sample weights and strata, should be utilized. UWt: Unweighted, Wt: Weighted. doi:10.1371/journal.pone.0150783.g001 females. However, for males older than 30 the mean threshold levels at 3 kHz, 4 kHz, and 6 kHz were significantly worse than the levels for females in the same age group. As seen in Table 3, the difference between the hearing thresholds for males and females at 3 kHz, 4 kHz, and 6 kHz began in the 30–39 age group and increased with increasing age until the 60–69 age group; however, the differences in these three hearing thresholds decreased in the 70–79 and 80–85 age groups for both genders. Among the latter three frequencies, the gender difference of the mean threshold level at 4 kHz was the most significant. g The four-tone (0.5 kHz, 1 kHz, 2 kHz, and 3 kHz) averages are shown in Fig 3. The average between males and females was similar except for the 30–39, 40–49, 50–59, and 60–69 age groups. The four-tone (0.5 kHz, 1 kHz, 2 kHz, and 3 kHz) averages are shown in Fig 3. The average between males and females was similar except for the 30–39, 40–49, 50–59, and 60–69 age groups. Fig 4 shows the mean audiograms for the Korean population as a function of gender, fre- quency, and age. As the age of the male or female participants increased, the hearing thresholds at all frequencies increased. The higher the frequencies, the greater the hearing loss. Fig 4 shows the mean audiograms for the Korean population as a function of gender, fre- quency, and age. As the age of the male or female participants increased, the hearing thresholds at all frequencies increased. The higher the frequencies, the greater the hearing loss. Results The blue and red columns show the distribution of 15,606 participants, and the cyan and pink columns represent 33,011,778 members of the Korean population, according to gender and age groups. The proportion of the participants is different from the proportion of the study population. This figure shows PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 5 / 13 Discussion This study used three years of pooled data: 2010, 2011, and 2012. The KNHANES 2010–2012 sampling weights ranged from 75.66 to 45,812.38 persons, and the median and mean values were 4,792.564 and 6470.565, respectively. The number of participants in 2010, 2011, and 2012 were 6,532, 6,302, and 5,817, respectively. The total Korean non-institutionalized population in 2010, 2011, and 2012 was 39,514,595, 40,324,566, and 38,978,241, respectively. The KNHANES guideline for data processing suggests that, in order to adjust sampling weights, the sample weights should be divided by 3; that is, the number of years of data pooled. Thus, the adjusted and pooled number representing the total Korean non-institutionalized population 2010–2012 was 39,605,801. While several papers addressing hearing thresholds for the Korean population have been published, they have only focused on certain groups, including visitors at health screening cen- ters, adolescents, male firefighters, the elderly, and residents of specific regions [1–4, 15]. Kim et al. presented hearing thresholds of 462 Korean adolescents who used a personal music player [3]. While no gender differences for the thresholds at all frequencies was found in the 12–19 age group in the present study, the authors did find gender differences at 4 kHz for the same age group. In addition, the three-tone average hearing thresholds in their study were much worse than the three-tone average thresholds in our study. It is assumed that the differences in the findings of these two studies resulted from the exclusion of participants with a history of noise exposure. Kang et al. compared the hearing threshold of male firefighters with hearing data from a screened and unscreened male population [15]. They found that male firefighters that were younger than 45 had worse hearing than the screened population. It is difficult to directly compare their results with the results from our study, as they sup- plied median hearing thresholds. Bahng and Lee reported the hearing thresholds of 263 people whose ages ranged from 60 to 84 [1]. They found that the hearing thresholds gradually wors- ened with increasing age, and they showed gender differences in hearing thresholds at 1 kHz, 2 kHz, 3 kHz, 4 kHz, 6 kHz, and 8 kHz. Kim et al. reported on the hearing thresholds of 6,028 people who lived in three provinces in Korea [2]. They found that hearing loss was more preva- lent in males than females over 65. PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 Age-Related Gender Difference of Mean Hearing Threshold Level Fig 2. Gender difference of the mean hearing threshold in proportion to age at 0.5 kHz (A), 1 kHz (B), 2 kHz (C), 3 kHz (D), 4 kHz (E), and 6 kHz (F) frequencies (highly-screened population, N = 33,011,778 ears). At 0.5 kHz, 1 kHz, and 2 kHz, no difference in the mean threshold level was found between males and females. However, at 3 kHz, 4 kHz, and 6 kHz, the mean threshold levels for males are worse than the levels for females. Among the three frequencies, the most significant difference of the mean threshold level between males and females with the same age occurs at 4 kHz. doi:10.1371/journal.pone.0150783.g002 Kim et al. reported gender differences with hearing thresholds for 1,116 visitors at a health screening center that were otologically normal, and they reported no occupational or non- occupational noise exposure [4]. They found that males had a significantly greater rate of change in their hearing thresholds at 4 kHz and 8 kHz than females. The hearing threshold at 6 kHz was not checked in that study. While gender-related differences of mean threshold at each frequency were evaluated in the present study, the threshold change at each frequency by age was analyzed. In [4], the threshold change at 8 kHz was larger than the change at 4 kHz for males. However the gender difference of mean hearing threshold at 4 kHz by age groups was not stated. The study subjects were healthy clients who visited the health screening center. In that study, males who had a history of military service were excluded. Therefore, the sample size of males (N = 214) was remarkably smaller than the sample size (N = 902) of females. In contrast, in this present study, the study subjects were members of the general population and the sample size of males and females was similar. The authors provided mean thresholds and 95% confidence intervals by age groups. In this present study, while all the mean thresholds increased depending on age at all frequencies, they did not consistently increase in [4], proba- bly because the sample size in certain age groups was too small. Hoffman et al. reported hearing results from the US National Health and Nutrition Exami- nation Survey (NHANES), 1999–2004 [16]. Their analysis was given in median (95% confi- dence intervals in an unscreened US population). Discussion 6 / 13 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 Age-Related Gender Difference of Mean Hearing Threshold Level PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 7 / 13 In this present study, hearing thresholds in a highly-screened Korean population were obtained in mean value. To compare both studies, the data were divided in the same age groups. And then median thresholds at 0.5, 1, 2, 3, 4 and 6 kHz from our data were calculated. Across age and sex groups, difference of median thresh- olds between the NHANES 1999–2004 and KNHANES 2010–2012 at 0.5, 1, 2, 3, 4 and 6 kHz (8 kHz was not tested in this study) were less than 3 dB except three thresholds at 3 kHz for females aged 55 to 64 years and 6 kHz for females aged 35 to 44 and 45 to 54 years. So the median values of most frequencies in both studies were very similar. See the S2 Table of sup- porting files. There have been controversies about gender differences in relation to hearing thresholds in the literature. Some studies have reported that there was no difference in hearing thresholds between males and females [17]. In contrast, other studies have found that hearing was worse in males than it was in females [1, 18–22]. In the present study, the remarkable finding is that 4 kHz is the frequency that showed the largest gender difference for mean hearing threshold (Fig 2E). Fig 2D and 2F shows that there is less of a gender difference for the mean threshold at 3 kHz and 6 kHz than at 4 kHz. The exclusion of subjects exposed to regular or occupational noise exposure was the minimum criteria used to eliminate the effect of noise on hearing thresholds. The real amount of exposure an individual has to noise cannot be investigated. It is assumed that the most remarkable reason for the gender difference at the 4 kHz threshold is due to socio-environmental factors, not biological differences; men are exposed to more noise than women. This hypothesis is supported by the tendency that gender difference for hearing thresholds decreased in retired people (>70 years). Fig 4 shows the mean pure-tone audiogram results by age and gender. As age increased, the mean threshold at each frequency increased in spite of gender. We thought that this pattern resulted from the aging effect. The hearing threshold for males was worse than it was for females. However, increased hearing thresholds at 6 kHz were noted, irrespective of age and 8 / 13 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 Age-Related Gender Difference of Mean Hearing Threshold Level Fig 3. The four-tone average of 0.5 kHz, 1 kHz, 2 kHz, and 3 kHz (PTA4) (highly-screened population, N = 33,011,778 ears). Both genders showed similar four-tone average values. However, with increasing age, the gender difference of PTA4 tends to increase. PTA4: four-pure tone average. doi:10.1371/journal.pone.0150783.g003 Age-Related Gender Difference of Mean Hearing Threshold Le doi:10.1371/journal.pone.0150783.g003 doi:10.1371/journal.pone.0150783.g003 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 9 / 13 Age-Related Gender Difference of Mean Hearing Threshold Level Fig 4. Mean audiograms for the Korean population as a function of gender, frequency, and age (highly-screened population, N = 33,011,778 ears). (A) Male; (B) Female. As age increased, the mean threshold at each frequency increased in both genders. *: The case in which the mean threshold is statistically significantly larger for males than for females. †emThe case in which the mean threshold is statistically significantly smaller for males than for females. Fig 4. Mean audiograms for the Korean population as a function of gender, frequency, and age (highly-screened population, N = 33,011,778 ears). (A) Male; (B) Female. As age increased, the mean threshold at each frequency increased in both genders. *: The case in which the mean threshold is statistically significantly larger for males than for females. †emThe case in which the mean threshold is statistically significantly smaller for males than for females. doi:10.1371/journal.pone.0150783.g004 gender, as compared to the other frequencies. This outcome might be due to the known cali- bration problem in the TDH-39 earphone of the GSI SA-203 audiometer, which could give rise to a roughly 5 dB increase at 6 kHz compared to the average thresholds for most other audio- metric frequencies [16, 23]. The information from this present study, provided in Table 3 and Fig 4, can be used as ref- erence data to estimate the pre-accidental hearing threshold based on age and gender. Table 3. Mean Hearing Threshold Level and Standard Deviation as a Function of Gender, Frequency, and Ag tion, and Comparison between Mean Thresholds of both Genders (M: F = 15,575,206: 17,436,572, weighted). Bold: Statistically significant (p<0.05, using Bonferroni correction) PTA4: Four-tone average threshold = (0.5 kHz + 1 kHz + 2 kHz + 3 kHz threshold)/4. Standard Deviations here are based on the distribution of participants (sample). Standard Errors for estimates of the population mean are provided in the supporting information. doi:10.1371/journal.pone.0150783.t003 Supporting Information Supporting Information S1 Table. Detailed numeric data for Figs 2 and 3. (CSV) S2 Table. Comparison between KNHANES 2010–2012 and NHANES 1999–2004. (DOCX) S3 Table. Detailed values for Table 3 including 95% confidence interval. (XLSX) S3 Table. Detailed values for Table 3 including 95% confidence interval. (XLSX) Conclusions Gender difference in mean hearing threshold was observed at the frequencies of 3 kHz, 4 kHz, and 6 kHz from the data obtained from the KNHANES 2010–2012. The most significant gen- der-related hearing threshold difference was observed at 4 kHz. Our results will be useful for formulating healthcare-related hearing policies, and they can be used as basic reference data for disability ratings for hearing loss due to various causes. Table 2. Demographic Data of Highly-Screened Participants in the Korea National Health and Nutri- tion Examination Survey (KNHANES), 2010–2012. Processed as Simple random sampling design (unweighted) Complex survey design (weighted) Total No. of participants 15,606 33,011,778 Sex Male 6,399 (41.0%) 15,575,206 (47.2%) Female 9,207 (59.0%) 17,436,572 (52.8%) Mean age ± SD (year- old) 45.8 ±19.0 40.9 ±17.8 Age range (years) 12–85 12–85 doi:10.1371/journal.pone.0150783.t002 Table 2. Demographic Data of Highly-Screened Participants in the Korea National Health and Nutri- tion Examination Survey (KNHANES), 2010–2012. PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 10 / 13 Age-Related Gender Difference of Mean Hearing Threshold Level Table 3. Mean Hearing Threshold Level and Standard Deviation as a Function of Gender, Frequency, and Age Group in a Highly-Screened Popula- tion, and Comparison between Mean Thresholds of both Genders (M: F = 15,575,206: 17,436,572, weighted). Age group No. Participants No. Representing population Mean Hearing Thresholds (dB) 0.5 kHz 1 kHz 2 kHz 3 kHz 4 kHz 6 kHz PTA4 Male: Female Male: Female Male: Female Male: Female Male: Female Male: Female Male: Female Male: Female Male: Female 12–19 992: 904 2,431,315: 2,192,738 7.2: 6.9 2.0: 1.9 1.7: 2.1 1.9: 1.6 1.9: 2.0 9.0: 10.6† 3.2: 3.1 20–29 602: 960 2,709,851: 2,715,782 7.0: 6.8 3.1: 2.4 2.9: 3.3 2.0: 1.8 4.7: 3.1* 11.3: 11.5 3.7: 3.6 30–39 991: 1696 2,885,434: 3,289,781 8.6: 8.5 5.0: 4.1* 5.9: 5.0 5.9: 4.0* 11.0: 5.6* 17.7: 14.0* 6.4: 5.4* 40–49 996: 1515 2,933,533: 3,341,372 10.2: 10.5 7.1: 6.8 8.9: 8.5 12.9: 8.3* 20.9: 10.0* 27.4: 20.9* 9.8: 8.5* 50–59 1004: 1633 2,329,503: 2,749,101 12.7: 13.4 10.7: 10.9 13.8: 12.8 21.4: 14.7* 29.7: 16.8* 37.4: 29.6* 14.6: 13.0* 60–69 983: 1327 1,352,532: 1,618,758 17.4: 18.5 16.3: 17.0 22.5: 20.7 33.1: 24.1* 41.9: 26.5* 52.5: 41.3* 22.3: 20.1* 70–79 726: 1020 796,380: 1,295,721 23.3: 26.6† 22.9: 25.7† 30.9: 30.0 42.6: 35.4* 50.8: 38.8* 63.1: 56.2* 29.9: 29.4 80–85 105: 152 136,659: 233,319 31.8: 32.0 31.5: 32.6 39.5: 37.7 51.6: 44.5 57.8: 47.4* 74.10: 67.6 38.6: 36.7 Standard Deviation (dB) 12–19 ±7.0: ±6.7 ±6.7: ±5.8 ±7.2: ±7.2 ±7.9: ±7.9 ±8.6: ±9.7 ±10.0: ±12.4 ±5.6: ±5.3 20–29 ±7.6: ±8.2 ±6.5: ±7.6 ±7.7: ±9.9 ±10.0: ±13.7 ±12.9: ±17.7 ±14.6: ±19.0 ±6.1: ±7.3 30–39 ±9.8: ±12.1 ±10.2: ±13.3 ±12.4: ±16.4 ±17.7: ±20.7 ±19.4: ±21.6 ±21.1: ±22.0 ±10.3: ±13.2 40–49 ±13.7: ±17.9 ±15.0: ±19.5 ±18.4: ±20.4 ±20.8: ±21.9 ±20.8: ±20.5 ±21.6: ±20.6 ±14.4: ±17.8 50–59 ±7.4: ±6.6 ±6.8: ±6.3 ±7.8: ±6.8 ±8.2: ±7.3 ±8.5: ±8.8 ±10.4: ±10.8 ±6.1: ±5.1 60–69 ±7.3: ±8.0 ±6.8: ±8.0 ±6.8: ±8.8 ±7.3: ±9.0 ±8.9: ±10.4 ±10.8: ±12.8 ±5.2: ±6.8 70–79 ±10.1: ±12.6 ±10.2: ±13.0 ±11.4: ±13.9 ±12.2: ±14.8 ±13.5: ±16.4 ±16.9: ±19.9 ±9.2: ±11.8 80–85 ±15.8: ±16.0 ±16.4: ±17.0 ±16.5: ±16.4 ±17.3: ±18.2 ±18.0: ±19.7 ±19.9: ±19.3 ±14.7: ±15.0 Table 3. Mean Hearing Threshold Level and Standard Deviation as a Function of Gender, Frequency, and Age Group in a Highly-Screened Popula- tion, and Comparison between Mean Thresholds of both Genders (M: F = 15,575,206: 17,436,572, weighted). Bold: Statistically significant (p<0.05, using Bonferroni correction) PTA4: Four-tone average threshold = (0.5 kHz + 1 kHz + 2 kHz + 3 kHz threshold)/4. Standard Deviations here are based on the distribution of participants (sample). Standard Errors for estimates of the population mean are provided in the supporting information. References 1. Bahng J, Lee J. Hearing Thresholds for a Geriatric Population Composed of Korean Males and Females. J Audiol Otol. 2015; 19(2):91–6. doi: 10.7874/jao.2015.19.2.91 PMID: 26413575 2. Kim HN, Kim SG, Lee HK, Ohrr H, Moon SK, Chi J, et al. Incidence of presbycusis of Korean popula- tions in Seoul, Kyunggi and Kangwon provinces. J Korean Med Sci. 2000; 15(5):580–4. PMID: 11068997 3. Kim MG, Hong SM, Shim HJ, Kim YD, Cha CI, Yeo SG. Hearing threshold of Korean adolescents asso- ciated with the use of personal music players. Yonsei Med J. 2009; 50(6):771–6. doi: 10.3349/ymj. 2009.50.6.771 PMID: 20046416 4. Kim S, Lim EJ, Kim HS, Park JH, Jarng SS, Lee SH. Sex Differences in a Cross Sectional Study of Age-related Hearing Loss in Korean. Clin Exp Otorhinolaryngol. 2010; 3(1):27–31. doi: 10.3342/ceo. 2010.3.1.27 PMID: 20379399 5. Kim Y, Park S, Kim N-S, Lee B-K. Inappropriate Survey Design Analysis of the Korean National Health and Nutrition Examination Survey May Produce Biased Results. J Prev Med Public Health. 2013; 46 (2):96–104. doi: 10.3961/jpmph.2013.46.2.96 PMID: 23573374 6. Korea Centers for Disease Control and Prevention. Guideline for the evaluation of the fourth Korea National Health and Nutrition Examination Survey. Cheonwon: Korea Centers for Disease Control Prevention; 2009. p. 19–34. 7. National Center for Health Statistics. The National Health and Nutrition Examination Survey (NHANES): analytic and reporting guidelines. Atlanta: Centers for Disease Control and Prevention; 2012. p. 1–14. 8. Park HA. The Korea national health and nutrition examination survey as a primary data source. Korean J Fam Med. 2013; 34(2):79. doi: 10.4082/kjfm.2013.34.2.79 PMID: 23560205 9. Joo YH, Han KD, Park KH. Association of Hearing Loss and Tinnitus with Health-Related Quality of Life: The Korea National Health and Nutrition Examination Survey. PLoS One. 2015; 10(6):e0131247. doi: 10.1371/journal.pone.0131247 PMID: 26121026 10. Jun HJ, Hwang SY, Lee SH, Lee JE, Song JJ, Chae S. The prevalence of hearing loss in South Korea: data from a population-based study. Laryngoscope. 2015; 125(3):690–4. doi: 10.1002/lary.24913 PMID: 25216153 11. Oh IH, Lee JH, Park DC, Kim M, Chung JH, Kim SH, et al. Hearing loss as a function of aging and dia- betes mellitus: a cross sectional study. PLoS One. 2014; 9(12):e116161. doi: 10.1371/journal.pone. 0116161 PMID: 25549095 12. Swanepoel de W, Mngemane S, Molemong S, Mkwanazi H, Tutshini S. Hearing assessment-reliability, accuracy, and efficiency of automated audiometry. Telemed J E Health. 2010; 16(5):557–63. doi: 10. 1089/tmj.2009.0143 PMID: 20575723 13. Author Contributions Conceived and designed the experiments: SB. Performed the experiments: SB. Analyzed the data: SB. Contributed reagents/materials/analysis tools: YP JK. Wrote the paper: SS. Acknowledgments We would like to thank the 150 residents of the Otorhinolaryngology Departments of 47 train- ing hospitals in South Korea and the members of the Division of Chronic Disease Surveillance 11 / 13 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 Age-Related Gender Difference of Mean Hearing Threshold Level at the Korea Centers for Disease Control and Prevention for participating in this survey. We would also like to express our gratitude for the dedicated work they provided. PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 References Mahomed F, Swanepoel de W, Eikelboom RH, Soer M. Validity of automated threshold audiometry: a systematic review and meta-analysis. Ear hear. 2013; 34(6):745–52. doi: 10.1097/01.aud. 0000436255.53747.a4 PMID: 24165302 14. Committee on Hearing and Equilibrium guidelines for the evaluation of results of treatment of conduc- tive hearing loss. Otolaryngol Head Neck Surg. 1995; 113(3):186–7. PMID: 7675477 15. Kang TS, Hong OS, Kim KS, Yoon CS. Hearing among male firefighters: a comparison with hearing data from screened and unscreened male population. J Expo Sci Environ Epidemiol. 2015; 25(1):106– 12. doi: 10.1038/jes.2014.72 PMID: 25352160 16. Hoffman HJ, Dobie RA, Ko CW, Themann CL, Murphy WJ. Americans hear as well or better today com- pared with 40 years ago: hearing threshold levels in the unscreened adult population of the United States, 1959–1962 and 1999–2004. Ear hear. 2010; 31(6):725–34. doi: 10.1097/AUD. 0b013e3181e9770e PMID: 20683190 17. Abdel-Hamid O, Khatib OM, Aly A, Morad M, Kamel S. Prevalence and patterns of hearing impairment in Egypt: a national household survey. East Mediterr Health J. 2007; 13(5):1170–80. PMID: 18290411 12 / 13 PLOS ONE | DOI:10.1371/journal.pone.0150783 March 7, 2016 Age-Related Gender Difference of Mean Hearing Threshold Level 18. Corso JF. Age and sex differences in pure-tone thresholds. Survey of hearing levels from 18 to 65 years. Arch Otolaryngol. 1963; 77:385–405. PMID: 14041404 19. Moscicki EK, Elkins EF, Baum HM, McNamara PM. Hearing loss in the elderly: an epidemiologic study of the Framingham Heart Study Cohort. Ear hear. 1985; 6(4):184–90. PMID: 4043571 20. Kell RL, Pearson JCG, Taylor W. Hearing thresholds of an island population in north Scotland. Int J Audiol. 1970; 9(2–4):334–49. 21. Ciorba A, Benatti A, Bianchini C, Aimoni C, Volpato S, Bovo R, et al. High frequency hearing loss in the elderly: effect of age and noise exposure in an Italian group. J Laryngol Otol. 2011; 125(8):776–80. doi: 10.1017/S0022215111001101 PMID: 21729437 22. Baraldi Gdos S, de Almeida LC, Borges AC. Hearing loss in aging. Braz J Otorhinolaryngol. 2007; 73 (1):58–64. PMID: 17505600 23. Schlauch RS, Carney E. The challenge of detecting minimal hearing loss in audiometric surveys. Am J Audiol. 2012; 21(1):106–19. doi: 10.1044/1059-0889(2012/11-0012) PMID: 22271907 13 / 13
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A mesh morphing computational method for geometry optimization of assembled mechanical systems with flexible components
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International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 https://doi.org/10.1007/s12008-022-00850-z International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 https://doi.org/10.1007/s12008-022-00850-z International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 https://doi.org/10.1007/s12008-022-00850-z ORIGINAL PAPER Abstract In this paper an interactive computational methodology was developed assuming that shape and size optimization of flexible components can significantly improve energy absorption or storage ability in assembled systems with flexible components (AS-FC). A radial basis functions mesh morphing formulation in non-linear numerical finite element analysis, including contact problems and flow interaction, was adopted as optimal design method to optimize shape and size design parameters in AS-FC. Flexible components were assembled in finite element environment according to functional ISO-ASME tolerances specification; non-linear structural analysis with flow interaction analysis was performed. The results of the study showed that the proposed method allows to optimize the shape and size of the flexible components in AS-FC maximizing the system’s ability to absorb or store energy. The potentiality of the method and its forecasting capability were discussed for the case study of an automotive crash shock in which the specific energy absorption was increased by over 40%. The case studied refers to a simple flexible component geometry, but the method could be extended to systems with more complex geometries. Keywords Radial basis functions · ISO-ASME tolerances specification · Specific energy absorption · Shape and size optimization · Crash shock absorber Keywords Radial basis functions · ISO-ASME tolerances specification · Specific energy absorption · Shape and size optimization · Crash shock absorber A mesh morphing computational method for geometry optimization of assembled mechanical systems with flexible components Michele Calì1 · Rita Ambu2 Received: 30 October 2020 / Accepted: 28 January 2022 / Published online: 22 February 2022 © The Author(s) 2022 2 Materials and methods The computational optimization of shape and size of flexible components in assembled mechanical systems traditionally occurs through iterative processes in which geometric modi- fications are carried out at the first steps. In general, no single absolute best solution for geometry optimization of flexible components exists. Often the best solution is chosen as a compromise by experimentally testing different prototypes. In the present approach, the RBF, a class of interpola- tion mathematical functions, was used directly in the FEA environment to perform shape and size optimization of flexi- ble components via MM. Using RBF-MM on the discretized domain in CAE applications, it was possible to apply prede- fined displacements to a set of generated points, which are called source points, directly to the computational model in FE environment. In this way RBF-MM formulation allows to define, directly in the FE environment, a parametric model of flexible parts. In Fig. 1a, the workflow of the pro- posed methodology is schematized, while Fig. 1b shows the traditional method based on iterative processes in which geo- metric parametrization and modifications are the basis of the process. In this work, the AS-FC with parts much less rigid than others were specifically considered for the study of the influ- ence that the shape and the size of the flexible components have on the assembled system performance. The analysis was carried out though a proper combination of compu- tational methodologies whereby a mesh morphing (MM) formulation in non-linear numerical FEA, including con- tact problems and flow interaction was applied. In particular, through the evaluation of specific energy absorption (SEA), the proposed computational method allowed to optimize the shape and size of flexible components. The system’s ability to absorb energy was maximized respecting the correct values of ISO-ASME tolerances specification with which flexible components are assembled in the system directly in the FEA environment. Through the formulation illustrated in the following para- graph RBF-MM allows to carry out the geometric modifica- tions of shape and size of flexible components in non-linear numerical FEA, including non-linear sliding contacts and flow interaction. It is observable, in fact, that usually the deformation of flexible components in assembled mechanical systems occurs within a viscous fluid in order to increase its energy absorption performance [18]. 1 Introduction blies, where different parts interact with each other, occurs almost exclusively by means of experimental testing [6–8]. The design of AS-FC requires to consider particular precau- tions for an optimal performance under operation. Standards for dimensioning and tolerancing [1, 2] consider non-rigid parts which deform substantially from their manufactured condition and provide procedures for the geometrical specifi- cations of these parts, which generally apply in the restrained assembled condition. The behaviour of AS-FC is complex and their design specifications can be improved only through effective computational methodologies that study the inter- action among the parts [3–5]. This is confirmed by the fact that the dynamic characterization of many of these assem- Frequent is also the usage of AS-FC to dampen or absorb transient mechanical vibrations of components such as gears, supports, transmissions, chain drives, linkages, bearings, brakes, fasteners, sensors, cables, etc. in different mechan- ical systems (actuators, engines, transmission and transport systems, manufacturing systems, etc.) [9]. This is important since vibrations are a common source of undesirable effects such as wear, fatigue, noise, etc. in industrial, military, naval, and aerospace applications. Nowadays another widely used category of AS-FC is that of compliant mechanisms. They are flexible structures that gain mobility from deflection of flexible components rather than kinematic pairs [10]. In particular, in the field of robotics, it’s possible to find many recent studies concerning design methods [11] and topological optimization of com- pliant mechanisms [12]. Mainly two different types of AS-FC can be distinguished, namely extended assembled systems made up of several parts allhavingcomparablestiffness(aeronautical,automotiveand robotic structures) [13] and assembled systems, in which few 12 3 International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 576 finally, in Sect. 5, conclusion with closing remarks and future research developments are reported. parts are much less rigid than others (supports, dampers, iso- lators, etc.) [14]. For both of these types the design is not limited to its geometric dimensioning and modelling; interference and contact problem require the utilization of proper computa- tional approaches for the analysis of assembly functional behaviour. Several research studies demonstrated the need to consider tolerances in the calculation and simulation stages of the AS-FC development [15, 16]. In fact, the correct modelling of AS-FC requires consideration of geometrical and dimensional manufacturing inaccuracies as well as the deformations caused by external loads. The above defects subsequently affect the functional requirement and the effi- ciency of the AS-FC [17]. 123 2.1 Parametrization with RBF MM A parametric radius allowed to parameterize the limit- ing source points and to use the different geometric models obtained in finite element simulations. The boundary condi- tions fixed then the min and max values of the radius. The flexible parts of assembled systems were geometri- cally parameterized with the desired tolerances using an appropriate number of source points. Through source points, the geometric parametrization based on RBF-MM imple- mented shape modifiers, amplified by parameters, directly on the computational domain. New geometric configura- tions resulted from the displacement of a set of mesh regions (walls, boundaries or discrete points within the vol- ume) through the use of algorithms, based on RBFs, which smoothly propagated the imposed displacement to the sur- rounding volume. A NURBS surface obtained through an extrusion with a circular profile with a radius equal to the value of the parame- ter radius allowed, in this example, to evaluate the geometric precision of the morphing performed (Fig. 2g). Figure 2h, i show the final meshed model and the solid model respectively. Parametrization with RBF MM was employed in litera- ture to obtain shape-based optimization [24], but its use was recently also explored for advanced studies, such as steady [25] and unsteady fluid structure interaction problems, evo- lutive shape-based optimizations [26, 27]. The number and position of source points were established in such a way to ensure the functionality of the system, in relation to the established tolerances [19–21]. The advantages of the proposed approach can be so synthesized: there is no need to regenerate the grid; the robustness of the procedure is preserved; it has the capability to support different mesh types thanks to its meshless nature and the high parallelizable smoothing process. The morph- ing action integrated in a solver updating the computational domain interactively during the progress of the computation can be executed in RBF Morph across three steps: (1) setup: it consists of the manual definition of the morphing targets, i.e. the portions of the FEA mesh that will be updated, and morphing sources, i.e. the portions of the FEA mesh con- trolled, and the definition of the required movements (design parameters) of the points driving the shape deformation; (2) fitting: the solution of RBF system obtained by collecting the morphing sources; (3) smoothing: it is obtained by propagat- ing the displacement prescribed on sources to the volume mesh target. 2.1 Parametrization with RBF MM Evaluating the accuracy derived by morphing the source points were properly incremented introducing further driving points in the areas where unwanted deformation between the morphed surface and theoretic surfaces of the flexible component had maximum values. The surface of deformable parts was discretized by tri- angular membrane elements; their structural deformation calculation, according to the method of Arcaro [22, 23], assumed homogeneous and orthotropic linear elastic mate- rial. Figure 2 shows, as an example, the choice and the localization of the source points related to the study of a fillet in the edge of a parallelepiped. The solid straight parallelepiped in Fig. 2a was meshed with tetrahedral elements. A set of source points, equally spacedeachother, was definedonaboxshapedboundingvol- umeinordertolimitthespaceoftheMMaction(Fig.2b).The moving points of parallelepiped located on the part surface showed the domain of the RBF setup (Fig. 2c, d). The limit- ing source points are marked in red while the moving centres, belonging to the part, are highlighted in green (Fig. 2e, f). 2 Materials and methods To address these challenges the method proposed herein allows, through an appro- priate interactively integration of the CAE tools (MM, non-linear structural analysis and flow interaction), the optimization of the shape and size of flexible compo- nents. Fig. 1 Comparison between: a the proposed methodology and b the traditional geometric optimization approach The main original contribution of the proposed approach is that shape and size of flexible component can be analysed and optimized by directly modifying the calculation grid in FE simulation environment without the CAD model under- going any modification. This allows to quickly evaluate the influence of shape and size changes on AS-FC performance also taking into account assigned tolerances. For validation, the forecasting capability of the method is presented and verified for the case study of an automotive crash shock absorber. The paper is organized as follows: in Sect. 2 materials and methods of the proposed methodology are described; Sect. 3 illustrates the application and validation of the method to a crash shock absorber for car bumpers; in Sect. 4 the main results obtained with the methodology are discussed; Fig. 1 Comparison between: a the proposed methodology and b the traditional geometric optimization approach 123 577 International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 578 Table 1 RBF with global and compact support radial RBF with global support ’(r) Spline type (Rn) rn, nodd Thin plate spline (TPSn) rnlog(r), neven Multiquadric (MQ) √ 1 + r2 Inverse multiquadric (IMQ) 1 √ 1+r2 Inverse quadratic (IQ) 1 1+r2 Gaussian (GS) e−r2 RBF with compact support ’(r)  f(ξ), ξ ≤1, ξ  r Rsup Wendland (C0) (1 −ξ)2 Wendland (C2) (1 −ξ)4(4ξ + 1) Wendland (C4) (1 −ξ)6 35 3 ξ2 + 6ξ + 1  where x is the vector indicating the position of a generic node of the surface and/or volume mesh xk, is the ith source node position vector and ∥•∥is the Euclidean norm. The RBF fitting solution is possible when the RBF coeffi- cients vector γi and the weights of the polynomial corrector vector βi are found so that, at source points, the interpolant function has the specified (known) values of displacement gi, whilst the polynomial terms give a null contribution, namely the following relations are simultaneously verified: s(xsi)  gsi, 1 ≤i ≤N (2) N  i1 γi p(xsi)  0 (3) (2) (3) assuming that source points are not contained in the same plane. RBF Morph operates the smoothing of mesh nodes with the formulation of the interpolant: for all polynomials q with a degree less than or equal to that of polynomial h. A unique RBF interpolant exists if the RBF is conditionally positive definite. Moreover, if the order is less than or equal to 2, a linear polynomial applies: ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩ sx(x)  N  i1 γ x i ϕ(x −xsi) + βx 0 + βx 1 x + βx 2 y + βx 3 z sy(x)  N  i1 γ y i ϕ(x −xsi) + β y 0 + β y 1 x + β y 2 y + β y 3 z sz(x)  N  i1 γ z i ϕ(x −xsi) + βz 0 + βz 1x + βz 2 y + βz 3z (9) h(x)  β0 + β1x + β2y + β3z (4) (4) allowing for rigid body translations recover. In this case, the interpolant has the form: allowing for rigid body translations recover. In this case, the interpolant has the form: s(x)  N  i1 γiϕ(∥x −xki∥) + β0 + β1x + β2y + β3z (5) (5) Common radial functions are summarized in Table 1. International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 The distance function (global supported and bi-harmonic in 3D), used by default, performs very well in volume morphing since reaching very good quality. and γi and βi values are obtained by this solution of the system system According to Eq. (9), every node of the volume mesh to be morphed has a new position computed in a meshless way using as input just its original position and the RBF expansion (Eq. 10):  M Ps PT s 0  γ β    gs 0  (6) (6) where U is the interpolation matrix whose elements are derived by calculating all the radial interactions between source points as follows: xnodenew  xnode + ⎡ ⎣ sx(xnode) sy(xnode) sz(xnode) ⎤ ⎦ (10) (10) Mi j  ϕ xsi −xsj  , 1 ≤i ≤N, 1 ≤j ≤N (7) (7) 2.2 RBF MM theory The mathematical solution of the RBF problem consists in calculatingthecoefficientsofalinearsystemoforderequalto thatofthenumberofsourcepoints[28,29].Thedisplacement of an arbitrary node of the grid is imposed as the sum of the radial contributions of each source node. In this way, the morphing of the mesh can be applied quickly while keeping the grid topology unchanged in terms of total number and type of elements. Fig. 2 Parametrization of an edge fillet of a cube with RBF MM The solution is unique with compatibility for rigid motions when the RBF interpolant is composed by a radial function containing the RBF ϕ and a multivariate polynomial correc- tor vector h of order m –1, where m is the order of ϕ. If N is the total number of source points, the RBF interpolant is: s(x)  N  i1 γiϕ(∥x −xi∥) + h(x) (1) (1) Fig. 2 Parametrization of an edge fillet of a cube with RBF MM 123 123 International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 3 CASE STUDY: the crash shock absorber The crash shock absorber (Fig. 4) is a mechanical energy storage device in which a piston (or a leaf spring) moves (and/or deforms) within charged viscoelastic polymer blend to develop a damper force over an axial displacement. In this loop in which CFD and Structural analyses exchanged geometrical data a proper Mesh updating with RBF allowed to edit shape and size of flexible components in order to maximize the system’s ability to absorb or store energy. The geometric shape of its three assembled components (sheath, stem and leaf spring) determines this axial dis- placement between the point “A” of the stem and the point “B” of the sheath (Fig. 5a) and the maximum energy the crash absorber can dissipate (green area in Fig. 5b). The leaf spring sliding and deforming within charged viscoelas- tic polymer blends inside the sheath produces the parabolic force response dissipating energy. In the FE model frictional contact formulation using CONTA175 and TARGE169 elements was adopted. Static and dynamic frictional coefficients were set as 0.2. Thus, contact pair component (also flexible) could slide and sepa- rate. A functional dimensioning of the components was car- ried out according to the standards ASME 14.5 [1] in order to provide, through geometric dimensioning and toleranc- ing (GD&T), those variations allowed for manufacturing in relation with the assembling conditions. 2.3 Structural-CFD set-up analysis and P is a constraint matrix that arises balancing the poly- nomial contribution: Using the commercial tools ANSYS Mechanical® with the ACT RBF Morph™plugin extension and ANSYS- CFD® (ANSYS Inc., Canonsburg, PA, USA) a fluid-dynamics anal- ysis coupled with finite element structural analysis of the flexible components of assembled systems was carried out through a 2-way Structural-CFD simulation, synthesized in Fig. 3. Ps  ⎛ ⎜⎜⎜⎝ 1 xs1 ys1 zs1 1 xs2 ys2 zs2 ... ... ... ... 1 xsN ysN zsN ⎞ ⎟⎟⎟⎠ (8) (8) 123 123 International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 579 Fig. 3 Workflow of 2-way structural-CFD analysis cess. SEA can be expressed as Eq. (11) and evaluated during the structural-CFD analysis. A higher SEA indicates higher exploitation of material usage for energy-absorption and effi- ciency of the AS-FC design. SE A  ∫d 0 F(x)dx m (11) (11) m Fig. 3 Workflow of 2-way structural-CFD analysis The above-mentioned method was used to maximize the SEA of an automotive crash shock absorber. In the next sec- tion the values of the main parameters were evaluated and the potential of the proposed method was discussed. The process start with the CFD analysis of the baseline of flexible components at the design conditions. A mapping pro- cedure was then applied to transfer the fluid-dynamic loads to FE model of flexible components. The structural analysis solution, in terms of flexible components deformations, was used in order to update the fluid dynamic domain according to the estimated deformed shape. The CFD computation was restarted on the new configuration and the cycle continued until the flexible components deformed shape (for a steady condition) was reached. 2.4 RBF parameters Setting a maximum stroke of 100 mm, a maximum crush- ing speed of 600 mm/s and a maximum acceptable value for the internal material stress of 300 MPa, a fluid–structure interaction analysis of the device was performed by using the commercial tools ANSYS Mechanical® with RBF Morph™ plugin extension and ANSYS-CFD®. Assuming the mate- rial properties as linearly elastic and transversely isotropic, a “morphing” study was performed by varying the transver- Taking into account the results obtained from the Structural- CFD analysis for the baseline geometrical configuration of flexible components, the changes in shape and size were applied to the geometry of the flexible components accord- ing to the geometrical specifications and tolerances. The RBF solutions related to these changes were generated using the ’Surfs’ and ’Encap’ features of RBF Morph ™[30]. In par- ticular, for each shape modification a set of surfaces “Surfs” was used to fix the nodes that are required not to change their position, a cylindrical “Encap” to deform the surfaces inside a proper domain, limiting the morphing action while respecting the tolerances. Fig. 4 Car rear bumper crash shock absorber 2.5.1 Y absorption The evaluation of the ratio of the energy absorbed by the system to the mass of the flexible components measured the specific energy absorption (SEA) during the crushing pro- Fig. 4 Car rear bumper crash shock absorber 3 International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 580 Fig. 5 a Crash shock absorber FE model; b Dissipated energy sal dimensions (width “l” and height “h”) of the leaf spring (Table 2). The MM formulation, applied at the same time to the constrained parts, allowed them to be modified while maintaining the coupling tolerance values shown in Fig. 6. The discretized model is made of 256116 s order tetrahe- Fig. 5 a Crash shock absorber FE model; b Dissipated energy Fig. 6 Functional dimensioning: a leaf spring; b stem; c sheath Fig. 5 a Crash shock absorber FE model; b Dissipated energy sal dimensions (width “l” and height “h”) of the leaf spring (Table 2). The MM formulation, applied at the same time to the constrained parts, allowed them to be modified while maintaining the coupling tolerance values shown in Fig. 6. The discretized model is made of 256116 s order tetrahe- drons (TET 10) elements SOLID 187 with 584359 nodes. In the leaf spring-sheath-stem contact zone a denser mesh with patch conformity was employed using element “CONTA 174” and “TARGA 170” (Fig. 7). These elements allowed to simulate the friction forces in the contacts. Using Remote Point formulation axial F external loads were applied on the device ends. Geometric parameterization based on RBF-MM implemented shape modifiers directly on the computational domain. The new geometric configurations resulted from the Fig. 6 Functional dimensioning: a leaf spring; b stem; c sheath displacement of a mesh region, through the use of algorithms, based on RBFs, which smoothly propagated the imposed dis- placement to the surrounding volume. The RBF interpolation functions drove the MM of the discretized domain of the computational models by apply- ing predefined displacements in the set of 160 nodes (source Table 2 Geometric variations of leaf spring transversal dimensions Width « l»[mm] Height « h»[mm] Volume [mm3] Mass [g] Baseline values 15 3 10,800 87.1 Variation [%] −20÷40 −33÷66 5760÷22,800 46.4÷183.8 Variation absolute −3÷ + 6 −1÷ + 2 5760÷22,800 46.4÷183.8 Optimum values 18 4 12,960 104.5 12 3 3 International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 581 Fig. 2.5.1 Y absorption 7 Modified shape with morphing action shapeandsizedesignparametersofflexiblecomponentsused in AS-FC. The potentiality of the method and its forecasting capability were demonstrated for the case study of an auto- motive crash shock. In the proposed method these important features were combined: the definition of the FEA mesh portions to be updated, and the interactive definition of the required move- ments (design parameters) of the points driving the shape deformation (source points); the propagation of the displace- ment prescribed on source points to the volume mesh target with smoothing effect. Additionally, the paper focused on the specific energy absorption (SEA) maximization. In the system studied, an increase in SEA of over 40% was obtained. Two different geometrical modifications (Surfs and Encap) were simulated and the achieved results allowed for an optimal dimension- ing. Fig. 7 Modified shape with morphing action points) near the contact zones between leaf spring and stem. During the MM the nodes on the external profile moved according to the defined constrain varying l and h dimension (Fig. 6). Then, the RBF action was extended in the entire volume, smoothing the grid up to the prescribed surrounding limits. The obtained RBF solution was sent to the ANSYS Mechanical solver and a relation force–displacement was consequently obtained. The results obtained invite to extend the use of the pro- posed method also to further developments, e.g. for the optimization of mechanical components that dampen or absorb vibrations such as gears, supports, transmissions, chain drives, linkages, bearings, brakes, fasteners, sen- sors, cables, etc. in different mechanical systems (actuators, engines, transmission and transport systems, manufacturing systems, etc.). References 1. ASME Y14.5 (2018). Dimensioning and tolerancing. The Ameri- can Society of Mechanical Engineers, New York 4 Results Funding Open access funding provided by Università degli Studi di Catania within the CRUI-CARE Agreement. This paper belongs to a research path funded by Università degli Studi di Cata- nia (PIA.CE.RI.2020–2022 Linea 2 – Interdepartmental project GOSPEL–Code 61722102132). After validating the FE structural-fluidynamics model with an experimental quasi-static crushing test, the study con- tributed to enhance the understanding of the influence of geometry changes on dissipated energy. The optimum values of leaf spring transversal dimensions reported in Table 2 allowed to increase of almost 1 kJ (by over 40%) the device capability of storing energy (light blue area in Fig. 5b). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, 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 indi- cate 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 permitteduse,youwillneedtoobtainpermissiondirectlyfromthecopy- right holder. To view a copy of this licence, visit http://creativecomm ons.org/licenses/by/4.0/. Thanks to the described computational methodology, the performance of assembled system composed of flexible com- ponents was improved. The method, based on RBF-MM formulation, allowed to find the optimal geometry of the deformable components with respect to the original con- straints and geometry of the assembly. 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Proceedings of the Institution of Mechanical Engineers, Part B: Journal of Engineering Manufac- ture, 09544054211025775 (2021). 12 3 582 International Journal on Interactive Design and Manufacturing (IJIDeM) (2022) 16:575–582 20. Buhmann, M.D.: Radial basis functions. Cambridge University Press, New York (2003) 4. Falgarone H., Thiébaut F., Coloos J., Mathieu L.: Variation simula- tion during assembly of non-rigid components. Realistic assembly simulation with ANATOLEFLEX software. Procedia Cirp, 43, 202–207 (2016) 21. Sastry, S.P., Zala, V., Kirby, R.M.: Thin-plate-spline curvilinear meshingona calculus-of-variationsframework.Procedia Eng.124, 135–147 (2015) 5. Korbi,A.,Tlija,M.,Louhichi,B.,BenAmara,A.:ACADmodelfor tolerance analysis of non-rigid Planar parts assemblies. Procedia CIRP 70, 126–131 (2018) 22. Arcaro, V.F.: Analysis of orthotropic membrane structures, UNI- CAMP/FEC, http://www.arcaro.org/tension/main.htm (2004) 23. 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ELF5 modulates the estrogen receptor cistrome in breast cancer
PLOS genetics
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RESEARCH ARTICLE OPEN ACCESS Citation: Piggin CL, Roden DL, Law AMK, Molloy MP, Krisp C, Swarbrick A, et al. (2020) ELF5 modulates the estrogen receptor cistrome in breast cancer. PLoS Genet 16(1): e1008531. https://doi. org/10.1371/journal.pgen.1008531 Abstract Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endoge- nous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endo- crine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 pat- terns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interact- ing with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance. Editor: Matthew L. Freedman, Dana Farber Cancer Institute, UNITED STATES Received: July 8, 2019 Accepted: November 20, 2019 Published: January 2, 2020 ELF5 modulates the estrogen receptor cistrome in breast cancer Catherine L. Piggin1,2¤a, Daniel L. Roden1,2, Andrew M. K. LawID1,2, Mark P. Molloy3¤b, Christoph Krisp3¤c, Alexander SwarbrickID1,2, Matthew J. Naylor1,2,4, Maria Kalyuga1,2, Warren Kaplan1,2, Samantha R. OakesID1,2, David Gallego-OrtegaID1,2, Susan J. Clark1,2, Jason S. CarrollID5, Nenad BartonicekID1,2, Christopher J. OrmandyID1,2* 1 Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Victoria Street Darlinghurst Sydney, NSW, Australia, 2 St Vincent’s Clinical School, Faculty of Medicine, UNSW Sydney, Australia, 3 Australian Proteome Analysis Facility, Macquarie University, Sydney, Australia, 4 School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, Australia, 5 Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre Robinson Way, Cambridge, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤a Current address: Department of Biomedicine (DBM), University Hospital, Basel, Switzerland ¤b Current address: Northern Clinical School Royal North Shore Hospital, University of Sydney, Sydney, Australia ¤c Current address: University Medical Center Hamburg-Eppendorf, Institute of Clinical Chemistry and Laboratory Medicine, Mass Spectrometric Proteome Analysis, Hamburg, Germany * c.ormandy@garvan.org.au ELF5 and endocrine resistance GNT1063559), Cancer Council NSW (PG11-07, RG17-02, RG19-07), Cancer Institute NSW (DG00625), RT Hall Trust, Mostyn Family Foundation, Cue Clothing Co., Estee Lauder Australia and Sutton’s Motors Sydney. These sponsors played no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. GNT1063559), Cancer Council NSW (PG11-07, RG17-02, RG19-07), Cancer Institute NSW (DG00625), RT Hall Trust, Mostyn Family Foundation, Cue Clothing Co., Estee Lauder Australia and Sutton’s Motors Sydney. These sponsors played no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Author summary Two thirds of breast cancers are initially treated with endocrine therapy because they are likely to rely on estrogen for their proliferation. Understanding why this therapy ulti- mately fails in 2/3 of cases offers the chance of durable treatment. In 2012 we hypothesised that normal developmental cell fate decisions taken by mammary progenitor cells persist in tumours that are maintained by instances of a cancerous progenitor, and that a change in the relative influence of the two major transcription factors that drive progenitor cell fate, ER to specify the hormone sensing lineage, and ELF5 to specify the ER- alveolar line- age, may allow a cancer to shift control of proliferation from estrogen to ELF5. Here we show that these transcription factors are often co located at super enhancers, enhancers and at the promoters of differentially regulated genes. When the levels of ELF5 were increased this caused ER and its pioneer factor FOXA1 to move to new regions of the genome associated with resistance to hormonal therapy. We also showed that ELF both regulated and bound directly to members of the ER-transcriptional complex. These find- ings provide the first indication of the mechanisms by which an increase in ELF5 could drive the acquisition of endocrine resistance. Competing interests: The authors have declared that no competing interests exist. Editor: Matthew L. Freedman, Dana Farber Cancer Institute, UNITED STATES Editor: Matthew L. Freedman, Dana Farber Cancer Institute, UNITED STATES Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pgen.1008531 Copyright: © 2020 Piggin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Raw data is available via ArrayExpress E-MTAB-7641and Proteome exchange PXD013349. Funding: This work was supported by grants from the NHMRC Australia (GNT1068753, GNT1043400, 1 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 ELF5 binding to the genome overlaps with FOXA1 and ER binding We used doxycycline-inducible expression of human ESE2B, the ELF5 isoform expressed in mammary gland [22], combined with ChIP-seq from MCF-7 and T47-D breast cancer cells, to search for a transcriptional mechanism that allows ELF5 to modulate estrogen-driven gene expression. A series of quality control measures showed that our expression system [19] did not induce ELF5 levels beyond physiological levels (S1A Fig) and at the time points used ER and FOXA1 levels remained unchanged by induction of ELF5 (S8B and S8C Fig). MCF-7 ChIP-seq data set (ELF5, FOXA1, ER and H3K4Me3) had sufficient read depth with a low pro- portion of errors (S1B Fig), high quality scores (S1C Fig), low duplication levels (S1D Fig), and consistent GC content (S1E Fig). Ten percent of reads were called in peaks by MACS (S1F Fig) and signal strength was comparable across the four replicates, and the four ChIPs (S1G Fig). A principal components analysis showed that requiring binding peaks called by MACS [23] in three of the four replicates provided high discriminatory power without overly limiting peak numbers (S1H Fig) and a consensus of a peak in 3 of 4 replicates was used to define genomic binding sites. We found 28363 ELF5 peaks, many in the distal intergenic regions but about half in promoter regions (Fig 1A), with reduced frequency as distance from the transcriptional start site (TSS) increased (Fig 1B). An analysis using the MEME Suite [24], of DNA sequences at ELF5 binding peaks found that a consensus ELF5 binding motif was present with high prob- ability, as were consensus binding motifs for CTCF, FOXA1 and ER (Fig 1C). GREAT [25] was used to identify the function of genes potentially regulated by ELF5. Using the Molecular Signature Data Base (MSigDB) gene sets, perturbations such as response to treatment with estrogen, tamoxifen, EGF and chemotherapeutic drugs were significantly enriched (Fig 1D and S2A Fig), as were processes including peptidyl-proline modification and mammary mor- phogenesis, and pathways such as signal transduction and extracellular matrix, the interferon response and nuclear receptor action. The ELF5 peaks in MCF-7 cells overlapped very sub- stantially (80%) with ELF5 ChIP-seq peaks in T47-D cells (S2B Fig), showing a similar effect of ELF5 in a different cell line. Meme Suite also identified three sequences with homology to Alu transposable elements that overlapped ELF5 binding peaks and recurred in a variety of combinations (S3A Fig). ELF5 and endocrine resistance In 2012 we proposed that luminal breast cancer cells may retain some of the cell-fate plastic- ity of the progenitor cell population seen in normal development, and that the expressed can- cer phenotype results, at least in part, from the balance between ELF5 and ER influence [19]. Cancer cells may become insensitive to estrogen when ELF5 increases. Conditions that upre- gulate ELF5 expression, for example continuous endocrine therapy [19], or increased exposure to RANKL once resident in bone [17], may drive phenotypic conversion to an endocrine-resis- tant state via the remaking of the cell-fate decisions taken by the tumour initiating cells. To do this ELF5 must modulate ER-driven gene expression, but the mechanism is unknown. Here we report the result of a search for potential interactions between ELF5 and ER, using ChIP- seq, RIME and RNA-seq, in MCF-7 breast cancer cells, the model which alone has provided most of our understanding of the mechanisms of endocrine resistance [21]. We show that induction of ELF5 alters the ER cistrome and that this alteration is similar to that caused by the acquisition of endocrine resistance. We also show that ELF regulates, and interacts with, members of the ER transcriptional complex. These findings provide the first steps toward elu- cidation of the molecular mechanisms by which ELF5 modulates ER driven gene expression, to promote endocrine resistance. Introduction Estrogen receptor (ER) positive breast cancer is initially treated using endocrine therapy to withdraw estrogen, destroy its receptor, or alter ER-driven transcription [1]. ER+ breast cancer exhibits a unique and significant long-term risk of distant recurrence, characterized by renewed metastatic activity and resistance to endocrine therapy [2]. Resistance occurs via mul- tiple mechanisms [3, 4]. Mutations causing constitutive ER activity or hypermethylation of ER enhancers occur in response to estrogen withdrawal [5, 6]. Activation of signalling events downstream from ER can enhance or replace ER activation [7]. Repositioning of the ER tran- scriptional complex can occur [8–13], altering both the expression of individual genes and transcriptional programs [9]. These events ultimately regulate the basic cell-cycle and cell- death machinery, and disruptions here can also cause endocrine resistance [4]. Interventions at all of these points provide opportunities to treat endocrine-resistant disease. ELF5 is an ETS transcription factor that drives a cell fate decision by mammary progenitor cells, causing them to establish the ER negative (ER-) cell lineage responsible for alveolar devel- opment and milk production [14]. The hormones of pregnancy act on the ER+ hormone sens- ing cells to cause secretion of RANKL and other paracrine regulators, which cause the expansion of the stem cell compartment [15], and as they differentiate to produce progenitor cells, epigenetic mechanisms cause ELF5 to be expressed [16], which is induced further by RANKL, so driving further progenitor cell differentiation to make the mature milk-producing cells of the alveoli [17, 18]. Forcing ELF5 expression causes ER+ breast cancer cells to adopt gene expression patterns more like those seen in the ER- subtypes [19]. ELF5 levels increase when MCF-7 breast cancer cells are made resistant to endocrine therapies, and these cells also become dependent on ELF5 for their proliferation [19]. In mice, forcing ELF5 expression results in mammary tumor angiogenesis and an increase in lung metastasis via regulation of the innate immune system [20]. Thus ELF5 is associated with the two key aspects of progres- sion to the lethal phenotype in ER+ breast cancer, acquisition of endocrine resistance and renewed metastatic activity. This conclusion is supported by clinical data. In luminal A breast cancer patients treated with tamoxifen, high ELF5 expression strongly predicted early treat- ment failure and disease progression [20]. 2 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 Factors binding with ELF5 included ER-transcription complex mem- bers GREB1, TRPS1, SRC2, TLE3, MLL3, JUN, CBP, endocrine receptors RARG, RARA, RXRB, orphan nuclear receptor NR2C1, co-repressor RCOR1, co-activators NCOA2 (SRC2) and p300, and chromatin remodeller SMARCE 1 (S4A Fig). A very different pattern occurred at super enhancers (S4B Fig). MED1 is often used to define super enhancers and MED1 is fre- quently found to be a member of the 20+ transcription factor set including many members of the ER transcriptional complex such as ER, FOXA1, MLL3, GREB1, SRC2,TLE3 etc. NIPBL and Cohesin are also frequent members of this set, and are involved in enhancer-promoter communication. Fig 1E shows two instances of enhancers or super enhancers located near the PIP and RUNX1 genes. Near PIP ELF5 bound to three sites that also bound FOXA1 and a third site that bound ER. All three sites showed overlapping binding of a large number of tran- scription factors. The super enhancer near RUNX1 bound ELF5, and Diffbind [12] showed increased levels of FOXA1 and ER binding (blue squares), when ELF5 levels were induced. Enhancers in this region also bound both ELF5 and FOXA1 (S4C Fig). Enhancers showed pat- terns similar to promoters or super enhancers, suggesting either mis-annotation or dual func- tionality. Thus ELF5 binds to super enhancers together with a large number of transcription factors, but at the promoters of differentially expressed genes with a much smaller group. Many of the transcription factors with overlapping binding with ELF5 are members of the ER transcriptional complex. ELF5 and endocrine resistance different subtypes (S3E Fig). In transcriptionally active sites, such as highly occupied target binding regions (HOT) [27], enhancers and super enhancers, the odds ratio diverged from 1, and in genes that showed differential expression in response to ELF5 induction, there was sig- nificant enrichment of mammalian interspersed repeats (MIRs) (Odds ratio = 2, p = 8E-281) (S3E Fig). Thus ELF5 binds to the genome at ETS sites, many of which are located in repetitive elements [28]. These elements are rich in transcription factor binding sites and are proposed to act as transposable enhancers that drive genome evolution [29], by placing the binding of key transcription factors such as MYC, SP1 p53, RAR, ER, and now ELF5, into new genomic contexts [30]. Thus ELF5 is an additional transcriptional influence driving genome evolution by MIR elements and these sites remain active in the regulation of gene expression. y g g p We compiled public data from 134 MCF-7 ChIP-seq experiments (SDoc2 Text) and used correlation coefficients to identify sets of transcription factors that co-bind with ELF5, within genomic sub-regions such as enhancers, super enhancers [31], or genes with differential expression. The sets were visualized using UpSet [32] for the most frequent examples (S4A– S4C Fig), showing the number of times co-binding of the indicated set of transcription factors was observed. ELF5 binds to the genome with different groups of transcription factors at geno- mic loci with different functions. In the promoters of genes showing differential expression in response to elevated ELF5 the most frequent binding pattern was ELF5 in combination with a few transcription factors. For example, in 119 instances of genomic ELF5 binding to the pro- moters of differentially expressed genes, ELF5 bound alone with FOXA1 in 7 instances, with FOS alone in 6 instances, and with up to 6 transcription factors in 60 instances, most unique in their combinations. Factors binding with ELF5 included ER-transcription complex mem- bers GREB1, TRPS1, SRC2, TLE3, MLL3, JUN, CBP, endocrine receptors RARG, RARA, RXRB, orphan nuclear receptor NR2C1, co-repressor RCOR1, co-activators NCOA2 (SRC2) and p300, and chromatin remodeller SMARCE 1 (S4A Fig). A very different pattern occurred at super enhancers (S4B Fig). MED1 is often used to define super enhancers and MED1 is fre- quently found to be a member of the 20+ transcription factor set including many members of the ER transcriptional complex such as ER, FOXA1, MLL3, GREB1, SRC2,TLE3 etc. NIPBL and Cohesin are also frequent members of this set, and are involved in enhancer-promoter communication. Fig 1E shows two instances of enhancers or super enhancers located near the PIP and RUNX1 genes. Near PIP ELF5 bound to three sites that also bound FOXA1 and a third site that bound ER. All three sites showed overlapping binding of a large number of tran- scription factors. The super enhancer near RUNX1 bound ELF5, and Diffbind [12] showed increased levels of FOXA1 and ER binding (blue squares), when ELF5 levels were induced. Enhancers in this region also bound both ELF5 and FOXA1 (S4C Fig). Enhancers showed pat- terns similar to promoters or super enhancers, suggesting either mis-annotation or dual func- tionality. Thus ELF5 binds to super enhancers together with a large number of transcription factors, but at the promoters of differentially expressed genes with a much smaller group. Many of the transcription factors with overlapping binding with ELF5 are members of the ER transcriptional complex. We compiled public data from 134 MCF-7 ChIP-seq experiments (SDoc2 Text) and used correlation coefficients to identify sets of transcription factors that co-bind with ELF5, within genomic sub-regions such as enhancers, super enhancers [31], or genes with differential expression. The sets were visualized using UpSet [32] for the most frequent examples (S4A– S4C Fig), showing the number of times co-binding of the indicated set of transcription factors was observed. ELF5 binds to the genome with different groups of transcription factors at geno- mic loci with different functions. In the promoters of genes showing differential expression in response to elevated ELF5 the most frequent binding pattern was ELF5 in combination with a few transcription factors. For example, in 119 instances of genomic ELF5 binding to the pro- moters of differentially expressed genes, ELF5 bound alone with FOXA1 in 7 instances, with FOS alone in 6 instances, and with up to 6 transcription factors in 60 instances, most unique in their combinations. ELF5 binding to the genome overlaps with FOXA1 and ER binding Fur- ther investigation using RepeatMasker showed that mammalian inter-dispersed repeats (MIR) 3 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 ELF5 and endocrine resistance Fig 1. Genomic binding of ELF5 is co-located with ER and FOXA1, and occurs at promoter, enhancer and super Fig 1. Genomic binding of ELF5 is co-located with ER and FOXA1, and occurs at promoter, enhancer and super enhancer regions. Panel A, proportion of ELF5 binding to promoter regions at the indicated distance from the transcription start site (TSS) and to non-promoter regions. Panel B, distribution of ELF5 binding relative to the TSS. Panel C, positional distribution in base pairs (bp) (CentriMO plot) of the indicated transcription factor (TF) binding motifs relative to all ELF5 ChIP-binding sites centred at 0. Total probability for all curves in the figure is 1. Panel D Functional annotation by GREAT of ELF5 binding sites. Panel E, Transcription factor binding at enhancers and super enhancers near PIP and RUNX1 genes comparing ELF5, FOXA1 and ER binding with and without induction of ELF5 using doxycycline (DOX), in relation to the binding of the indicated transcription factors reported by ChIP-seq experiments in MCF-7 cells. Variation among replicates indicated by color shading. Blue boxes under FOXA1Dox and ERDox tracks indicate peaks called as statistically significantly increased (FDR<0.05) by DiffBind. https://doi.org/10.1371/journal.pgen.1008531.g001 of the SINE class were most the frequently found repetitive element at ELF5 peaks (S3B Fig), and that a consensus ETS motif was present within these repeats (S3C Fig). SINE elements were enriched close to Elf5 peaks but not at a distance (S3D Fig). Using Repbase to quantify this finding [26], we found that ELF5 peaks frequently overlapped a repetitive element of 4 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 Panel A, GO ontologies enriched in the RNA-seq data set by induction of ELF5, font size relative to enrichment score. Panel B, GSEA of RNA-seq data using the MSigDB oncogenic signatures gene sets. Heatmap shows all enriched sets arranged by nominal enrichment score, with the sets where pairs showed opposite enrichment highlighted in yellow and shown in detail above. Panel C, GSEA of a set of genes formed from proteins that interact with ER, overlaid on a volcano plot of all genes in the RNA-seq analysis. Genes with increased expression (left hand side- trailing edge) or decreased expression (right hand side- leading edge) of the enrichment are highlighted and listed, in blue, in their corresponding order. Panel D, enriched gene sets related to the indicated perturbations in MCF7 cells. MCF7 up or down indicates that the perturbation up- or down-regulates genes in the set. The top 10 MCF7 gene set overlaps with significantly up- regulated genes (left) and down-regulated genes (right) from the MCF7-ELF5 RNA-seq experiment are shown. https://doi.org/10.1371/journal.pgen.1008531.g002 Fig 2. ELF5 transcriptional effects measured by RNA-seq. Panel A, GO ontologies enriched in the RNA-seq data set by induction of ELF5, font size relative to enrichment score. Panel B, GSEA of RNA-seq data using the MSigDB oncogenic signatures gene sets. Heatmap shows all enriched sets arranged by nominal enrichment score, with the sets where pairs showed opposite enrichment highlighted in yellow and shown in detail above. Panel C, GSEA of a set of genes formed from proteins that interact with ER, overlaid on a volcano plot of all genes in the RNA-seq analysis. Genes with increased expression (left hand side- trailing edge) or decreased expression (right hand side- leading edge) of the enrichment are highlighted and listed, in blue, in their corresponding order. Panel D, enriched gene sets related to the indicated perturbations in MCF7 cells. MCF7 up or down indicates that the perturbation up- or down-regulates genes in the set. The top 10 MCF7 gene set overlaps with significantly up- regulated genes (left) and down-regulated genes (right) from the MCF7-ELF5 RNA-seq experiment are shown. https://doi.org/10.1371/journal.pgen.1008531.g002 breast cancer cells in response to induction of ELF5 [19] was very significantly (padj = 1.6E-5 GSEA) enriched, demonstrating that the effects seen in MCF-7 cells also occurred in another cell line. ELF5 and endocrine resistance Fig 2. ELF5 transcriptional effects measured by RNA-seq. Panel A, GO ontologies enriched in the RNA-seq data set by induction of ELF5, font size relative to enrichment score. Panel B, GSEA of RNA-seq data using the MSigDB oncogenic signatures gene sets. Heatmap shows all enriched sets arranged by nominal enrichment score, with the sets where pairs showed opposite enrichment highlighted in yellow and shown in detail above. Panel C, GSEA of a set of genes formed from proteins that interact with ER, overlaid on a volcano plot of all genes in the RNA-seq analysis. Genes with increased expression (left hand side- trailing edge) or decreased expression (right hand side- leading edge) of the enrichment are highlighted and listed, in blue, in their corresponding order. Panel D, enriched gene sets related to the indicated perturbations in MCF7 cells. MCF7 up or down indicates that the perturbation up- or down-regulates genes in the set. The top 10 MCF7 gene set overlaps with significantly up- regulated genes (left) and down-regulated genes (right) from the MCF7-ELF5 RNA-seq experiment are shown. https://doi.org/10.1371/journal.pgen.1008531.g002 breast cancer cells in response to induction of ELF5 [19] was very significantly (padj = 1.6E-5 Fig 2. ELF5 transcriptional effects measured by RNA-seq. Panel A, GO ontologies enriched in the RNA-seq data set by induction of ELF5, font size relative to enrichment score. Panel B, GSEA of RNA-seq data using the MSigDB oncogenic signatures gene sets. Heatmap shows all enriched sets arranged by nominal enrichment score, with the sets where pairs showed opposite enrichment highlighted in yellow and shown in detail above. Panel C, GSEA of a set of genes formed from proteins that interact with ER, overlaid on a volcano plot of all genes in the RNA-seq analysis. Genes with increased expression (left hand side- trailing edge) or decreased expression (right hand side- leading edge) of the enrichment are highlighted and listed, in blue, in their corresponding order. Panel D, enriched gene sets related to the indicated perturbations in MCF7 cells. MCF7 up or down indicates that the perturbation up- or down-regulates genes in the set. The top 10 MCF7 gene set overlaps with significantly up- regulated genes (left) and down-regulated genes (right) from the MCF7-ELF5 RNA-seq experiment are shown. https://doi.org/10.1371/journal.pgen.1008531.g002 breast cancer cells in response to induction of ELF5 [19] was very significantly (padj 1 6E 5 Fig 2. ELF5 transcriptional effects measured by RNA-seq. ELF5 driven changes in gene expression We used RNA-seq to examine the effects of induction of ELF5 on gene expression. Analysis of our RNA-seq data using Limma-Voom found 256 up-regulated genes and 291 down-regulated genes using the cut off values of FDR <0.05 and FC >1.5. The GO consortia functions of development, morphogenesis, differentiation and cell cycle were most prominently dimin- ished by induction of ELF5, while functions of amino acid biology, metabolic, catabolic, and translation were enhanced (Fig 2A). The set of genes showing differential expression in T-47D 5 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 ELF5 and endocrine resistance Using GSEA with the MSigDB oncogenic signature sets we observed 3 conditions that pro- duced changes in gene expression similar to induction of ELF5 (Fig 2B). Genes that went up or down in response to long term estrogen deprivation also went up or down in response to ELF5, consistent with ELF5 promoting an estrogen insensitive phenotype. Similarly, induction of ELF5 replicated the effects of induction of MYC. MYC is a direct transcriptional target of ELF5 and showed a fold change of 1.43 with FDR of 2E-4. ELF5 also produced effects seen in a model of EIF4GI function, a translation initiating factor linking nutrient starvation to inhibi- tion of MTOR and cell proliferation [33]. Using GSEA we examined the expression of a set of genes producing proteins identified as interacting with ER [34]. From this set 18 genes had increased expression (named on the left-hand side Fig 2C) and 49 showed decreased expres- sion (named on the right-hand side Fig 2C). Thus induction of ELF5 caused changes in the expression of genes encoding many members of the ER transcriptional complex. The Enrichr tool [35], showed that perturbations that opposed the changes in gene expression caused by the induction of ELF5 were dominated by treatment with various estrogens (Fig 2D). Using the MSigDB Hallmark sets, functions such as late estrogen response, interferon action, epithe- lial to mesenchymal transition and aspects of proliferation were significantly depleted, while early estrogen responses were increased together with many aspects of metabolism (S6A Fig), and similar effects on sets of estrogen regulated genes were found in the MSigDB C2 _all gene sets (S6B Fig). The GSEA was visualized using the Enrichment Map plugin for Cytoscape [36, 37] (S5 Fig, a scalable PDF that can be zoomed once downloaded), which presents a compre- hensive picture of these transcriptional responses to induction of ELF5. Overall the RNA-seq analysis shows that induction of ELF5 opposed estrogen-driven patterns of gene expression, consistent with the promotion of an estrogen insensitive phenotype. breast cancer cells in response to induction of ELF5 [19] was very significantly (padj = 1.6E-5 GSEA) enriched, demonstrating that the effects seen in MCF-7 cells also occurred in another cell line. 6 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 ELF5 causes FOXA1 and ER to bind to new regions of the genome in the same pattern caused by the acquisition of endocrine resistance The Enrichr tool [35] was used to identify transcription factor motifs in the promoters of genes showing altered gene expression in response to ELF5. These promoters were enriched for ER and FOXA1 binding motifs as well as ETS motifs (S6C–S6F Fig). We examined the co- occurrence of binding peaks in our ChIP data called present by MACS for ELF5, FOXA1 and ER using Venn analysis (S7A Fig with UpSet analysis in S7B Fig). There was a large overlap in the binding of these transcription factors, with 1244 genomic sites showing overlapping-bind- ing of ELF5, FOXA1 and ER, 7703 with overlapping-binding of ELF5 and FOXA1, and 194 with overlapping-binding of ER and ELF5. Induction of ELF5 caused FOXA1 to bind to 5875 new genomic sites and ER to bind to 1616 new sites. The probability that an ELF5 motif was present at sites of new binding was increased, but not where binding was lost (S7C and S7D Fig). There was no difference in the increase or decrease of FOXA1 binding sites at enhancers compared to super enhancers, and whether they were proximal or distal to the regions they regulate (S7E Fig), nor was there any variation in the sequence of the ELF5 binding motif at different genomic regions (S7F Fig). We used DiffBind to statistically test these associations of ELF5 with FOXA1 and ER. MACS simply calls peaks present or absent, but DiffBind deter- mines if a peak has shown a statistically significant increase or decrease in binding. At a false discovery rate of <0.05, induction of ELF5 caused ER and FOXA1 binding to increase at 404 and 503 sites (Fig 3A, top panel, UpSet analysis and corresponding Venn), and to decrease at 5 and 293 sites respectively (Fig 3A lower panel). The patterns of FOXA1 and ER genome bind- ing prior to, and following, induction of ELF5 are shown in Fig 3B, with enriched sites indi- cated in green. GREAT showed that the predominant function of these altered sites was estrogen action and endocrine resistance (Fig 3C). Using genome DNAase1 sensitivity data PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 7 / 24 ELF5 and endocrine resistance Fig 3. Induction of ELF5 alters FOXA1 and ER binding to the genome. Panel A, UpSet presentation of DiffBind output that identified statistically significant increases in FOXA1 or ER binding in response to induction of ELF5 with doxycycline (Dox). Corresponding Venn diagram for increased (enriched) peaks is shown. Lower panel, decreased peaks. Panel B, FOXA1 peaks that were increased by induction of ELF5 (+D, red) are plotted, centred on the FOXA1 peak, with the corresponding binding of FOXA1 at that location without induction of ELF5 (-D, blue). The resulting differential binding is shown in green. The binding of ER at these FOXA1 sites is shown with (+D, red), and without (-D, blue), with differential binding in green. Histograms show cumulative intensity on the y axis over the corresponding genomic region on the x axis. Color scale denotes log 10 of MACS (-D and +D columns) or DiffBind scores (E columns). Panel C, GREAT analysis of binding site function for increased binding sites for FOXA1 and ER. Blue indicates sets involved in estrogen action. Panel D, FOXA1 and ER binding sites that are specific in MCF-7 cells that were either sensitive to, or made resistant to, endocrine therapy, with corresponding ELF5 binding intensity. Histograms compare overall intensity with (+D) and without (-D) induction of ELF5 and green shows the enrichment (E) in response to ELF5. Color scale denotes log 10 of MACS (-D and +D columns) or DiffBind scores (E columns). Fig 3. Induction of ELF5 alters FOXA1 and ER binding to the genome. Panel A, UpSet presentation of DiffBind output that identified statistically significant increases in FOXA1 or ER binding in response to induction of ELF5 with doxycycline (Dox). Corresponding Venn diagram for increased (enriched) peaks is shown. Lower panel, decreased peaks. Panel B, FOXA1 peaks that were increased by induction of ELF5 (+D, red) are plotted, centred on the FOXA1 peak, with the corresponding binding of FOXA1 at that location without induction of ELF5 (-D, blue). The resulting differential binding is shown in green. The binding of ER at these FOXA1 sites is shown with (+D, red), and without (-D, blue), with differential binding in green. Histograms show cumulative intensity on the y axis over the corresponding genomic region on the x axis. Color scale denotes log 10 of MACS (-D and +D columns) or DiffBind scores (E columns). Panel C, GREAT analysis of binding site function for increased binding sites for FOXA1 and ER. Blue indicates sets involved in estrogen action. Panel D, FOXA1 and ER binding sites that are specific in MCF-7 cells that were either sensitive to, or made resistant to, endocrine therapy, with corresponding ELF5 binding intensity. Histograms compare overall intensity with (+D) and without (-D) induction of ELF5 and green shows the enrichment (E) in response to ELF5. Color scale denotes log 10 of MACS (-D and +D columns) or DiffBind scores (E columns). Fig 3. Induction of ELF5 alters FOXA1 and ER binding to the genome. Panel A, UpSet presentation of DiffBind output that identified statistically significant increases in FOXA1 or ER binding in response to induction of ELF5 with doxycycline (Dox). Corresponding Venn diagram for increased (enriched) peaks is shown. Lower panel, decreased peaks. Panel B, FOXA1 peaks that were increased by induction of ELF5 (+D, red) are plotted, centred on the FOXA1 peak, with the corresponding binding of FOXA1 at that location without induction of ELF5 (-D, blue). The resulting differential binding is shown in green. The binding of ER at these FOXA1 sites is shown with (+D, red), and without (-D, blue), with differential binding in green. Histograms show cumulative intensity on the y axis over the corresponding genomic region on the x axis. Color scale denotes log 10 of MACS (-D and +D columns) or DiffBind scores (E columns). Panel C, GREAT analysis of binding site function for increased binding sites for FOXA1 and ER. Blue indicates sets involved in estrogen action. Panel D, FOXA1 and ER binding sites that are specific in MCF-7 cells that were either sensitive to, or made resistant to, endocrine therapy, with corresponding ELF5 binding intensity. Histograms compare overall intensity with (+D) and without (-D) induction of ELF5 and green shows the enrichment (E) in response to ELF5. Color scale denotes log 10 of MACS (-D and +D columns) or DiffBind scores (E columns). Fig 3. Induction of ELF5 alters FOXA1 and ER binding to the genome. Panel A, UpSet presentation of DiffBind output that identified statistically significant increases in FOXA1 or ER binding in response to induction of ELF5 with doxycycline (Dox). Corresponding Venn diagram for increased (enriched) peaks is shown. Lower panel, decreased peaks. Panel B, FOXA1 peaks that were increased by induction of ELF5 (+D, red) are plotted, centred on the FOXA1 peak, with the corresponding binding of FOXA1 at that location without induction of ELF5 (-D, blue). The resulting differential binding is shown in green. The binding of ER at these FOXA1 sites is shown with (+D, red), and without (-D, blue), with differential binding in green. Histograms show cumulative intensity on the y axis over the corresponding genomic region on the x axis. Color scale denotes log 10 of MACS (-D and +D columns) or DiffBind scores (E columns). Panel C, GREAT analysis of binding site function for increased binding sites for FOXA1 and ER. Blue indicates sets involved in estrogen action. Panel D, FOXA1 and ER binding sites that are specific in MCF-7 cells that were either sensitive to, or made resistant to, endocrine therapy, with corresponding ELF5 binding intensity. Histograms compare overall intensity with (+D) and without (-D) induction of ELF5 and green shows the enrichment (E) in response to ELF5. Color scale denotes log 10 of MACS (-D and +D columns) or DiffBind scores (E columns). Fig 3. Induction of ELF5 alters FOXA1 and ER binding to the genome. Panel A, UpSet presentation of DiffBind output that identified statistically significant increases in FOXA1 or ER binding in response to induction of ELF5 with doxycycline (Dox). Corresponding Venn diagram for increased (enriched) peaks is shown. Lower panel, decreased peaks. Panel B, FOXA1 peaks that were increased by induction of ELF5 (+D, red) are plotted, centred on the FOXA1 peak, with the corresponding binding of FOXA1 at that location without induction of ELF5 (-D, blue). The resulting differential binding is shown in green. The binding of ER at these FOXA1 sites is shown with (+D, red), and without (-D, blue), with differential binding in green. Histograms show cumulative intensity on the y axis over the corresponding genomic region on the x axis. Color scale denotes log 10 of MACS (-D and +D columns) or DiffBind scores (E columns). Panel C, GREAT analysis of binding site function for increased binding sites for FOXA1 and ER. Blue indicates sets involved in estrogen action. Panel D, FOXA1 and ER binding sites that are specific in MCF-7 cells that were either sensitive to, or made resistant to, endocrine therapy, with corresponding ELF5 binding intensity. https://doi.org/10.1371/journal.pgen.1008531.g003 ELF5 and endocrine resistance [38] that defined regions of open chromatin in MCF-7 cells, we found that of the 503 FOXA1 sites enriched by induction of ELF5, only 139 (28%) occurred at regions of open chromatin, indicating that most of the enriched FOXA1 sites occurred at previously closed chromatin. Thus induction of ELF5 causes FOXA1 to bind to closed chromatin, consistent with its estab- lished pioneering activity. The increased FOXA1 binding sites caused by induction of ELF5 overlapped significantly with FOXA1 binding sites increased by the acquisition of endocrine resistance [39] (Chi- squared 8E-16) compared to endocrine sensitive MCF-7 (Fig 3D). Comparison to ER sites showed a similar trend (p = 0.1). There was only 1 decreased FOXA1 site that overlapped with the decreased FOXA1 sites in resistant MCF-7 cells, and there were only 7 ER sites in total that were decreased by induction of ELF5, making it unlikely that the ELF5-induced decrease in FOXA1 or ER binding is involved in endocrine resistance. These results demonstrate that induction of ELF5 increases FOXA1 binding to the same sites that the acquisition of endocrine resistance does, providing a genomic mechanism by which increased ELF5 can drive the acquisition of endocrine resistance. ELF5 drives changes in gene expression via the alteration of FOXA1 and ER binding to the genome To determine whether genes that bound ELF5, FOXA1 and ER in various combinations showed differential gene expression in response to induction of ELF5, we combined our RNA- seq and ChIP-seq data, by plotting the positions of each gene in a gene set formed based on the ChIP data, in their order based on their differential expression in the RNA-seq data, and then calculated an overall enrichment score (NES) and p value for clustering (enrichment) at either end of the differential expression spectrum. This technique does not require the use of arbi- trary cut offs to define changed level of expression. These plots were characterised by genes showing both increased and decreased gene expression, indicated by accumulation of genes at either end of the enrichment plot. For gene sets formed based on the presence of various com- binations of ELF5, ER and FOXA1 binding, we frequently observed enrichments, showing a transcriptional effect of these transcription factors (Fig 4A). We used a similar approach to determine whether the sets of genes that showed increased FOXA1 or ER genomic binding in response to induction of ELF5, also showed altered gene expression. Where FOXA1 binding increased we observed increased gene expression, and where FOXA1 genomic binding was depleted, reduced gene expression was observed (Fig 4B). Thus the change in FOXA1 and ER binding caused by ELF5 resulted in altered gene expression. Six genes, (CD36 [40], CUEDC1 [41], LAMP3 [42], SDCBP [43], LTBP2 [44], PIP [45]), of the set of 26 differentially expressed genes with enriched or depleted binding of FOXA1 or ER in response to induction of ELF5, are implicated endocrine resistance, and nearly all genes in this set appear in one or more gene sets derived from estrogen action, breast cancer subtype or endocrine resistance. Fig 4C shows ChIP-seq and RNA-seq results for one example, PIP. Increased FOXA1 binding was called by DiffBind at two sites (pink highlights), the first of which corresponded to a region binding a large suite of transcription factors associated with the ER transcriptional complex, such as GRHL2, MLL3, TEAD etc. Though not called by Diff- Bind, examination of the plots shows increased ER binding also at this site and elsewhere. PIP RNA-seq (top panels) showed a clear increase in transcript reads. Histograms compare overall intensity with (+D) and without (-D) induction of ELF5 and green shows the enrichment (E) in response to ELF5. Color scale denotes log 10 of MACS (-D and +D columns) or DiffBind scores (E columns). https://doi.org/10.1371/journal.pgen.1008531.g003 https://doi.org/10.1371/journal.pgen.1008531.g003 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 8 / 24 ELF5 binds directly to members of the ER transcriptional complex We conducted an unbiased search for ELF5 binding partners using rapid immunoprecipita- tion mass spectrometry (RIME), [34, 46] identifying 74 interactions (S1 Table). ELF5 was PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 9 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 ELF5 and endocrine resistance Fig 4. Changes in ELF5, FOXA1 and ER binding to the genome are reflected in gene expression. Panel A, Expression of genes identified by GREAT as ELF5, ER or FOXA1 transcriptional targets, showing their position in the distribution of differential expression (DE) in the RNA-seq data in response to induction of ELF5. GSEA-type visualization, nominal enrichment score (NES) and corresponding FDR (padj) are shown (R package fgsea). Panel B, as for panel A but showing genes for which FOXA1 or ER binding was increased or decreased by induction of ELF5. Panel C, coding-region of an ELF5-regulated gene involved in endocrine resistance (PIP) showing differential gene expression by RNA-seq, (exon splicing in blue) and increased FOXA1 binding by ChIP-seq at 2 locations with fold change (FC) shown, in response to induction of ELF5 with doxycycline (+) compared to vehicle (-). Binding of the indicated transcription factors to this locus from reported ChIP-seq experiments is shown together with the chromHMM model [61]. https://doi.org/10.1371/journal.pgen.1008531.g004 https://doi.org/10.1371/journal.pgen.1008531.g004 identified as one of the three top-ranking proteins by Mascot score using the very stringent cri- teria of present in all five replicates and absent in all five controls, along with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and protein transport protein SC16A (Fig 5A). DNA-PKcs is involved in the DNA damage response but also interacts with ER [47] and mod- ulates ER transcriptional activity [48]. Several proteins known to interact with DNA-PKcs were identified at lower stringency, including XRCC5/Ku80 (3/5 replicates), DNA topoisom- erase 2-beta (TOP2B, 2/5 replicates), with DNA topoisomerase 2-alpha (TOP2A) and poly (ADP-ribose) polymerase 1 (PARP1) present in 1/5 replicates and the second Ku sub-unit (XRCC6/Ku70) present in 2/5 replicates but also in one IgG control experiment (S1 Table). Comparison to proteins known to interact with ER [34] revealed extensive overlap and included the key ER transcription complex members GRHL2 and KDM1A (Fig 5B and S2 Table) which were also identified as interacting with ELF5 [49] in mouse trophoblasts (S3 Table). Immunoprecipitation experiments also demonstrated the interaction between DNA-PKcs and ELF5 (S8A Fig). We used the Duolink Proximity Ligation Assay (PLA) to further test the interaction with DNA-PKcs. We observed the presence of multiple nuclear signals in response to induction of ELF5. Average signal number increased and the distribution of nuclear PLA signals per cell shifted, demonstrating an interaction between ELF5 and DNA-PKcs in MCF-7 cells (Fig 5C). This effect was also seen using a second antibody recognising a different epitope on DNA-PKcs. We then searched for an effect of knockdown of DNA-PKcs on ELF5 driven transcription. We assembled a panel of qPCR assays for ELF5-regulated genes identified by RNA-seq that also had an ELF5 binding peak within the proximal promoter. The up-regulation by ELF5 of PIP, VTCN1 and GRHL3 in MCF7 cells was confirmed, as was the down-regulation of DKK1, MATN3, SNAI2, FILIP1L and LYN. Despite their identification in the RNA-seq experiment, ELF5 did not cause consistent changes in the expression of GDF15, STAT1, or SPDEF in this experiment. For 4 of these 8 ELF5-regulated genes, DNA-PKcs knockdown altered the ability of ELF5 to modulate their expression (Fig 5D and S9 Fig). For PIP, VTCN1 and GRHL3, loss of DNA-PKcs caused further upregulation of expression by ELF5, as well as an increase in the baseline expression (-Dox) for VTCN1 and GRHL3, suggesting an inhibitory effect of DNA-PKcs on ELF5 activity. DKK1 showed the opposite effect. We were able to find suitable antibodies to demonstrate this effect for VTCN1 by western blot (S8B and S8C Fig). Thus ELF5 interacts with DNA-PKcs and this interaction influences a portion of the transcriptional output of ELF5. ELF5 and endocrine resistance PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 10 / 24 ELF5 and endocrine resistance Fig 5. Identification of ELF5 binding partners. Panel A, Overlap of proteins identified in the ELF5 RIME replicates 1–5 following removal of non- specific interactors. Three proteins (ELF5, DNA-PKcs and SC16A) were identified in all replicates and no IgG controls. Gene lists show functional analysis using GSEA of interactors present in 2 of 5 replicates and not present in the IgG controls, full gene list provided in S1 Table. Panel B, overlap between ELF5-interacting proteins identified in at least one MCF7 ELF5 RIME replicate and those identified in mouse trophoblast cells [49], and ER RIME in MCF7 cells [34], revealing GRHL2 and KDM1A as potential common interactors. Gene lists show functional analysis using GSEA of interactors also present in ER RIME and not present in the IgG controls, full gene list provided in S2 Table. Panel C, top, proximity ligation assay (PLA) data showing increased signal count per nuclei between ELF5 and DNA-PKcs (DPK). Lower panel bar chart shows statistical analysis of the shift in the distribution of PLA signals per nucleus. Inset shows an example of increased PLA signal with induction of ELF5. Panel D, Effect of DNA-PKcs knockdown on ELF5-driven changes in expression of the indicated genes. Graphs show the mean calibrated normalised relative quantity values from three biological replicates with error bars showing 95% confidence interval. The associated table, vertically aligned with the corresponding samples in the graph, provides the exact mean normalised quantity value (Qty, row 1). Row 2 of the table indicates the effects of ELF5 induction on the target gene expression; the fold changes (FC) for the vertically aligned doxycycline induced (+D) or not induced (-D) sample pairs are shown, with red typeface indicating a significant upregulation, green a significant down regulation (one-way ANOVA). Rows 3 and 4 of the table indicate the effect of DNA-PKcs (PK) knockdown on the target gene expression. Row 3 compares the siPK-D sample (indicated by the orange box) with each of the untransfected (Unt) -D and the non-targeting (siNT) -D control samples, with a red asterisk indicating a significant upregulation and NS = no significant difference, one-way ANOVA). Similarly, row 4 compares the siPK+D sample (indicated by the orange box) with each of the untransfected (Unt) +D and non-targeting (siNT) +D samples Fig 5. Identification of ELF5 binding partners. Panel A, Overlap of proteins identified in the ELF5 RIME replicates 1–5 following removal of non- specific interactors. Three proteins (ELF5, DNA-PKcs and SC16A) were identified in all replicates and no IgG controls. Gene lists show functional analysis using GSEA of interactors present in 2 of 5 replicates and not present in the IgG controls, full gene list provided in S1 Table. Panel B, overlap between ELF5-interacting proteins identified in at least one MCF7 ELF5 RIME replicate and those identified in mouse trophoblast cells [49], and ER RIME in MCF7 cells [34], revealing GRHL2 and KDM1A as potential common interactors. Gene lists show functional analysis using GSEA of interactors also present in ER RIME and not present in the IgG controls, full gene list provided in S2 Table. Panel C, top, proximity ligation assay (PLA) data showing increased signal count per nuclei between ELF5 and DNA-PKcs (DPK). Lower panel bar chart shows statistical analysis of the shift in the distribution of PLA signals per nucleus. Inset shows an example of increased PLA signal with induction of ELF5. Panel D, Effect of DNA-PKcs knockdown on ELF5-driven changes in expression of the indicated genes. Graphs show the mean calibrated normalised relative quantity values from three biological replicates with error bars showing 95% confidence interval. The associated table, vertically aligned with the corresponding samples in the graph, provides the exact mean normalised quantity value (Qty, row 1). Row 2 of the table indicates the effects of ELF5 induction on the target gene expression; the fold changes (FC) for the vertically aligned doxycycline induced (+D) or not induced (-D) sample pairs are shown, with red typeface indicating a significant upregulation, green a significant down regulation (one-way ANOVA). Rows 3 and 4 of the table indicate the effect of DNA-PKcs (PK) knockdown on the target gene expression. Row 3 compares the siPK-D sample (indicated by the orange box) with each of the untransfected (Unt) -D and the non-targeting (siNT) -D control samples, with a red asterisk indicating a significant upregulation and NS = no significant difference, one-way ANOVA). Similarly, row 4 compares the siPK+D sample (indicated by the orange box) with each of the untransfected (Unt) +D and non-targeting (siNT) +D samples. Fig 5. Identification of ELF5 binding partners. Panel A, Ove Fig 5. Identification of ELF5 binding partners. ELF5 levels are higher in hormone resistant brain metastases compared to their matched primary tumour To determine if these effects of ELF5 could be seen in metastases from endocrine resistant breast cancer cases we interrogated gene expression profiles of matched primary and 11 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 https://doi.org/10.1371/journal.pgen.1008531.g005 ELF5 and endocrine resistance metastatic breast cancer from brain [50]. This tumour collection contained 7 ER+ cases, 2 of which, X4 and X72, showed a large increase in ELF5 expression in the metastasis compared to the matched primary (Fig 6A). We used DESeq to search for an enriched ELF5 signature in these metastases. First, we treated all 7 of the ER+ patients as replicates, and used DESeq to rank the genes by differential expression between primary and matched metastasis. GSEA showed significant enrichment of both the set of 497 ELF5-induced genes from MCF-7 cells, and a subset of 235 genes with an ELF5 ChIP binding site within 10kb from MCF-7 (p-values of 0.00025 and 0.00038 respectively) (S7C Fig). When the patients were considered individu- ally, three of the seven metastasis expression profiles were significantly enriched for ELF5 induced genes (X4, X62 and X72, p-values 0.00754, 0.00047 and 0.00048 respectively). When we reversed the analysis, using the MCF-7 experiments as replicates to create a ranked gene list and the differential gene expression between one primary and its matched metastasis to create the gene-sets, the results were also significant (p-values 0.00064, 0.00115 and 0.04704 respectively). This provides evidence that the effects of elevated ELF5 in MCF-7 cells also occur in three breast cancer metastases that have become resistant to endocrine therapy, con- sistent with elevation of ELF5 providing an additional mechanism driving acquired endocrine resistance. In summary our experimental investigation shows that induction of ELF5 alters patterns of estrogen-driven gene expression via transcriptional mechanisms, which includes a gain of new FOXA1 and ER binding sites, regulation of the expression of members of the ER complex and direct interaction with members of the ER transcriptional complex. This finding provides a mechanistic explanation for the role of ELF5 in the modulation of estrogen action and the acquisition of resistance to endocrine therapy. Panel A, Overlap of proteins identified in the ELF5 RIME replicates 1–5 following removal of non- specific interactors. Three proteins (ELF5, DNA-PKcs and SC16A) were identified in all replicates and no IgG controls. Gene lists show functional analysis using GSEA of interactors present in 2 of 5 replicates and not present in the IgG controls, full gene list provided in S1 Table. Panel B, overlap between ELF5-interacting proteins identified in at least one MCF7 ELF5 RIME replicate and those identified in mouse trophoblast cells [49], and ER RIME in MCF7 cells [34], revealing GRHL2 and KDM1A as potential common interactors. Gene lists show functional analysis using GSEA of interactors also present in ER RIME and not present in the IgG controls, full gene list provided in S2 Table. Panel C, top, proximity ligation assay (PLA) data showing increased signal count per nuclei between ELF5 and DNA-PKcs (DPK). Lower panel bar chart shows statistical analysis of the shift in the distribution of PLA signals per nucleus. Inset shows an example of increased PLA signal with induction of ELF5. Panel D, Effect of DNA-PKcs knockdown on ELF5-driven changes in expression of the indicated genes. Graphs show the mean calibrated normalised relative quantity values from three biological replicates with error bars showing 95% confidence interval. The associated table, vertically aligned with the corresponding samples in the graph, provides the exact mean normalised quantity value (Qty, row 1). Row 2 of the table indicates the effects of ELF5 induction on the target gene expression; the fold changes (FC) for the vertically aligned doxycycline induced (+D) or not induced (-D) sample pairs are shown, with red typeface indicating a significant upregulation, green a significant down regulation (one-way ANOVA). Rows 3 and 4 of the table indicate the effect of DNA-PKcs (PK) knockdown on the target gene expression. Row 3 compares the siPK-D sample (indicated by the orange box) with each of the untransfected (Unt) -D and the non-targeting (siNT) -D control samples, with a red asterisk indicating a significant upregulation and NS = no significant difference, one-way ANOVA). Similarly, row 4 compares the siPK+D sample (indicated by the orange box) with each of the untransfected (Unt) +D and non-targeting (siNT) +D samples. https://doi.org/10.1371/journal.pgen.1008531.g005 https://doi.org/10.1371/journal.pgen.1008531.g005 12 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 Discussion FOXA1 acts as a pioneer factor to regulate ER binding [8] and plays a crucial role in endocrine resistance [8, 12]. The genomic distribution of FOXA1/ER binding sites is significantly altered in endocrine resistant cell lines and poor-prognosis breast cancers [12]. Here we show that a very similar redistribution of FOXA1 binding can be caused by an increase in ELF5 levels, directly implicating ELF5 in this mechanism of endocrine resistance. Moreover, increased ELF5, and the resulting changes in FOXA1 or ER binding, regulated a set of genes with reported roles in driving endocrine resistance. For example, CD36 increased proliferation and migration of breast cancer cells and antagonised tamoxifen effects [40]. CUEDC1 is a tran- scriptional target of ER regulated via the associated CUTE enhancer discovered by CRISPR screening [51] and its product is essential for ER-driven MCF-7 cell proliferation [41]. LAMP3 is a regulator of autophagy that is induced by tamoxifen and causes insensitivity to this drug. Its expression is predictive of early disease progression. LTBP2 modulates the TGF beta path- way and is downregulated by estrogen and upregulated by TGFbeta and acquisition of the invasive phenotype. Elevated LTBP2 predicts early disease progression [44]. SDCBP is nega- tively correlated with ER in breast cancers and is down regulated by estrogen treatment. In ER-negative cancers it positively regulates cell cycle control [43]. PIP is expressed in ER+ and apocrine breast cancers and is regulated by androgens. Its product is an aspartyl protease required for RTK activation of FAK and other kinases by integrins and is involved in cancer cell invasion. It is required for proliferation in response to estrogen, but also for proliferation of tamoxifen resistant T-47D cells [45, 52]. We used RIME to search for a direct physical interaction of ELF5 with FOXA1, ER and members of the ER transcriptional complex. Limited evidence for an interaction with the his- tone demethylase LSD1/KDM1A was found. We discovered and verified interaction of ELF5 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 13 / 24 https://doi.org/10.1371/journal.pgen.1008531.g006 Ethics statement All animal experimentation was approved by the Garvan/St Vincent’s animal experimentation ethics committee, approval 17–23. ELF5 and endocrine resistance with ER-complex member DNA-PKcs. Whether ELF5 acts by participation in a complex with ER, or by sequestering and operating the complex with its key members in preference to ER, remains to be determined. One key unifying feature of our interaction studies is the identifica- tion of members of the CoREST complex. DNA-PKcs is a member of the CoREST complex and represses ER-driven transcription [48]. LSD-1 also participates in the CoREST complex but when bound to ER it can activate transcription associated with demethylation of H3K9me2 [53], indicating that these complexes can both repress and activate transcription. We found an increase in ELF5 levels, and enrichment of the ELF5 transcriptional signature, in the endocrine resistant brain metastases of ER+ primary tumours. This is consistent with our proposal that in some cases of endocrine resistance, increased levels of ELF5 are driving the acquisition of insensitivity to estrogen. This is unlikely to be the sole cause of acquired endocrine resistance but may play a contributing role in many cases. We reported that RANKL induces ELF5 levels in progenitor cells, forcing their differentiation toward the alveo- lar lineage [17]. If this mechanism continues in breast cancer then adjuvant treatment with therapeutics such as denosumab during endocrine therapy may reduce the incidence of endo- crine resistance, and a signal consistent with this action was reported by the ABCSG-18 trial (San Antonio Abs. S2-02 Can. Res. 76:2016). In conclusion we have demonstrated that ELF5 can modulate estrogen action via modula- tion of the genomic regions that ER and FOXA1 occupy to cause changes in ER-driven gene expression and in particular changes in the expression of genes implicated in the acquisition of endocrine resistance. ELF5 caused alterations in the expression of ER transcriptional complex members, and altered interactions with members of the ER-coactivator complex, characterised by their participation in the CoREST complexes. Together this establishes a multi-faceted mechanism by which ELF5 can modulate and repress estrogen-driven gene expression. This provides a mechanism by which increased ELF5 expression could drive progression of ER + breast cancer to become resistant to endocrine therapy. We have previously shown that increased ELF5 can drive mammary cancer metastasis, establishing that ELF5 can cause the acquisition of the two defining features of progressive ER+ breast cancer, endocrine resistance and renewed metastasis. Stable cell lines and culture MCF-7 cells expressing ELF5 Isoform 2 tagged with V5 were created and maintained as described [19]. Puromycin (Sigma-Aldrich, St Louis, Missouri, USA) at 1ug/mL was used to maintain selection. Doxycycline (Dox, Sigma-Aldrich) at 0.1ug/mL in water was used daily to induce ELF5. ELF5 and endocrine resistance Fig 6. ELF5 expression is increased in endocrine resistant metastases. Panel A, levels of ESR1 and ELF5 expression in ER+ matched primary (P) and brain metastases (M) normalised from data produced by Vareslija and colleagues [50]. Panel B, GSEA determined enrichment (NES) of gene sets comprising genes differentially expressed in MCF-7 cells due to induction of ELF5 (DE) or a subset with an ELF5 ChIP binding site with 10kb (DEgenes ChIP) Panel C genes at the leading edge of the enrichments shown in panel B asterisks Fig 6. ELF5 expression is increased in endocrine resistant metastases. Panel A, levels of ESR1 and ELF5 expression in ER+ matched primary (P) and brain metastases (M) normalised from data produced by Vareslija and colleagues [50]. Panel B, GSEA determined enrichment (NES) of gene sets comprising genes differentially expressed in MCF-7 cells due to induction of ELF5 (DE) or a subset with an ELF5 ChIP binding site with 10kb (DEgenes_ChIP). Panel C, genes at the leading edge of the enrichments shown in panel B, asterisks indicate a gene that is also a transcriptional target of ELF5 by ChIP-seq. Fig 6. ELF5 expression is increased in endocrine resistant metastases. Panel A, levels of ESR1 and ELF5 expression in ER+ matched primary (P) and brain metastases (M) normalised from data produced by Vareslija and colleagues [50]. Panel B, GSEA determined enrichment (NES) of gene sets comprising genes differentially expressed in MCF-7 cells due to induction of ELF5 (DE) or a subset with an ELF5 ChIP binding site with 10kb (DEgenes_ChIP). Panel C, genes at the leading edge of the enrichments shown in panel B, asterisks indicate a gene that is also a transcriptional target of ELF5 by ChIP-seq. Fig 6. ELF5 expression is increased in endocrine resistant metastases. Panel A, levels of ESR1 and ELF5 expression in ER+ matched primary (P) and brain metastases (M) normalised from data produced by Vareslija and colleagues [50]. Panel B, GSEA determined enrichment (NES) of gene sets comprising genes differentially expressed in MCF-7 cells due to induction of ELF5 (DE) or a subset with an ELF5 ChIP binding site with 10kb (DEgenes_ChIP). Panel C, genes at the leading edge of the enrichments shown in panel B, asterisks indicate a gene that is also a transcriptional target of ELF5 by ChIP-seq. PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 14 / 24 ChIP-seq peak calling Peaks were called using MACS 2.0.10. Consensus peaks were present in three out of four repli- cates (or two out of two for H3K4me3). Differential binding to peak regions was defined with R package DiffBind with default parameters [12]. ELF5 and endocrine resistance ChIP-seq sample preparation MCF7-ELF5-Isoform2 cells were seeded in 15cm plates, and doxycycline (or vehicle) treat- ment was commenced 24 hours after plating. After 48 hours of doxycycline treatment, cells were cross-linked for 10 minutes at room temperature using 1% formaldehyde diluted in cell growth medium. After 10 minutes, formaldehyde was quenched with 0.2M glycine. Plates were then placed on ice and washed x 2 with cold PBS. Cross-linked cells were collected in 2mL PBS using a cell scraper and pellets containing approximately 20 million cells were stored at -70˚C. Four independent replicates for ER, FOXA1, ELF5 and H3K4me3 ChIP-seqs and 6 input replicates (one per lane) were performed according to the protocols described in [54], with or without Dox treatment. ELF5 was IP with a mixture of anti V5 and anti ELF5 (Santa- Cruz N20) antibodies. ELF5 samples were treated with Dox only. DNA purification following IP was performed using phenol:chloroform:isoamyl alcohol and Phase Lock Gel tubes. Librar- ies were prepared using the TruSeq ChIP Sample Preparation Kit (A) from Illumina, with AMPure XP bead double-sided size selection. Samples were multiplexed in 3 pooled libraries (library 1 FOXA1 samples and input, library 2 ER samples and input, library 3 ELF5, H3K4me3 samples and input) and sequenced in 3 lanes on the Illumina HiSeq 2500. RNA-sequencing (MCF7-ELF5 cells) MCF7-ELF5-Isoform2 cells treated with Dox or vehicle from 24 hours after plating for 48 hours. Sequencing used the Illumina HiSeq2000 using v3 SBS reagents and 100bp paired-end reads. Alignment was done with STAR (v 2.4.0d) [56] against the human genome (hg38) with gencode v20 annotations. Transcript counts were summarised and transcripts per million (TPM) calculated using RSEM (v 1.2.18) [57]. Counts were normalised using TMM [58] and transformed using voom [59]. Differential expression analysis was carried out using limma [60]. When performing GSEA for comparison of MCF-7 RNA-seq with T-47D and MCF-7 microarrays, FDR cut-off for DE genes from ELF5 treated T-47D and MCF-7 microarrays were 0.05. Functional annotation of peak regions Functional analyses used the Genomic Regions Enrichment of Annotations Tool (GREAT) [25]. Motif analysis Enrichment of transcription factor DNA binding motifs under peaks was performed with MEME-ChIP, using default parameters [55]. Genomic regions with repetitive elements were adopted from Repbase [26]. ChIP-seq MCF7 or T-47D -ELF5-Isoform2 cells treated with Dox or vehicle from 24 hours after plating for 24 (T-47D) or 48 (T-47D and MCF-7) hours then cross-linked for 10 minutes at room tem- perature using 1% formaldehyde. Four independent replicates for ER, FOXA1, ELF5 and H3K4me3 ChIP-seq were performed according to the protocols described in [8] and [19]. PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 15 / 24 ELF5 and endocrine resistance method = "blind" and sharingMode = "fit-only". GSEA was performed with R package fgsea, with 105 permutations, as described above. Proximity ligation assays (PLAs) Cells were seeded on glass coverslips in 12-well plates (Corning) and treated after 24 hours with doxycycline or vehicle for 48 hours. Three biological replicates were performed. Quantitative PCR All qPCR reactions were run on the Applied Biosystems ABI7900 qPCR machine (Thermo Fisher). Two to three technical replicates were run for each sample, as well as negative controls (no template, no reverse transcriptase, water). Standard curves using a 1:10 dilution series were run for every assay. Complete methods are available as supplementary material (SDoc1 Text). Raw data is avail- able via ArrayExpress E-MTAB-7641and Proteome exchange PXD013349. Rapid Immunoprecipitation of Endogenous Protein (RIME) MCF7-ELF5-Isoform2 cells treated with Dox or vehicle from 24 hours after plating for 48 hours, cross-linked using 1% methanol-free formaldehyde (Thermo Fisher). RIME was per- formed as described [46] with modifications (see extended methods) using a 1:1 mix of anti- ELF5 and anti-V5 antibodies. siRNA transfection ON-TARGETplus human PRKDC SMART pool siRNA and ON-TARGETplus non-targeting siRNA #1 (Dharmacon, Lafayette, Colorado, USA) were used. All transfections were per- formed using Lipofectamine RNAiMAX (Thermo Fisher). After 24 hours, the medium was changed and puromycin and Dox treatment was started on day 2 and cells were collected on day 4. Patient data analysis Raw counts of matched primary and metastatic breast cancer RNA-seq data were downloaded from https://github.com/npriedig/jnci_2018. Ranked list of genes was calculated with DESeq using patients as replicates, or estimating dispersion for individual patients from the rest with PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 16 / 24 S2 Fig. Additional aspects of ELF5 genomic binding. Panels A, GREAT functional analysis of ELF5 genomic binding using MSigDB gene sets as indicated. Panel B, overlap of MCF-7 ChIP peaks with those observed in T-47D cells [19]. (TIF) ELF5 and endocrine resistance S3 Fig. ELF5 binds to repetitive elements. Panel A, sequence of motifs at ELF5 binding sites with identity to Alu repeats (DFAM) with red blue and green color bars showing the most fre- quent arrangements of these motifs and their enrichment (E) p value. Panel B, RepeatMasker analysis of repeat sequences at ELF5 binding sites showing number and type detected. Panel C, consensus ETS motif under ELF5 binding sites at repeats. Panel D, distribution of the indi- cated repeat types around ELF5 binding sites at the indicated window sizes. Panel E, odds ratios for finding the indicated repeat types under all transcription factor binding sites (wgEn- code TfbsV3), under FoxA1, ER and ELF5 with or without DOX treatment, then at ELF5 bind- ing sites within highly occupied target regions (HOT), enhancers (E), super enhancers (SE) or in the vicinity of differentially expressed genes (DE). Error bars represent standard error. (TIF) S3 Fig. ELF5 binds to repetitive elements. Panel A, sequence of motifs at ELF5 binding sites with identity to Alu repeats (DFAM) with red blue and green color bars showing the most fre- quent arrangements of these motifs and their enrichment (E) p value. Panel B, RepeatMasker analysis of repeat sequences at ELF5 binding sites showing number and type detected. Panel C, consensus ETS motif under ELF5 binding sites at repeats. Panel D, distribution of the indi- cated repeat types around ELF5 binding sites at the indicated window sizes. Panel E, odds ratios for finding the indicated repeat types under all transcription factor binding sites (wgEn- code TfbsV3), under FoxA1, ER and ELF5 with or without DOX treatment, then at ELF5 bind- ing sites within highly occupied target regions (HOT), enhancers (E), super enhancers (SE) or in the vicinity of differentially expressed genes (DE). Error bars represent standard error. (TIF) S4 Fig. UpSet analysis of transcription factors significantly co-located with ELF5. UpSet analysis, using the transcription factors whose binding is most frequently co-located with ELF5, to identify patterns of co-binding at differentially expressed (DE) genes, (Panel A 119 genomic loci), super enhancers (Panel B 259 loci) and enhancers (Panel C 2644 loci). Numbers above the transcription factor sets show the number instances of that specific set. Black dots indicate the presence within the set of the indicated transcription factor. (TIF) S5 Fig. ELF5-induced gene expression analysed by GSEA and Cytoscape. Supporting information S1 Fig. Quality control measures for the ChIP-seq experiments. MCF-7 cells stably infected with either an empty retroviral doxycycline-inducible vector, or one expressing isoform 2 of human ELF5 tagged with V5 [19] (Panel A), were treated with 0.1ug/ml doxycycline for 48 hours prior to cross-linking and processing for ChIP-seq using antibodies precipitating ELF5, ER, FOXA1 or H4K4me3 together with inputs. Four independent replicates for each ChIP (2 for H3K4me3) were conducted. A set of standard quality control measures were evaluated. Panel A, ELF5 expression level before and after induction with DOX compared to levels of induction achieved in T-47D cells with R5020 [19], or in mammary gland by 18 days of preg- nancy in mice. Panel B, Read depth and errors, panel C, paired read score, panel D sequence duplication, panel E GC content, panel F, reads in peaks and panel G, signal strength. Panel H, principle components analysis (PCA), showed the presence of a peak in 3 of 4 replicates cor- rectly grouped the ChIP replicates and separated all of the FOXA1 plus and minus dox repli- cates. S2 Fig. Additional aspects of ELF5 genomic binding. Panels A, GREAT functional analysis of ELF5 genomic binding using MSigDB gene sets as indicated. Panel B, overlap of MCF-7 ChIP peaks with those observed in T-47D cells [19]. (TIF) S2 Fig. Additional aspects of ELF5 genomic binding. Panels A, GREAT functional analysis of ELF5 genomic binding using MSigDB gene sets as indicated. Panel B, overlap of MCF-7 ChIP peaks with those observed in T-47D cells [19]. (TIF) 17 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 ELF5 and endocrine resistance summits that overlap enhancers and superenhancers. Color scale denotes log 10 of MACS score. Panel F, consensus ETS motifs under ELF5 binding sites in the indicated genomic regions. (TIF) S8 Fig. ELF5 interacts with DNA PKcs to regulate VTCN1. Panel A, immunoprecipitation of ELF5 co-precipitates DNA-PKcs. Samples were prepared using the RIME protocol and immunoprecipitated with a combination of ELF5 and V5 antibodies or IgG control. Blots for V5 and DNA-PKcs are shown. Lane 1 is the input or total lysate (In), lane 2 is the ELF5-V5 immunoprecipitation (IP) and lane 3 is the IgG control immunoprecipitation (IgG). Lanes 4 and 7 are supernatants from the immunoprecipitations (Sup, representing unbound protein), while lanes 5–6 and 8–9 are supernatants from the first (W1) and third (W3) bead washes (indicating no residual unbound protein after the final third wash). RIME 5 is replicate 5 of the ELF5-V5 RIME experiments, while additional replicates 1 and 2 did not form part of the ELF5-V5 RIME dataset. Panel B and C, Western blots for MCF-7 (panel B) and T-47D (panel C) cell lines, stably modified with doxycycline-inducible pHUSH-ELF5 isoform 2 or isoform 3 vector (empty vector as a control). Cells were untransfected (Unt), transfected with a non-tar- geting siRNA (NT) or transfected with siRNA targeting DNA-PKcs (PK). Cells were also treated with doxycycline (Dox, indicated by + symbol) or vehicle (-). Each box represents an individual blot and is shown with the corresponding beta-actin (b-actin) loading control. (TIF) S9 Fig. Knockdown of DNA-PKcs alters ELF5-driven gene expression. Panels A-H, Effect of DNA-PKcs knockdown on ELF5-driven changes in expression of the indicated genes in MCF- 7 and T47-D cells. Fold change is indicated in red where DNA-PKcs exerted a significant sup- pression of gene expression and in green where DNA-PKcs enhanced expression. Cells were untransfected (Unt), transfected with a non-targeting siRNA (NT) or transfected with siRNA targeting DNA-PKcs (PK). Cells were also treated with doxycycline (Dox, +) or vehicle (-). Graphs show the mean calibrated normalised relative quantity values from three biological replicates with 95% confidence interval. The associated table, vertically aligned with the corre- sponding samples in the graph, provides the exact mean normalised quantity value (Qty, row 1). Scalable .pdf showing complete Cytoscape representation of the RNA-seq data. Each circle (node) is sized to indicate the relative number of genes in the set and coloured to show enrichment score in response to ELF5. Nodes with overlaps in their gene content are linked by green lines and are clustered according to the degree of overlap. Download and zoom to see the detail. (TIF) S6 Fig. ELF5-induced gene expression analysed by RNA-seq. Panel A, GSEA of MSigDB Hallmark gene sets coloured according to enrichment score as indicated by the scale. Panel B, example GSEA plots from the MSigDB C2-all sets showing significant enrichment. Panel C, enriched ChIP sets (ranked by Enrichr combined score) identified in the regulatory regions of the top 100 differentially expressed MCF7-ELF5 RNA-seq genes (filtered for absolute fold- change >1.5 and ranked by FDR). The identifier for each ChIP set contains the name of the transcription factor followed by the PubMed ID, the type of experiment (ChIP-seq or ChIP- chip), the cell line or tissue, and the species. The top 10 sets (of 37 sets with an FDR <0.05) are shown. Analysis was performed using the Enrichr ChIP enrichment analysis (ChEA) tool. Panel D, enriched ChIP sets identified in the regulatory regions of down-regulated genes. Panel E, enriched ChIP sets identified in the regulatory regions of up-regulated genes. Panel F, enriched transcription factor motifs in ELF5 regulated genes from the TRANSFAC and JASAPR databases. No enriched motifs were identified for the down-regulated RNA-seq genes. S7 Fig. Characteristics of FOXA1 binding sites enriched or depleted by induction of ELF5. Panel A. Venn diagram for co-occurrence of binding peaks called present by MACS for ELF5, FOXA1 and ER. Panel B, matching UpSet plot for co-occurrence of binding peaks called pres- ent by MACS. Panel C, motif probability analysis of sequences under FOXA1 binding sites that were increased by induction of ELF5. Panel D), motif probability analysis of sequences under FOXA1 binding sites that were decreased by induction of ELF5. Panel E, ELF5, FOXA1, ER and H3K4me3 peaks are plotted, before and after induction of ELF5, centred on all FOXA1 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 18 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 Acknowledgments The authors acknowledge the support of Ms Gillian Lehrbach, Garvan Tissue Culture Facility Sydney for advice with breast cancer cell tissue culture, Dominic Kaczorowski for DNA sequencing and Aaron Statham for DNA sequencing quality control, from the Kinghorn Cen- ter for Clinical Genomics, Sydney, and Helen Speirs from The Ramaciotti Centre for Geno- mics UNSW Sydney for RNA sequencing. ELF5 and endocrine resistance identified to interact with ELF5 by RIME in MCF-7 cells. (PDF) S3 Table. Proteins interacting with ELF5 compared to proteins known to interact with ELF5 in trophoblasts. Comparison of proteins found to interact with ELF5 by RIME in breast cancer cells compared to those identified as interacting with ELF5 [49] in mouse trophoblast stem cells by mass spectrometry (MS). (PDF) identified to interact with ELF5 by RIME in MCF-7 cells. (PDF) S3 Table. Proteins interacting with ELF5 compared to proteins known to interact with ELF5 in trophoblasts. Comparison of proteins found to interact with ELF5 by RIME in breast cancer cells compared to those identified as interacting with ELF5 [49] in mouse trophoblast stem cells by mass spectrometry (MS). (PDF) S1 Document. Complete Methods. Full description of methods used in PDF format. (DOCX) S2 Document. MCF ChIP experiments used. Hyperlinks to the MCF-7 ChIP data used in PDF format. (CSV) S3 Table. Proteins interacting with ELF5 compared to proteins known to interact with ELF5 in trophoblasts. Comparison of proteins found to interact with ELF5 by RIME in breast cancer cells compared to those identified as interacting with ELF5 [49] in mouse trophoblast stem cells by mass spectrometry (MS). Row 2 of the table indicates the effects of ELF5 induction on the target gene expression; the fold changes for the vertically aligned +Dox and -Dox sample pairs are shown, with red type- face indicating a significant upregulation (one-way ANOVA), green a significant downregula- tion and black a non-significant fold change. Rows 3 and 4 of the table indicate the effect of DNA-PKcs knockdown on the target gene expression. Row 3 compares the siPK -Dox sample (indicated by the orange box) with each of the Unt -Dox and the siNT -Dox samples, with a red asterisk indicating a significant upregulation and a green asterisk a significant downregula- tion (NS = no significant difference, one-way ANOVA). Similarly, row 4 compares the siPK +Dox sample (indicated by the orange box) with each of the Unt +Dox and siNT +Dox sam- ples. (TIF) S1 Table. Proteins interacting with ELF5 discovered by RIME. Proteins are listed by the level of stringency required to include them, by progressive relaxing the requirements for pres- ence in the replicates, as indicated. (PDF) S1 Table. Proteins interacting with ELF5 discovered by RIME. Proteins are listed by the level of stringency required to include them, by progressive relaxing the requirements for pres- ence in the replicates, as indicated. (PDF) S2 Table. Proteins interacting with ELF5 compared to proteins known to interact with ER. Comparison to proteins known to interact with ER in MCF-7 cells [34] compared to those 19 / 24 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008531 January 2, 2020 S1 Document. Complete Methods. Full description of methods used in PDF format. (DOCX) S1 Document. Complete Methods. Full description of methods used in PDF format. (DOCX) S2 Document. MCF ChIP experiments used. Hyperlinks to the MCF-7 ChIP data used in PDF format. (CSV) S2 Document. MCF ChIP experiments used. Hyperlinks to the MCF-7 ChIP data used in PDF format. (CSV) Author Contributions Conceptualization: Catherine L. Piggin, Alexander Swarbrick, Matthew J. Naylor, Maria Kalyuga, Warren Kaplan, Samantha R. Oakes, David Gallego-Ortega, Susan J. Clark, Jason S. Carroll, Nenad Bartonicek, Christopher J. Ormandy. Data curation: Catherine L. Piggin, Daniel L. Roden, Andrew M. K. Law, Mark P. Molloy, Christoph Krisp, Samantha R. Oakes, David Gallego-Ortega, Jason S. Carroll, Nenad Barto- nicek, Christopher J. Ormandy. Formal analysis: Mark P. Molloy, Christoph Krisp, Nenad Bartonicek, Christopher J. Ormandy. References 1. Osborne CK, Schiff R. Mechanisms of endocrine resistance in breast cancer. 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Linear models and empirical bayes methods for assessing differential expression in microar- ray experiments. Stat Appl Genet Mol Biol. 2004; 3:Article3. https://doi.org/10.2202/1544-6115.1027 PMID: 16646809. 61. Taberlay PC, Statham AL, Kelly TK, Clark SJ, Jones PA. Reconfiguration of nucleosome-depleted regions at distal regulatory elements accompanies DNA methylation of enhancers and insulators in can- cer. Genome research. 2014; 24(9):1421–32. https://doi.org/10.1101/gr.163485.113 PMID: 24916973; PubMed Central PMCID: PMC4158760. 24 / 24
https://openalex.org/W4287990754
https://zenodo.org/records/3733182/files/Acta%20historiae%202019%20-%2038%20-%201-2-26-29.pdf
English
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MARCUS AURELIUS SEVERINUS AND HIS ATTEMPT TO REFORM THE SURGERY
Zenodo (CERN European Organization for Nuclear Research)
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Acta hist. med. stom. pharm. med. vet. / 2019 / 38 / 1–2 / 26–29 Acta hist. med. stom. pharm. med. vet. / 2019 / 38 / 1–2 / 26–29 Original scientifi c article UDC: 616-089(450)”16” 61:929 Марко Аурелије Северино DOI: 10.5281/zenodo.3733182 UDC: 616-089(450)”16” 61:929 Марко Аурелије Северино DOI: 10.5281/zenodo.3733182 Antonio Perciaccante Department of Medicine, “San Giovanni di Dio” Hospital via Fatebenefratelli 34, 34170 Gorizia, Italy E-mail: antonioperciaccante@libero.it Antonio Perciaccante Department of Medicine, “San Giovanni di Dio” Hospital via Fatebenefratelli 34, 34170 Gorizia, Italy E-mail: antonioperciaccante@libero.it MARCUS AURELIUS SEVERINUS AND HIS ATTEMPT TO REFORM THE SURGERY Abstract: Th is paper will analize the attempt of a 17th century Italian surgeon and anatomist Marcus Aurelius Severinus (1580-1656) to reform operative surgical procedures. His contribution to surgery was twofold: both on a methodological and technical level. Severinus was one of the fi rst physicians to theorize and practice an active surgery he called “del medicar crudo” (“to medicate crudely”). He performed the newest surgical techniques, and professed a concept of an early intervening surgery, when the pathological process didn’t show signs of spontaneous resolution. Subsequently, he showed boldness in his surgical practice, as reported in some extracts of his works “De recondita abscessum natura” and “De effi caci Medicina”. However, Severinus’eff orts weren’t met with support from medical community. At that time, surgeons were more careful to avoid pain and further risks to the patients, and they preferred to wait, rather then to attempt a premature surgical approach. However, Severinus was vindicated about 250 years later, when the discovery of anesthesia and infectious prophylaxis allowed for elevation of surgery to a scientifi c status, as well as application of his principles. Keywords: General surgery, barber surgeons, operative surgical procedures Non MeSh: Marcus Aurelius Severinus Keywords: General surgery, barber surgeons, operative surgical procedures Non MeSh: Marcus Aurelius Severinus Until the 18th century, surgery was practiced mostly by the barber-surgeons. Th ey were unskilled practitioners, who learned through apprenticeship and observa- tion, and performing minor procedures, such as bloodletting and teeth extractions, and treating wounds. [1] Due to lack of adequate anesthetic and antiseptic supports, surgery was considered brutal and dangerous, and it was considered “a lesser profes- sion” compared to classical medicine. [2 p1] 26 Perciaccante A., Marcus Aurelius Severinus and his Attempt to Reform the Surgery Despite these diffi culties, the Middle Ages was characterized by surgical pio- neers, who contributed to raising the ethical, scientifi c and technical status of the sur- gery: Andreas Vesalius (1514-1564) and Ambroise Paré (1510-1590). Until authori- zation to perform dissections on human corpses was issued, knowledge of anatomy was based on Greek and Roman misconceptions based on the animals’ dissection. Vesalius’ studies on human anatomy, collected in his main work “De Humani Cor- poris Fabrica Libri Septem”, [3] defi nitely changed medicine. He fi rst suggested the hands-on approach of human dissection by physicians and surgeons.l French army surgeon Ambroise Paré (1510-1590) also infl uenced surgery’s de- velopment, making wounds cauterization less dangerous and painful. Indeed, using a mixture of turpentine, rose oil and egg yolk, he developed a technique to treat gunshot wounds, which was alternative to the previous cauterizing technique based on use of boiling oil. Moreover, Paré reduced the bleeding risk, reintroducing the ligating of blood vessels during the amputations. [4]i Here, I want to point out a further important, but little known fi gure, under- lying his contribution to surgery both at technical and methodological level: Marcus Aurelius Severinus (1580, Tarsia, Italy - 1656, Naples, Italy).t Aft er some intention of studying law, Severinus given the preference to medi- cine, and on February 1606, he obtained the bachelor in “Alma Philosophia e Sacra Medicina” at the Collegium Medicorum of Salerno (Italy), one of the most ancient and renowned Collegium in Europe. [5] Disregarding Aristotle, he was follower of the atomist Democritus, and corresponded with Tommaso Campanella and William Harvey. A renowned and distinguished surgeon and anatomist, Severinus was author of an important scientifi c book titled “Zootomia democritea”, which received world- wide recognition as the fi rst comparative anatomy book. Keywords: General surgery, barber surgeons, operative surgical procedures Non MeSh: Marcus Aurelius Severinus i Trained by the anatomist Giulio Iasolino (1538-1622), Severinus was accom- plished surgeon and anatomist, and he carried out his main surgical work at the “Ospedale degli Incurabili” in Naples. He had a brilliant academic career, and he was professor of anatomy, and later fi lled the chair of surgery in University of Naples. [5] Severinus’ contribution to surgery was twofold: on a methodological and tech- nical level. He was very critical about surgery and surgeons of that time, which were discredited in the 17th century. Severinus attributed this discredit to the separation of medicine and surgery, and he strongly criticized the surgeons’ detachment from the study of the classical medicine. Severinus’ opinion was that the surgeons were negligents toward the study of the ancient Greek and Roman medicine. Due to lack of anatomy and physiology’s knowledge, the surgeons’ activity was limited, and the use of medicaments were preferred to surgical interventions. [6]i Severinus was one of the fi rst physicians to theorize and practice an active sur- gery he called “del medicar crudo” (“to medicate crudely”). He performed the new- est surgical techniques, and professed an early intervening surgery when the patho- logical process didn’t show signs of spontaneous resolution. He insisted that “active 27 Acta hist. med. stom. pharm. med. vet. / 2019 / 38 / 1–2 / 26–29 Herculean” surgery was less dangerous and brought more glory than the haphazard, eff eminate and compromising surgery. [6] Herculean” surgery was less dangerous and brought more glory than the haphazard, eff eminate and compromising surgery. [6] According with his idea of an innovative surgery, he showed boldness in his surgical practice, as reported in some extracts of his works “De recondita absces- sum natura” and “De effi caci Medicina”. [5] In these books, Severinus illustrated and discussed several cases of ulcers, plagues, tumors, ernia, and their treatment. He de- scribed his innovative surgical technique to treating an inguinal hernia, using “a large needle threaded with very strong new silk... a little ivory or bone plate, rectangular in shape and about one inch in thickness”. A further interesting example of Severinus’ surgical practice is represented by the trephining he performed to treat a Spanish nobleman, who suff ered from an in- tolerable headache. Keywords: General surgery, barber surgeons, operative surgical procedures Non MeSh: Marcus Aurelius Severinus Aft er the trephining, Severinus found a “fungous excrescences”, and destroyed it resolving the symptomatology, and likely performing on of the fi rst successful excision of brain tumor. [7 p62] However, Severinus’ boldness wasn’t fully appreciated at that time, and although farsighted, his idea was unsuccessful. It was obstacled by pain, bleeding and infection risks related to surgery. In “De effi caci medicina”, writing about the surgical treatment of the cutaneous ulcers in 1625, performed by cutting the varices surrounding the ulcers (although it wasn’t recommended), Severinus stated that: “I fi rst performed this surgical operation in Naples. Nevertheless, I was challenged from other doctors, which protested with the governors of hospital, because they believed that it (the surgical operation) could expose the patients to a serious risk.” [7 p125] At that time, surgeons were more careful to avoid pain and further risks to the patients, and they preferred a waiting attitude to a premature surgical approach. Th ose were the reasons why the Severinus’ attempt to reform surgery failed.it h Only about two hundred and fi ft y years later, the discoveries of anesthesia [8] and the infectious prophylaxis [9] changed the future of surgery and whole medicine. Th e scientifi c status of surgery was raised and the idea of an early and resolutive sur- gery stated by Severinus was accomplished. Rezime Operativne hirurške procedure su doživele jedan pokušaj reformacije u XVII veku, od strane italijanskog hirurga i anatoma Marka Aurelija Severina (1580- 1656). Njegov doprinos hirurgiji obuhvatao je dva nivoa: metodološki i tehnički. On je bio jedan od prvih hirurga koji je teorijski razmatrao i praktično primenjivao aktivnu hirurgiju, koju je sam nazivao “del medicar crudo” (lečenje na grubi način). Primenjivao je najsavremenije hirurške tehnike, pri čemu je osmislio koncept rane hirurške intervencije, pre nego što bi patološki proces pokazao znake spontanog razrešenja. Shodno tome, smelo je pristupao hirurškoj praksi, što je i demonstrirao u svojim delima “De recondita abscessum natura” i “De effi caci Medicina”. Međutim, njegove ideje nisu naišle na prihvatanje tadašnjih medicinskih krugova. U to vreme 28 Perciaccante A., Marcus Aurelius Severinus and his Attempt to Reform the Surgery su hirurzi nastojali da izbegnu bol i dalje rizike za pacijenta i radije su odabirali pasivniji pristup. Njegove ideje su naišle na prihvatanje nekih 250 godina kasnije, kada su proboji na polju anestezije i infektivne profi lakse omogućile da se hirurgija uzdigne na nivo nauke, što je i omogućilo da se njegovi principi primene u praksi. References: 1. Richardson RG. Th e story of surgery: an historical commentary. Shrewsbury, United Kingdom: Quiller Press; 2004. 2. Zimmerman LM, Veith I. Great ideas in the history of surgery. Baltimore: Th e Williams & Wilkins Company; 1961. Vesalius A. De humani corporis fabrica libri septem. Basilea: Ioannem Oporinum; 155 4. Paré A. Th e apologies and treatise of Ambroise Paré. Containing the voyages made into divers places, with many of his writings upon surgery. Textbook Publisher; 2003. 5. Accattatis L. Le biografi e degli uomini illustri delle Calabrie. Cosenza: Tipografi a Muni- cipale; 1869. 6. Reichert FL. Th e lure of medical history: Marcus Aurelius Severinus (1580-1656): a con- temporary of Harvey, and author of the fi rst work on comparative anatomy. Cal West Med. 1929; 30(3):183-85. 7. Severinus MA. De effi caci medicina libri III. Francofurtum ad Moenum: Beyer; 1646. 7. Severinus MA. De effi caci medicina libri III. Francofurtum ad Moenum: Beyer; 1646. 8. Bigelow HJ. Insensibility during surgical operations produced by inhalation. Boston Med Surg J 1846 35:309-17 8. Bigelow HJ. Insensibility during surgical operations produced by inhalation. Boston Med Surg J 1846 35:309-17 9. Lister J. On a new method of treating compound fracture, abscess, etc., with observations on the conditions of suppuration. Lancet 1867 89:326-9,357-9,507-9. 9. Lister J. On a new method of treating compound fracture, abscess, etc., with observations on the conditions of suppuration. Lancet 1867 89:326-9,357-9,507-9. Recieved: 27/09/2019 Reviewed: 21/10/2019 Accepted: 06/11/2019 29
https://openalex.org/W4200404095
https://journals.openedition.org/angles/pdf/3715
English
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Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and the Queer Afterlife of “BS” Johnson in
Angles
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Printed version Date of publication: 15 December 2021 Chris Clarke Electronic version URL: https://journals.openedition.org/angles/3715 DOI: 10.4000/angles.3715 ISSN: 2274-2042 Angles New Perspectives on the Anglophone World  13 | 2021 The Torn Object Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and the Queer Afterlife of “BS” Johnson in We Are Made of Diamond Stuff by Isabel Waidner Chris Clarke Angles New Perspectives on the Anglophone World  13 | 2021 The Torn Object Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and the Queer Afterlife of “BS” Johnson in We Are Made of Diamond Stuff by Isabel Waidner Chris Clarke Angles New Perspectives on the Anglophone World  13 | 2021 The Torn Object Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and the Queer Afterlife of “BS” Johnson in We Are Made of Diamond Stuff by Isabel Waidner Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and the Queer Afterlife of “BS” Johnson in We Are Made of Diamond Stuff by Isabel Waidner Chris Clarke Electronic reference Chris Clarke, “Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and the Queer Afterlife of “BS” Johnson in We Are Made of Diamond Stuff by Isabel Waidner”, Angles [Online], 13 |  2021, Online since 15 December 2021, connection on 29 December 2021. URL: http:// journals.openedition.org/angles/3715 ; DOI: https://doi.org/10.4000/angles.3715 This text was automatically generated on 29 December 2021. Angles est mise à disposition selon les termes de la Licence Creative Commons Attribution 4.0 International. 1 1 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... “Another Ballsup” 1 Jonathan Coe presents the “Coda” of his biography, Like a Fiery Elephant: The Story of B. S. Johnson, as “merely ‘a story about B. S. Johnson’” (2004: 421, Coe’s emphasis). This story, Coe implies, is to be treated differently from “The Story” announced in the text’s subtitle, which reconstructs “A Life” for its “one-man literary avant-garde of the 1960s” in various ways (3). Coe’s biography honours Johnson’s view that “Life is chaotic, fluid, random; it leaves myriads of ends untied, untidily” (1973: 14) by adopting a fragmentary form that divides the text into three parts: “A LIFE IN SEVEN NOVELS” (2004: 11), “A LIFE IN 160 FRAGMENTS” (33), and “A LIFE IN 44 VOICES” (387).1 Separated from these different constructions of “A LIFE”, the “Coda: Fragment 46, or Why Did B. S. Johnson Cut Holes in the Pages of Albert Angelo?” concerns a discarded opening section to Johnson’s 1964 novel. This fragment presents “an electric unspeakable, inexplicable knowing” (436) between the characters Samuel (Albert in the final novel) and Graham, and the apparent “ballsup” (441) that God has made Samuel’s other half “almost a man instead of wholly a woman” (441).2 For Coe, this deleted section of the novel provides a way to tie up the “loose ends” (422) in the “picture [of Johnson he] was trying to reconstruct” (422). The text, Coe suggests, reflects how, in the 1950s, Johnson had failed to “act upon” “powerful (and disturbing) homoerotic feelings” (447), “‘betray[ing]’ [his friend Michael Bannard] by taking the ‘normal way’” (447): “heterosexuality” (70). But no sooner does Coe emphasize the “homoerotic Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 2 undertones of this deleted section” (445), than he dispels the significance of his biographical reading because “we can never know something like that” (447): “Not for certain. It’s a good story, that’s all” (447). Coe closes his biography on this note of uncertainty, and consequently one could argue that his text reproduces the “self-doubt and vulnerability” (452) that he suggests characterise Johnson’s novels: “they quiver with nervous energy even now” (452). However, critical responses to the biography suggest that the “Coda” has the opposite effect. “Another Ballsup” Jennifer Hodgson, for example, contends that Coe’s attempt to “explain the ‘something inexplicable’ about Johnson’s suicide” “undercut[s]” the “biography’s own literary experimentation” (2013: 30).3 The uncertain status of Johnson’s discarded representation of same-sex desire — it is a part of, and yet apart from, “The Story of B. S. Johnson” — highlights the danger that the reclamation of the work of postwar experimental novelists could obstruct an engagement with the more difficult affects their texts dwell upon. This essay suggests that Coe’s equivocal retrieval and (re)displacement of the abandoned opening of Albert Angelo provides an opportunity to critically reflect on what we risk missing in appearing to recover the experimental novels of the postwar period. Where Johnson had once been “in danger of being forgotten as a postmodernist who wasn’t postmodern, and a realist who had rejected (conventional) realism” (Tew & White 2007: 6), Coe’s biography has sparked a reappraisal of his work and other forms of postwar narrative experimentation, with essay collections such as British Avant-Garde Fiction of the 1960s (Mitchell & Williams 2019) stimulating discussions on the literary innovators fleetingly alluded to in Coe’s biography: “(Alan Burns, Eva Figes, Ann Quin, Christine Brooke-Rose spring immediately to mind)” (Coe 2004: 3). While this broadening of the scholarly conversation on postwar experimental novels challenges the view that “Johnson was the dominant voice and leader” (Tew 2012: 59), reassessments of these experimentalists’ writings have tended, as Julia Jordan observes, to offer “recuperative gestures” (Jordan 2020: 4). Paradoxically, scholars’ attempts to reinstate these experimental writers in histories of the postwar British novel might make it harder to acknowledge those aspects of their texts that resist the work of recovery, such as the “ballsup” (Coe 2004: 441) hovering on the edge of the image of Johnson produced by Coe. 3 In her revisionary analysis of these literary innovators, Late Modernism and the Avant- Garde British Novel: Oblique Strategies (2020), Jordan argues that we can view these experimental novels as part of a “strain of oblique late modernism” (2020: 8), constituted by a “thematic and philosophical concern with, and compositional and formal use of, accident, error, and indeterminacy” (2). Jordan’s argument — that “a form of being best articulated by the accidental is constitutive of [this writing’s] distinctively ‘late’ status” — provides one way to appreciate the postwar experimental novel’s “affective distinctiveness” (6). “Another Ballsup” However, I want to suggest that we could equally understand the temporal splitting and historical ambivalence characterizing this work (and responses to it) as a repercussion of the way in which these experimental novels turn away from contemporary readers and linger on negative affects and irreparable losses. In particular, I argue that we can reconsider the anxieties surrounding the recovery of Johnson’s abandoned fragment — a “ballsup” (Coe 2004: 441) deleted from the published text of Albert Angelo — by exploring how this text evokes what Heather Love describes as the “backward feelings — shame, depression, and regret” (2007: 8) — “tied to the experience of social exclusion and to the historical ‘impossibility’ of same- 3 In her revisionary analysis of these literary innovators, Late Modernism and the Avant- Garde British Novel: Oblique Strategies (2020), Jordan argues that we can view these experimental novels as part of a “strain of oblique late modernism” (2020: 8), constituted by a “thematic and philosophical concern with, and compositional and formal use of, accident, error, and indeterminacy” (2). Jordan’s argument — that “a form of being best articulated by the accidental is constitutive of [this writing’s] distinctively ‘late’ status” — provides one way to appreciate the postwar experimental novel’s “affective distinctiveness” (6). However, I want to suggest that we could equally understand the temporal splitting and historical ambivalence characterizing this work (and responses to it) as a repercussion of the way in which these experimental novels turn away from contemporary readers and linger on negative affects and irreparable losses. In particular, I argue that we can reconsider the anxieties surrounding the recovery of Johnson’s abandoned fragment — a “ballsup” (Coe 2004: 441) deleted from the published text of Albert Angelo — by exploring how this text evokes what Heather Love describes as the “backward feelings — shame, depression, and regret” (2007: 8) — “tied to the experience of social exclusion and to the historical ‘impossibility’ of same- Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 3 sex desire” (4). In Feeling Backward: Loss and the Politics of Queer History (2007), Love traces “a tradition of queer backwardness” (8) in texts by writers including Walter Pater and Sylvia Townsend Warner, in order to attend to the way in which a “history of marginalization and abjection” (29) continues to “structure queer experience in the present” (29). “Another Ballsup” Love’s study is motivated by her concern that the political strategy of turning shame into pride and visibility “has meant that the painful and traumatic dimensions” (3) of texts marked by the losses of queer history “have been minimized or disavowed” (4). 4 Love’s reassessment of this “affirmative turn” (4) in gay, lesbian, and queer criticism suggests one way to engage with the problem that Coe’s recovery of the deleted section of Albert Angelo presents: namely, that “the critical compulsion to fix — at least imaginatively — the problems of queer life has made it difficult to fully engage with such difficulties” (3). While attending to the specific socio-historical context of Johnson’s work, I read his abandoned representation of same-sex desire through Love’s analysis of how late 19th- and early 20th-century authors cultivated a kind of “backward modernism” (7) in response to their “painful negotiation of the coming of modern homosexuality” (4). In doing so, I suggest that the value of our return to Johnson’s work lies in how its figures of backwardness — its “explorations of haunting and memory” (Love 2007: 7) and its articulation of feelings such as “regret”, “ressentiment”, and “despair” (4) — asks of readers to “refuse to write off the most vulnerable, the least presentable, and all the dead” (30). My reading of this strain of queer backwardness in Johnson’s work impresses, in Love’s words, how his texts “do not welcome contemporary critics — instead they turn away from us” (8). Consequently, it proposes a way to understand why the discarded opening of Albert Angelo has been omitted from discussions of his writing and to appreciate Johnson’s subsequent exploration of the problems with what he calls “tak[ing] the normal way” (Coe 2004: 70) in House Mother Normal (1971). Johnson’s destabilizations of notions of the (ab)normal accentuate experiences of loss, abjection, and marginalization, and they thereby point to his work’s resonance with recent literary contestations of damaging social norms. We Are Made of Diamond Stuff (2019) by Isabel Waidner, for example, reanimates Johnson’s character “House Mother Normal” as part of their text’s intervention against both “the appropriation of queer cultures by the straight mainstream” and “the normativity and elitism of much English-language and European avant-garde literature” (2019b). “House Mother Normal”, Waidner writes, “might be one of the villains in Diamond Stuff — but another villain might be B. S. Johnson himself” (2019b). “Another Ballsup” Since Diamond Stuff resists being “read as an extension of a genealogy of experimental literature” (Waidner 2019b) represented by Johnson, it highlights the need to critically reassess the affirmative history and identity that have accompanied Johnson’s reappearance. “A Nothing Teacher” 6 It may seem strange to examine backwardness in the work of Johnson, a writer intensely committed to “the evolution of the form in which he is working” (Johnson 1973: 16). “The novelist cannot”, Johnson proposes, “legitimately or successfully embody present-day reality in exhausted forms” (16), and “[n]o sooner is a style or technique established than the reasons for its adoption have vanished or become Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... irrelevant” (17). However, as Love argues, “[e]ven when modernist authors are making it new, they are inevitably grappling with the old: backwardness is a feature of even the most forward-looking modernist literature” (6). Indeed, Johnson’s aesthetic innovations respond to what he calls “exhausted” (1973: 13) and “clapped out” (13) literary forms, “failed” (19) and “inadequate” (19) writing conventions, as well as his sense that he “has something to say which [he] fail[s] to say satisfactorily in conversation, in person” (18). Johnson sets up his commitment to “writ[ing] truth in the form of a novel” (14) as a way to fix these literary and personal faults — he writes “to retaliate on those who have hurt [him]” and to “repay those people who have helped [him]” (18). However, the more his texts insist on presenting his authentic self, the more they call attention to the subject’s always partial and uncertain articulation in language as the parentheses of the lines below insinuate: The following tries to grope towards it, in another way: I have a (vision) of something that (happened) to me something which (affected) me something which meant (something) to me (Johnson 1973: 19) 7 Yet, despite critics’ frequent refutations of Johnson’s claim to present his truth, readings of the limitations of his truth-telling tend paradoxically to privilege his authorial agency by implying that Johnson alone lies behind his writing’s apparent failure. Patricia Waugh, for instance, suggests that Johnson’s misplaced focus on telling the truth shows how he “failed, tragically and inevitably, to get out from under a ‘net’ which increasingly closed in on him until his suicide” (1984: 98). Jordan suggests that this tendency to conflate the “literary-theoretical and biographical in the face of [Johnson’s] 1973 suicide” partly emanates from the way in which readers “take [Johnson] too much at his word” (2020: 132). “A Nothing Teacher” “[W]hat makes “the experience of reading [Johnson] an oddly intimate one”, Jordan observes, is his work’s “tone, in [Sianne] Ngai’s sense” of “‘a cultural object’s affective bearing’” (119). For Jordan, “[e]xcessiveness in all senses” — “overstatement, fatness, disproportion; the inelegance of feeling oneself to be overwhelming” — characterizes the tone of Johnson’s work and it creates “an excess of intimacy between author and reader” (119). I want to suggest that figures of backwardness in Johnson’s writing offer a way to attend to the socio-historical privations that underpin his work’s need to produce a “peculiar excess of identification” (122) — what Jordan discerns as its “affective potency” (117). In contrast to the apparent authority and autonomy of the “I” staged in his literary polemics and parts of his fiction, Johnson’s experimental novels dwell on failed forms of sociality and the backward feelings they evoke. While, for example, Albert Angelo presents its eponymous protagonist — an “‘architect manqué’” (Johnson 1964: 29) having to “earn [his] living by [substitute] teaching” (12) — as an “allornothinger” (105), the novel emphasizes that “it is usually nothing” (105), and we hear him think, during a difficult lesson, “Nothing. This class is a nothing / And I’m a nothing teacher” (72). My reading of backwardness in Johnson’s texts follows Carol Watts’ suggestion that the “lineaments [of his work] are to be read less in [its] authorial pronouncements, and more in the formal experiments of his writing” (2007: 87). For Watts, “the seeming egotism of that Johnsonian voice” (91), permeated with “its angers and points of impasse” (91), betrays an “encounter” with “non-identity” (82). Moreover, Watts helpfully suggests that “[t]he formal engagement with non-identity in Johnson’s work becomes a means of forcing an encounter with those excluded from the Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 5 conservative certainties of bourgeois life, even as they are made the object of those certainties” (88). The “nonidentity” Johnson’s writing asserts “with his reader” (Watts 2007: 82) — its backwardness — is crucial, I argue, because it orientates readers to the states of injury, or what Love calls “the wounds, the switchbacks, and the false starts”, that spur his text’s expressions of other more affirmative feelings and give them their appeal and “binding power” (32). “A Nothing Teacher” The diary’s allusion to “the Goddess”, as Coe clarifies, refers to The White Goddess by Robert Graves, which, as Johnson would put it in a review of the text, argues that “[t]he function of poetry […] is ritual invocation of the Muse Goddess; true poetry is concerned with the relationship between men and women” (Coe 2004: 113). 10 The diary’s “Bach link” reappears, in a different form, in the abandoned section of Albert Angelo through the representation of the first time Samuel and Graham touch: “when they sat back to back on a hot summer evening in the arena at a prom, and the points of contact seemed to burn together so that they thought they might never come apart” (Coe 2004: 441). In another echo of the diary extract, the narrator of the discarded section at once builds and breaks up this intimate attachment, referring to the relationship as another “ballsup” on God’s part and insisting that Graham was “not homosexual, just neuter” (441). In both texts, the stuttering articulation of same-sex desire is offset by uneven gendered identities, which simultaneously idealize and denigrate femininity. Graham, for example, is “almost a man instead of wholly a woman” and “hates” (441) women, while the extract from the diary plays with the idea of upsetting “the Goddess” (38). The diary’s allusion to “the Goddess”, as Coe clarifies, refers to The White Goddess by Robert Graves, which, as Johnson would put it in a review of the text, argues that “[t]he function of poetry […] is ritual invocation of the Muse Goddess; true poetry is concerned with the relationship between men and women” (Coe 2004: 113). 11 Where the misfiring of Johnson’s truth-telling presents the possibility of destabilizing these limited gender roles and relations, Coe’s return to the diary entry at the close of his biography offers to reverse the future into which he has been projected and complicate the image of Johnson he has constructed. At one point, Coe reflects on the apparent inevitability of writing Johnson’s biography: Johnson always had his eye on posterity, and I reckon he was certain that someone like me would come along and write this book one day. I have the resigned sense of having been chosen to fulfil a confident prophecy. “A Nothing Teacher” 9 The discarded opening of Albert Angelo and other texts by Johnson articulate the difficulties of bearing marginalized desires and subject positions in postwar Britain. We can attend to the lasting imprint of these backward feelings by returning to Coe’s biography and reconsidering key fragments for thinking about the missing section of Albert Angelo. The “Coda” re-examines what the biography labels as fragment 1, an extract from Johnson’s diary from 1961, which — for Coe — made his subject “flicker to life for the first time” (2004: 37): 9 The discarded opening of Albert Angelo and other texts by Johnson articulate the difficulties of bearing marginalized desires and subject positions in postwar Britain. We can attend to the lasting imprint of these backward feelings by returning to Coe’s biography and reconsidering key fragments for thinking about the missing section of Albert Angelo. The “Coda” re-examines what the biography labels as fragment 1, an extract from Johnson’s diary from 1961, which — for Coe — made his subject “flicker to life for the first time” (2004: 37): Homosexuality would be such an affront to the Goddess that I am tempted, merely to see if she would destroy me. […] Bach link with Michael: the constant, ever-fixed mark, the centre around which it is possible to build an intellectual life. (Qtd. in Coe 2004: 37-8) 10 The diary’s “Bach link” reappears, in a different form, in the abandoned section of Albert Angelo through the representation of the first time Samuel and Graham touch: “when they sat back to back on a hot summer evening in the arena at a prom, and the points of contact seemed to burn together so that they thought they might never come apart” (Coe 2004: 441). In another echo of the diary extract, the narrator of the discarded section at once builds and breaks up this intimate attachment, referring to the relationship as another “ballsup” on God’s part and insisting that Graham was “not homosexual, just neuter” (441). In both texts, the stuttering articulation of same-sex desire is offset by uneven gendered identities, which simultaneously idealize and denigrate femininity. Graham, for example, is “almost a man instead of wholly a woman” and “hates” (441) women, while the extract from the diary plays with the idea of upsetting “the Goddess” (38). “A Nothing Teacher” (Coe 2004: 84) 11 Where the misfiring of Johnson’s truth-telling presents the possibility of destabilizing these limited gender roles and relations, Coe’s return to the diary entry at the close of his biography offers to reverse the future into which he has been projected and complicate the image of Johnson he has constructed. At one point, Coe reflects on the apparent inevitability of writing Johnson’s biography: Johnson always had his eye on posterity, and I reckon he was certain that someone like me would come along and write this book one day. I have the resigned sense of having been chosen to fulfil a confident prophecy. (Coe 2004: 84) 12 Yet, in the Coda’s restaging of the moment in which Coe felt his preconception of Johnson — “[a] man utterly sure of himself” (2004: 36) — come undone, we can see how the biography, rather than simply bringing Johnson back to life, instead offers, in Love’s words, a way to imagine the past as something “dissonant, beyond our control and capable of touching us in the present” (2007: 9-10). This disruption of Coe’s Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 6 received image of Johnson accentuates how the biography registers other kinds of historical discord. Where Coe had imagined that the story of B. S. Johnson would “fit a predetermined mould” — “embattled working-class modernist, with fiery temper and tunnel vision, pits himself against a complacent and reactionary literary establishment” (2004: 41) — fragment 56, “extracts from Albert Angelo”, reveals how the biography’s title is a correction of a spelling mistake made in one of the school children’s essays collected in the novel: “He is a bit of a film Star he acted the part of Garula. / He walks like a firy elephant” (Coe 2004: 143). The image of Johnson as “fiery” seems limiting, then, precisely insofar as it implicitly reaffirms Coe’s emphasis on a male subject’s volatile agency, and thereby eclipses the semantic indeterminacy of the child’s use of “firy” and its links to the educational failings explored in the novel: what the text refers to as its “social comment on teaching” (Johnson 1964: 176). “A Nothing Teacher” In reading Johnson and the subjects of his novels as “fiery”, we risk failing to attend, as Watts highlights, to how, “in the voices of the children of Albert Angelo”, there is “an acute sense of human cost”: “the potential and actual waste of lives and stories, caught within, and asserted against the grain of, the bland generalizations of a society that are violent in their effects” (Watts 2007: 87). Reading for backwardness in Johnson’s work thus offers a way to offset the argument that the “modernist subjectivism” of his novels helps to “abstract the subject form the social determinants of [their] resentment” (Bond 2005). “A Mortal Wound Above His Right Eye” 13 The disavowal of same-sex desire in the discarded section of Albert Angelo is doubled by its literal absence from the novel’s final form. However, these kinds of negative representation do have, as Love suggests, “a lot to tell us […]: they describe what it is like to bear a ‘disqualified’ identity, which at times can simply mean living with injury — not fixing it” (Love 2007: 4). Though the “social negativity” that we find in this ghost section of Albert Angelo might, as Love warns, be “branded as internally homophobic, […] or too depressing to be of use” (4), such a reading risks missing how the novel turns backward and impresses our ties with the “abject multitude against whose experience we define our own liberation” (10). The following reading of Albert Angelo and its abandoned section traces how these texts’ figures of backwardness expose the limitations of the gender relations instituted by the postwar education system, before considering how the imprint of this lost attachment accentuates the novel’s appeal to attend to other experiences of social exclusion. 14 The discarded section sets up the fragile “mystical oneness, connection” (Coe 2004: 442) between Samuel and Graham by presenting the moment in which “their eyes met and a strange thing happened. It was as if they had exchanged minds for a second, synchronously” (437). The scene evokes Johnson’s poem, “An Eye for Situation”, written six years before the publication of Albert Angelo, whose dedication reads, in a significant reversal, “for Bryan, from Michael”: 14 The discarded section sets up the fragile “mystical oneness, connection” (Coe 2004: 442) between Samuel and Graham by presenting the moment in which “their eyes met and a strange thing happened. It was as if they had exchanged minds for a second, synchronously” (437). The scene evokes Johnson’s poem, “An Eye for Situation”, written six years before the publication of Albert Angelo, whose dedication reads, in a significant reversal, “for Bryan, from Michael”: 14 The discarded section sets up the fragile “mystical oneness, connection” (Coe 2004: 442) between Samuel and Graham by presenting the moment in which “their eyes met and a strange thing happened. It was as if they had exchanged minds for a second, synchronously” (437). “A Mortal Wound Above His Right Eye” The novel intimates that the formation of distinct gendered subjects is implemented at the level of education, and it reflects on the limits and damaging consequences of this: They sit, large and awkward at the aluminium-framed tables and chairs, men and women, physically, whom you are today trying to help to teach to take their places in a society you do not believe in, in which their values already prevail rather than yours. Most will be wives and husbands, some will be whores and ponces: it’s all the same; any who think will be unhappy, all who don’t think will die. (Johnson 1964: 47) 17 The novel emphasizes the losses of this apparently rigid gender framework in its depiction of Albert calling the school register. Where at first students “chorus, ‘He’s not here’” when “Jackie Weir[’s]” (128) name is called, later repetitions of their refrain take on a disquieting and ambiguous echo: “after a couple of days, it was ‘Jackie Weir—He’s a queer’, mingled with ‘He’s not here’” (128). 18 In Albert Angelo, the shadow of Samuel’s “betrayal” of Graham also offers a critical perspective on the text’s misogyny, what is repeatedly referred to as Albert’s “betrayal” (124) by Jenny. The poems included and excluded from the novel highlight the divergence between these relationships. While Johnson’s poem “An Eye for Situation” is absent, another with a similar title, “An Eye for Place”, does appear, and it presents how I “anti-romantically” (Johnson 1964: 140) kissed you, in “Hungerford lane” (139) in order, as an earlier part of the novel reflects, “to prove it, the romance, the love” (49) — the speaker stopping “in the middle of a sentence” (49) and turning Jenny towards him. This interruption and turning mid-sentence is reenacted by the novel’s carefully orchestrated aposiopesis — “OH, FUCK ALL THIS LYING! […] —fuck all this lying” (163-7) — and the unravelling of Albert’s character implicitly realizes Graham’s prophecy that he would never become an architect. In the “Disintegration” (167) that follows, the narrator claims that “the end of this book” has 18 In Albert Angelo, the shadow of Samuel’s “betrayal” of Graham also offers a critical perspective on the text’s misogyny, what is repeatedly referred to as Albert’s “betrayal” (124) by Jenny. The poems included and excluded from the novel highlight the divergence between these relationships. “A Mortal Wound Above His Right Eye” The scene evokes Johnson’s poem, “An Eye for Situation”, written six years before the publication of Albert Angelo, whose dedication reads, in a significant reversal, “for Bryan, from Michael”: Recalling your intense obliqueness, the unsaid things we knew together, you would disapprove; whether you are right I cannot say, but you would call it treacherous weakness that I thus take the normal way. (Qtd. in Coe 2004: 70) Recalling your intense obliqueness, the unsaid things we knew together, Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 7 15 The poem’s difficult recollection of rejecting a deep and elusive connection parallels the breakdown of Samuel and Graham’s relationship in the discarded fragment.4 When Samuel “plunge[s]” into an “incongruous affair” (442) with a woman, Graham condemns Samuel to death at 29, a fate Albert Angelo alludes to through its famous future-seeing holes, which reveal an account of Christopher Marlowe’s death at the same age. The deleted section dwells on this breakup, describing Graham shouting about how “Samuel would never be an architect”, “he had treacherously betrayed all that was good in him and in the world, […] his small spark of spirit would prick and burn him still. And he would die at 29” (443). 16 This “betrayal” appears indirectly, if at all, in the final text of Albert Angelo. Albert occupies the flat Graham has vacated, and Albert’s relation to an absent other extends to his occupation as a substitute teacher: “always in supply there is this mysterious figure whom you are replacing. You try to build up your own conception of him from what the others let fall in conversation” (Johnson 1964: 38). This architectural language — the idea of “build[ing] up” people from others’ words — echoes Samuel’s desire to “communicate [to Graham] his enthusiasm about architecture” (Coe 2004: 439). At the same time, the narrator’s detailed descriptions of the schools in which he works calls attention to the institutions shaping the gender roles and relations that are possible and permissible in postwar Britain. “[T]all factory buildings, with heavy wire shields over their windows”, border the playground of a school, one of its walls “broken in its length only by a door, later than the rest of the building […], dividing the sexes” (Johnson 1964: 29). “A Mortal Wound Above His Right Eye” While Johnson’s poem “An Eye for Situation” is absent, another with a similar title, “An Eye for Place”, does appear, and it presents how I “anti-romantically” (Johnson 1964: 140) kissed you, in “Hungerford lane” (139) in order, as an earlier part of the novel reflects, “to prove it, the romance, the love” (49) — the speaker stopping “in the middle of a sentence” (49) and turning Jenny towards him. This interruption and turning mid-sentence is reenacted by the novel’s carefully orchestrated aposiopesis — “OH, FUCK ALL THIS LYING! […] —fuck all this lying” (163-7) — and the unravelling of Albert’s character implicitly realizes Graham’s prophecy that he would never become an architect. In the “Disintegration” (167) that follows, the narrator claims that “the end of this book” has Angles, 13 | 2021 Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 8 had a “definite effect of release” from “the influence of [the] memory of [Jenny], suffering the pain of her betrayal” (171). The novel even reveals the real name of the so-called “betrayer”. 19 However, the text’s punishing misogyny appears as another shaky facade when we consider how the actual holes in the novel unsettle Albert’s accusation that women “only want bits of [him]” rather than the “enormous totality” (105) (or whole) of himself. The holes in the novel’s pages look forward to someone inflicting “on him a mortal wound above his right eye […] from which he died instantly” (149-153). The effacement of the “right” (or so-called “normal”) eye takes on a deep symbolism when one considers how the poem, “An Eye for Place”, seems to have displaced “An Eye for Situation” in the novel. Furthermore, the “anti-romantic” (Johnson 1964: 140) kiss in Hungerford Lane presented in Albert’s poem for Jenny seems all too appropriate reading it alongside the moment in the abandoned section when Samuel and Graham walk up “Hungerford Lane together, under the arches’ groins” (Coe 2004: 444), and Samuel confesses “that he had brought the girl he loved here once” — “had shown her what Graham had shown him, and was guilty about it” (444). “A Mortal Wound Above His Right Eye” One is left with the sense that the “eye for place” (139) referred to in Albert Angelo always already involved another person, and that the “situation” the wounded “I” of this text confronts, but cannot fix, is that their love for another cannot find a place in this time because their relationship lies outside postwar social conventions. 20 The backward feelings that emanate from the missing relationship at the centre of Albert Angelo also accentuate how the literal holes in this text evoke other forms of social exclusion. The novel depicts how Albert and his fellow teacher and friend, Terry, frustrated with how the postwar education system is “desperately old-fashioned” and “waste[ful]”, “revolt, in desperation […] becom[ing] like delinquent teachers” (52). The pair wander through the city at night “to remind [themselves] that other people are suffering”, finding “a place for outcasts, misfits, where [they] feel something in common, however else we differ” (52). Throwing an empty milk bottle “overarm, as if it were a grenade, towards the playground” (126), Albert’s frustration mirrors that which he fears is being produced by the faltering education system. “ [T]he violence will out” if “we go on half-educating these kids” (133), Albert warns, before acknowledging how “the kids take breaking up [for the school holidays] rather literally” (147): “Last term windows were broken and a couple of doors kicked in” (147). The novel’s future-seeing holes appear directly beneath the lines on the page relating how “‘windows were broken’” (147); these rectangles of empty space in the novel correspond to the position of the words “were broken” (147) resting on top of them. Turning over the page, the text appears to have been subject to the student’s violence; its “window” of representation has been fissured, and the novel warns that there may be more to come as the holes in the pages look ahead to someone who “died instantly” (149-153) (Figure 1). Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 9 Figure 1: Hole in the page in B.S. Johnson’s Albert Angelo, 149 Figure 1: Hole in the page in B.S. Johnson’s Albert Angelo, 149 Source: https://www.pinterest.co.uk/pin/405464772671726512/ Source: https://www.pinterest.co.uk/pin/405464772671726512/ 21 While the holes in the pages amplify feelings of anger and resentment towards the failings of the postwar education system, they equally overlap with anxieties concerning others on the margins of society. Philip Tew notes how “in [Albert and Terry’s] nocturnal meandering they face the diasporic realities of another form of alterity, the nocturnal colonial presence in the underprivileged quarters of London, Cypriots, West Indians and Africans in particular” (2007: 205). The novel depicts Albert “try[ing] to give as many […] lessons as possible that do not involve reading and writing” (33) so as not to exclude the children who cannot speak English. Similarly, the pages directly preceding the violent murder revealed by the future-seeing holes present a fight between Albert and a “Giant negro” (151): “He was [coming], screaming something I don’t remember. I screamed something back that included fuck and indicated he wouldn’t be able to do it [kill him]” (151). The holes in the pages of Albert Angelo thereby impress the negative affects attached to different experiences of marginalization, while the absence they circle insinuates the ghost of those figures that, as Love notes, have been discarded by modernity: “the nonwhite and nonmonogamous, the poor and the genderdeviant, the fat, the disabled, the unemployed, the infected, and a host of unmentionable others” (10). 21 While the holes in the pages amplify feelings of anger and resentment towards the failings of the postwar education system, they equally overlap with anxieties concerning others on the margins of society. Philip Tew notes how “in [Albert and Terry’s] nocturnal meandering they face the diasporic realities of another form of alterity, the nocturnal colonial presence in the underprivileged quarters of London, Cypriots, West Indians and Africans in particular” (2007: 205). The novel depicts Albert “try[ing] to give as many […] lessons as possible that do not involve reading and writing” (33) so as not to exclude the children who cannot speak English. Similarly, the pages directly preceding the violent murder revealed by the future-seeing holes present a fight between Albert and a “Giant negro” (151): “He was [coming], screaming something I don’t remember. I screamed something back that included fuck and indicated he wouldn’t be able to do it [kill him]” (151). “(Some BS there, B. S.?)” 23 The shadowy figurations of an unrealized intimate connection and forms of social exclusion in Albert Angelo and its discarded section cast new light on Johnson’s subsequent critique of enforcing “the normal way” in his novel, House Mother Normal. Where, in the poem, “An Eye For Situation”, “the normal way” implies, as Coe suggests, “heterosexuality” (2004: 70), House Mother Normal attempts, as Johnson put it, to “say something about the things we call ‘normal’ and ‘abnormal’” (1973: 26), specifically in relation to the provision of elderly care in postwar Britain. The novel extends his work’s examination of notions of the (ab)normal by staging varying (de)compositions of characters’ bodies and memories, which register and expose violent impositions of so-called normality. The subversions of normality presented in House Mother Normal reappear in Isabel Waidner’s recent innovative novel, We Are Made of Diamond Stuff, in order to impress, as Waidner puts it, “that queer politics […] must be transformative of society at large” (2019b). Following the “eco-feminist Françoise d’Eaubonne”, Waidner contends “that ‘it’s not a question of integrating homosexuals into society, but of disintegrating society through homosexuality’” (2019b). Aligning the subversions of normality presented in House Mother Normal with the social disintegrations staged by Diamond Stuff, we can chart how figures of backwardness are a key aspect of the lasting connection between postwar narrative experimentation and contemporary innovative fiction. 24 Set inside “an old people’s home” (Coe 2004: 24), House Mother Normal presents, as Coe elucidates, “one single event” — a social evening hosted by the House Mother for the elderly residents — “from ten different points of view” (23). In the novel, the first- person interior monologues of the nine residents appear consecutively and are evenly weighted so that, “in every section, the same event (and the characters’ differing responses to it) occurs not just on the same page but at precisely the same point on that page” (Coe 2004: 24). Consequently, “the whole book becomes”, in Coe’s words, “polyphonic, fugal, a novel that can be read ‘vertically’ as well as ‘horizontally’” (24-25). Johnson heightens this twisting of readerly conventions through House Mother Normal’s concluding monologue, which reveals her exploitation and sexual abuse of those in her care. Figure 1: Hole in the page in B.S. Johnson’s Albert Angelo, 149 The holes in the pages of Albert Angelo thereby impress the negative affects attached to different experiences of marginalization, while the absence they circle insinuates the ghost of those figures that, as Love notes, have been discarded by modernity: “the nonwhite and nonmonogamous, the poor and the genderdeviant, the fat, the disabled, the unemployed, the infected, and a host of unmentionable others” (10). 21 22 Bringing Albert Angelo into a dialogue with its discarded section, we can recognise the painful substitutions and irreparable losses underpinning its conspicuous representation of an insecure virility and the politics of ressentiment it risks producing — an eye for an eye could describe the novel’s punishing approach to failed relationships and appears to underpin its misogyny. But the unspoken eye-to-eye relation that the novel refuses to bring to light gives a new significance to other Johnson texts, which also try to foreground an authoritative, masculine subject. The 22 Bringing Albert Angelo into a dialogue with its discarded section, we can recognise the painful substitutions and irreparable losses underpinning its conspicuous representation of an insecure virility and the politics of ressentiment it risks producing — an eye for an eye could describe the novel’s punishing approach to failed relationships and appears to underpin its misogyny. But the unspoken eye-to-eye relation that the novel refuses to bring to light gives a new significance to other Johnson texts, which also try to foreground an authoritative, masculine subject. The — an eye for an eye could describe the novel’s punishing approach to failed relationships and appears to underpin its misogyny. But the unspoken eye-to-eye relation that the novel refuses to bring to light gives a new significance to other Johnson texts, which also try to foreground an authoritative, masculine subject. The Angles, 13 | 2021 10 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... notion that one starts from “I”, “always with I” (Johnson 1966: 7), as Johnson’s next novel Trawl (1966) insists, takes on a critical ambiguity if we hear in the “I”s that echo in this opening line a nod backwards to an “electric unspeakable” (Coe 2004: 437) eye- to-eye meeting. “(Some BS there, B. S.?)” In this section, House Mother Normal offers a false justification for her actions by claiming that, “There are worse conditions and worse places, friend” (Johnson 1971: 197): “geriatric wards” (197) where people “are forgotten and wholly in the power of nurses who have been known to make them alter their wills” (198). But the novel’s final page subsequently upends even House Mother Normal’s disclosure of her abusive “normal” — her “I” is revealed to “be the puppet or concoction of a writer […], a writer who has me at present standing in post-orgasmic nude” (204) after a scene of bestiality. In light of the reading of backwardness in Albert Angelo and its discarded fragment, House Mother Normal’s grim order, “prepare, accept, worse times are a- coming, nothing is more sure” (204), appears to emanate as much from the ressentiment of the anonymous “I” that speaks on this final page as from the novel’s attentiveness to Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 11 11 lost and fragile relationships. The novel represents, for instance, “Gloria Ridge” repeatedly recalling an image of her “one true love” (103), but (poignantly) the colour of their lover’s hair changes with each new recollection, emphasizing the vulnerability and loss underpinning the memorization of human connections. 25 The lost relationships indirectly staged by texts such as Albert Angelo, its discarded fragment, and House Mother Normal evoke Love’s point that some aspects of “history only live on in the present through […] wounded attachments” (2007: 42). In order to avoid “severing” our ties to “traumatic — albeit important — parts of the past” (Love 2007: 42), we need to consider the aesthetic and affective discontinuities (as much as the continuities) between the work of Johnson and current manifestations of narrative experimentation. Waidner’s work is particularly helpful for exploring such literary- historical disjunctions. In a mock-academic essay that “peer-reviews” (Waidner 2019b) Diamond Stuff and Caspar Heinemann’s poetry collection Novelty Theory (2019), Waidner describes the relationship between their “own writing practice” and the “historical avant-garde” as “disidentificatory” (2019b). Drawn from José Esteban Muñoz’s analysis of the queer past, “disidentification”, Waidner elucidates, is “a practice enacted by a minority subject (me)”, who “must work with/resist the conditions of (im)possibility that the dominant culture (in this case, the avant-garde canon) generates” (2019b). “(Some BS there, B. S.?)” Far from offering a straightforward continuation of an “avant-garde canon”, Waidner’s adoption of a literary strategy of “disidentification” — the paradoxical way they “[a]lign […] [themselves] neither with, nor fully against” (2019b) Johnson’s work — reinforces the need to attend to the more backward aspects of Johnson’s experimental texts. 26 Diamond Stuff propels its critique of the inequities entwined with class, queerness, and citizenship in contemporary Britain by weaving a surreal and capricious narrative around an assemblage of texts, ranging from the sociology of Paul Willis and the queer theory of Jasbir Puar, to the science fiction of Samuel Delany and the poetry of Nisha Ramayya. Among the dialogues it opens with other texts, Diamond Stuff reimagines Johnson’s character “House Mother Normal” as a manager not of “a nursing home in ’70s London” (Waidner 2019a: 17) but of “a no-star hotel — the ‘New House of Normal’ — in present-day Ryde” (17) on the Isle of Wight. Diamond Stuff recalls how House Mother Normal “exploits and abuses those in her care” (17) — “the Pass the Parcel game” in which there is “no chocolate inside the parcel, only dog shit” (17) — before noting that “B. S. Johnson violates House Mother Normal in turn, putting her through a public masturbation I mean bestiality scene — with dog, Ralphie — not once, not twice but nine times over the course of the novel” (17). The repetition of this violation is, the narrator observes, “Ghastly, really, but funny. Funny’s important. It was a different time —. (Some BS there, B. S.?)” (17). While the text points to the temptation to conflate Johnson’s violations with those of his character — “Violent character is B. S. Johnson’s House Mother Normal” (17) — the narrator complicates their critique of Johnson’s work by acknowledging the troubling feelings it evokes (“ghastly”/”funny” (17)) and the temporal break between their times (signalled by the em dash). The novel thereby challenges the depiction of Johnson as a figure of “British avant-garde literature” (15) by insinuating that this recuperated image of the author may make it harder to appreciate the implications of the “BS” (17) of his work. “(Some BS there, B. S.?)” 26 Diamond Stuff propels its critique of the inequities entwined with class, queerness, and citizenship in contemporary Britain by weaving a surreal and capricious narrative around an assemblage of texts, ranging from the sociology of Paul Willis and the queer theory of Jasbir Puar, to the science fiction of Samuel Delany and the poetry of Nisha Ramayya. Among the dialogues it opens with other texts, Diamond Stuff reimagines Johnson’s character “House Mother Normal” as a manager not of “a nursing home in ’70s London” (Waidner 2019a: 17) but of “a no-star hotel — the ‘New House of Normal’ — in present-day Ryde” (17) on the Isle of Wight. Diamond Stuff recalls how House Mother Normal “exploits and abuses those in her care” (17) — “the Pass the Parcel game” in which there is “no chocolate inside the parcel, only dog shit” (17) — before noting that “B. S. Johnson violates House Mother Normal in turn, putting her through a public masturbation I mean bestiality scene — with dog, Ralphie — not once, not twice but nine times over the course of the novel” (17). The repetition of this violation is, the narrator observes, “Ghastly, really, but funny. Funny’s important. It was a different time —. (Some BS there, B. S.?)” (17). While the text points to the temptation to conflate Johnson’s violations with those of his character — “Violent character is B. S. Johnson’s House Mother Normal” (17) — the narrator complicates their critique of Johnson’s work by acknowledging the troubling feelings it evokes (“ghastly”/”funny” (17)) and the temporal break between their times (signalled by the em dash). The novel thereby challenges the depiction of Johnson as a figure of “British avant-garde literature” (15) by insinuating that this recuperated image of the author may make it harder to appreciate the implications of the “BS” (17) of his work. 27 In Diamond Stuff, House Mother Normal employs the unnamed narrator, who resembles the character “Eleven” (3) from the science fiction television series “Stranger 27 Angles, 13 | 2021 12 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 12 Things” (3), and Shae, for whom the stereotype of the “Reebok Working Class” (28) “fits but also doesn’t” (29) because Shae is “working-class and also queer (there’s no hiding it)” (29). “(Some BS there, B. S.?)” In addition, Shae’s clothes become characters — “The polar bears [on his “army green t-shirt”] are novelists (infantry soldiers), the reeboks are poets (intelligence operatives)” (15) — whose anarchic agency symbolize an alternative to the forms of resistance charted by Willis in Learning to Labour: How Working Class Kids Get Working Class Jobs (1977). In a moment that evokes Albert Angelo’s concern with the damaging consequences of “half-educating these kids” (Johnson 1964: 147), Diamond Stuff reflects on what Willis analysed as the pyrrhic victory of the “working class counter-school culture” (Willis 1977: 2). In “successfully resisting the norms of capitalism transmitted in school” (Waidner 2019a: 28), participants in this counter- school culture end up “having to take on lowly paid working-class jobs as adults” (28). The novel parodies how the characters consequently redouble their attempts to “escape their [supposed] working-class destin[ies]” by “putting their difference to work —.” (29): “Do you know why they’re grafting like this. (Queer. Queerdo.)” (29). In the kitchen of House Mother Normal’s hotel, for instance, the characters work gutting “raw” and “inky” squid, “purg[ing] entire beaches and tiny digestive tracts from maritime bodies” (9). Ironically, their work produces “Sandy refuse” and “raining black squid ink”, which threatens the existence of the narrator’s “permanent residence card” (40): “Squid ink gets on it, thing is disintegrating—.” (40). The abject and damaging detritus produced by the characters’ work exposes the incongruities and hypocrisies of the “Life in the UK test” (51), which is presented as a matter of acquiring “a particular form of educational capital, namely the ability to memorise information — kings, queens, invasions, a sanctioned version of British history and culture” (52). 28 Diamond Stuff uses parataxis, textual collage, and critically satirical parentheses and em dashes, amongst other rhetorical devices, to emphasize how the unpredictable performances of its characters constantly subvert the norms of capitalism, education, and Britishness, which its House Mother Normal symbolizes. If the novel invites us to see Shae and the narrator, the Reeboks and the polar bears, as backward children on this “‘derelict’” (103) island, House Mother Normal embodies a financial class “preoccupied with optional wage cuts, pulling tricks, cutting corners, profiteering” (59). With hotel “guests ‘disappear[ing]’ without paying” (59) — more people check in to the hotel than check out — House Mother Normal also appears to have “gone” (59): “Indisposed, Shae says (Disappeared.)” (63). “(Some BS there, B. S.?)” The unreliable presence of Johnson’s character in Diamond Stuff allows the text to pose the question, “Where is Normal?” (25), and this challenge to the construction of normality intersects with the novel’s challenge to promoting forms of social and political visibility. 29 In a parallel to the literary form of House Mother Normal, and its play on the discrepancies between different characters’ perceptions of events, Diamond Stuff skips between universes in order to imagine an alternative reality, in which all inequities have disappeared. In this “upside down world” (90), House Mother Normal (re)appears in “full drag” (91) and hosts the narrator’s “citizenship ceremony” (91): “I will be faithful and bear true allegiance to her Majesty Queen Mother Normal the First” (92). The novel’s satirical reversal of the deprivations that have given rise to a resurgent nationalism intensifies as the narrator remarks, “This is the part where working-class people including working-class migrants live in quality social housing in Central London” (92). The parentheses that follow impress how the representation of this ideal alternative universe serves to underline the novel’s critique of the punitive forms of Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 13 social normalization prevailing in “Tory Britain”: “(Stop it.) (Is this dimension selling me BS, I increasingly think so. Social housing in London? Micky Mouse citizenships, Queen Mother Normal the First? WORKING-CLASS GRADUATES)” (92-3). 30 This reference to “selling me BS” (93) implicitly iterates how Diamond Stuff challenges the temptation to align it with Johnson and the reappropriation of a postwar avant- garde, and instead insists that we engage with the experiences of abjection to which it points. Indeed, the novel collapses the sense of exhilaration that attends its ideal, mirror image of the world and emphasizes the seeming impossibility of its actualization when, in a parallel to its earlier enquiry regarding the location of “normal”, it turns backward, asking “(Where’s reality, I want to change it.)” (93). In its final pages, Diamond Stuff responds to this appeal to transform reality by presenting quotations from reviews of the Isle of Wight Zoo. The fragments of text evoke the architectural ruins of Albert Angelo — “I thought it was derelict. It looked like it probably closed 30 years ago. “(Some BS there, B. S.?)” It stands next to a crumbling hotel or casino” (103) — and echo the juxtapositions of children’s voices we hear in the essays written by Albert’s students: 30 “Half an hour of my life I can’t get back.” “This is a con.” “Symptomatic.” “The staff really do try.” “Desolate.” “Heartbreaking.” (Waidner 2019a: 105) “The staff really do try.” Desolate. “Heartbreaking.” (Waidner 2019a: 105) “Heartbreaking.” (Waidner 2019a: 105) 31 The novel closes with the acknowledgement of a time that an anonymous reviewer cannot “get back” and feelings of “heartbreak” and “desolation” (105). In doing so, it entwines its anarchic juxtapositions and self-satire with the backward feelings that come from writing within the constraints of the dominant culture’s “conditions of (im)possibility” (Waidner 2019b). In this respect, Diamond Stuff recalls how Johnson’s experimental texts similarly resist and contest forms of social normalization by turning backward toward the past and demanding that readers find a way to recognize its difficulties. 31 The novel closes with the acknowledgement of a time that an anonymous reviewer cannot “get back” and feelings of “heartbreak” and “desolation” (105). In doing so, it entwines its anarchic juxtapositions and self-satire with the backward feelings that come from writing within the constraints of the dominant culture’s “conditions of (im)possibility” (Waidner 2019b). In this respect, Diamond Stuff recalls how Johnson’s experimental texts similarly resist and contest forms of social normalization by turning backward toward the past and demanding that readers find a way to recognize its difficulties. “We Do Not Form a School” 32 This essay’s reading of the backwardness permeating Albert Angelo and its deleted fragment and Diamond Stuff’s disidentificatory writing practice suggests one way to understand why, as Jordan observes, Waidner’s “invocations of B. S. Johnson’s writing” are “magpieish” (2020: 31). While scholars’ “recent recastings of modernism” might, as Hodgson argues, go some way to establishing the sense of ‘historical continuity’ so dearly lacking in existing accounts of the British novel” (Hodgson 2013: 20), this emphasis on their work’s apparent connection to the present risks, as I have argued, attenuating our ability to engage with the ways in which Johnson’s writing turns away from us. If allusions to Johnson and his contemporaries’ experimental novels in recent innovative fiction seem fragmentary, then these disjointed literary responses to their work may offer a valuable counterpoint to the way in which dominant modes of writing literary history struggle, as Coe’s biography demonstrates, to respond to the damage articulated by this writing. Another unfinished text by Johnson, an essay entitled “Experimental British Fiction”, reiterates the problem of engaging with the damage and dissident desires presented by these writers’ texts. In this essay, Jordan notes, Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 14 Johnson “writes of a ‘we’” — the literary group to which he temporarily belonged — “and yet explicitly refuses the collectivization this might entail: ‘we do not form a school’” (Jordan 2015: 145). In light of the critique of the different kind of school we find in Albert Angelo, this simultaneous invocation and disavowal of a collective perhaps asks us to acknowledge how the literary experiments of writers such as Johnson, Figes and Burns respond in different ways to subjects who cannot be consoled. NOTES 1. Instead of telling stories, Johnson claimed to write “truth in the form of a novel” (1973: 14). Confronted with Johnson’s “untenable” “theories” (2004: 452), Coe turns to the “other extreme” (35), proposing a notion of the “biography as creative enterprise” (35), an “artwork” (35) that moulds “the chaos of reality” (35) into “appealing narrative shapes” (35). 2. All quotations from the abandoned opening section of Albert Angelo are made from the version of this text published by Coe in Fiery Elephant under the title, “46: Deleted Episode from Albert Angelo” (437-445). When I quote from other archival fragments collected by Coe, the page numbers refer to the location of that text in Coe’s biography. 3. Similarly, Vanessa Guignery suggests that the footnote in which Coe acknowledges the displacement of fragment 46 to the Coda has the effect of “arous[ing] the reader’s curiosity and introduc[ing] suspense” (135), and emphasizes how “a form of linearity persists in his biography, as opposed to Johnson’s more discontinuous novels” (135). 4. Jordan also remarks on how this poem figures “homosexual desire” as an “‘intense obliqueness, / the unsaid things we knew together’” (2020: 115) in the introduction to her analysis of the excessive tone of Johnson’s texts. Interestingly, Jordan attaches this “obliquity” to “someone” other than Johnson — “this is likely a reference to a homosexual advance from Johnson’s friend Michael Bannard” (115) — despite the poem’s admission that the feelings here, although unrealized, are shared — “the unsaid things we knew together”. Jordan’s subsequent claim that the “skew-whiff, the perverted, and the eccentric all hold some terror for Johnson, whose work is riddled with attempts to get on the perceptual and philosophical — as well as, perhaps, the sexual — straight and narrow” (115-6), only holds, I would suggest, if we implicitly privilege the excess of identification that Johnson’s writing is said to produce. BIBLIOGRAPHY Bond, Robert. “Pentonville Modernism: The Fate of Resentment in B. S. Johnson’s Albert Angelo.” Literary London: Interdisciplinary Studies in the Representation of London 3(1) 2005. http:// literarylondon.org/the-literary-london-journal/archive-of-the-literary-london-journal/ issue-3-1/pentonville-modernism-the-fate-of-resentment-in-b-s-johnsons-albert-angelo/ Coe, Jonathan. Like a Fiery Elephant: The Story of B. S. Johnson. London: Picador, 2004. Guignery, Vanessa. “Jonathan Coe’s Like a Fiery Elephant: The Story of B.S. Johnson —A Dialogue with Biography.” Biography 39 (2) 2016: 129-146. Hodgson, Jennifer. “‘Such a Thing as Avant-Garde Has Ceased to Exist’: The Hidden Legacies of the British Experimental Novel.” Twenty-First Century Fiction: What Happens Now. Ed. Siân Adiseshiah. Hampshire: Palgrave Macmillan, 2013. Johnson, B. S. Albert Angelo. London: Picador, [1964] 2004. Johnson, B. S. House Mother Normal. London: Picador, [1971] 2004. Johnson, B. S. “Introduction.” Aren’t You Rather Young To Be Writing Your Memoirs? London: Hutchinson, 1973. 11-31. Johnson, B. S. Trawl. London: Picador, [1966] 2004. Jordan, Julia. Late Modernism and the Avant-Garde British Novel: Oblique Strategies. Oxford: Oxford UP, 2020. Jordan, Julia. “Late Modernism and the Avant-Garde Renaissance.” In The Cambridge Companion to British Fiction since 1945. Ed. David James. Cambridge: Cambridge UP, 2015. 145-159. Love, Heather. Feeling Backward: Loss and the Politics of Queer History. Cambridge: Harvard UP, 2007. Mitchell, Kaye, and Nonia Williams, eds. British Avant-Garde Fiction of the 1960s. Edinburgh: Edinburgh UP, 2020. Tew, Philip. “Otherness, Post-Coloniality and Pedagogy in B. S. Johnson’s Albert Angelo and See the Old Lady Decently.” In Re-reading B. S. Johnson. Eds. Philip Tew and Glyn White. London: Palgrave Macmillan, 2007. 202-219. Tew, Philip, and Glyn White. “Introduction: Re-reading B. S Johnson.” In Re-reading B. S. Johnson. Eds. Philip Tew and Glyn White. London: Palgrave Macmillan, 2007. 3-13. Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 15 Waidner, Isabel. “Class, queers & the avant-garde in new British writing with Caspar Heinemann & Isabel Waidner.” AQNB 25 June 2019, https://www.aqnb.com/2019/06/25/class-queers-and- the-avant-garde-in-new-british-writing-with-caspar-heinemann-and-isabel-waidner/ Waidner, Isabel. We Are Made of Diamond Stuff. Manchester: Dostoyevsky Wannabe Originals, 2019. Watts, Carol. “‘The Mind Has Fuses’: Detonating B. S. Johnson.” In Re-reading B. S. Johnson. Eds. Philip Tew and Glyn White. London: Palgrave Macmillan, 2007. 80-94. Waugh, Patricia. Metafiction: The Theory and Practice of Self-Conscious Fiction. London: Methuen, 1984. Willis, Paul. Learning to Labour: How Working Class Kids Get Working Class Jobs. Aldershot: Ashgate, [1977] 1993. Waidner, Isabel. BIBLIOGRAPHY “Class, queers & the avant-garde in new British writing with Caspar Heinemann & Isabel Waidner.” AQNB 25 June 2019, https://www.aqnb.com/2019/06/25/class-queers-and- the-avant-garde-in-new-british-writing-with-caspar-heinemann-and-isabel-waidner/ Waidner, Isabel. We Are Made of Diamond Stuff. Manchester: Dostoyevsky Wannabe Originals, 2019. Watts, Carol. “‘The Mind Has Fuses’: Detonating B. S. Johnson.” In Re-reading B. S. Johnson. Eds. Philip Tew and Glyn White. London: Palgrave Macmillan, 2007. 80-94. Waugh, Patricia. Metafiction: The Theory and Practice of Self-Conscious Fiction. London: Methuen, 1984. Willis, Paul. Learning to Labour: How Working Class Kids Get Working Class Jobs. Aldershot: Ashgate, [1977] 1993. Willis, Paul. Learning to Labour: How Working Class Kids Get Working Class Jobs. Aldershot: Ashgate, [1977] 1993. ABSTRACTS In the discarded opening section of Albert Angelo (1964), recovered by Jonathan Coe in his biography of B. S. Johnson, the narrator laments how “God had made another ballsup”: the protagonist’s “other half” is “almost a man instead of wholly a woman”. This essay reads Johnson’s abandoned representation of same-sex desire through the “tradition of queer Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 16 backwardness” traced by Heather Love in Feeling Backward: Loss and the Politics of Queer History (2007). I argue that the abandoned fragment’s representation of “an unspeakable, inexplicable knowing” between two characters evokes the “backward feelings” which Love proposes are tied to “the historical ‘impossibility’ of same-sex desire” (Love 2007). In doing so, I show how Johnson’s literary afterlife lies in orientating readers to modernity’s denigrated “others”, such as sexual deviants marginalized for refusing to take what Johnson calls the “normal way”. Where the backward feelings in Johnson’s experimental novel illuminate his work’s resistance to various conditions of modern subjecthood, they can equally help us understand the resonance between his work and recent innovative fiction, which also contests punitive forms of social normalization. Isabel Waidner’s “critically British novel”, We Are Made of Diamond Stuff (2019), for example, reanimates Johnson’s perverse character “House Mother Normal” to propel its critique of the inequities of class, queerness, and national identity. We can appreciate what remains most subversive about Johnson’s experimental novels by charting how their backwardness reverberates with the concerns of queer experimental literature in the present. Dans la première section rejetée d'Albert Angelo (1964), retrouvée par Jonathan Coe dans sa biographie de B. S. Johnson, le narrateur se lamente sur le fait que « Dieu a fait une autre connerie » : l'autre moitié du protagoniste est « presque un homme au lieu d'être entièrement une femme ». Cet article étudie la représentation abandonnée par Johnson du désir homosexuel à travers la « tradition du sous-développement ou retard [backwardness] queer » décrite par Heather Love dans Feeling Backward: Loss and the Politics of Queer History (2007). Je démontre que la représentation dans le fragment abandonné d'un « savoir indicible et inexplicable » entre deux personnages évoque les « sentiments sous-développés » qui, selon Love, sont liés à « la prétendue ‘impossibilité’ historique du désir homosexuel » (Love 2007). INDEX Keywords: experimental novel, queer theory, queer politics, Johnson B. S., Waidner Isabel Mots-clés: roman expérimental, théorie queer, politique queer, Johnson B. S., Waidner Isabel ABSTRACTS Ce faisant, je montre comment l’après-vie littéraire de Johnson consiste à orienter les lecteurs vers les « autres » déconsidéré.e.s du monde moderne, comme les déviants sexuels marginalisés pour avoir refusé de suivre ce que Johnson appelle la « voie normale ». Si les sentiments d'arriération ou de sous-développement dans le roman expérimental de Johnson mettent en lumière la résistance de son œuvre à diverses conditions de la subjectivité moderne, ils peuvent également nous aider à comprendre les échos entre son œuvre et la fiction innovante la plus récente qui conteste également les formes punitives de normalisation sociale. Le « roman critique britannique » d’Isabel Waidner, We Are Made of Diamond Stuff (2019), par exemple, fait revivre le personnage pervers de Johnson, « House Mother Normal », pour alimenter sa critique des inégalités de classe, de l'homosexualité et de l'identité nationale. Pour appréhender ce qui reste le plus subversif dans les romans expérimentaux de Johnson, on peut tracer la manière dont leur sous-développement est en résonance avec les préoccupations de la littérature expérimentale queer d’aujourd’hui. CHRIS CLARKE Chris Clarke is a writer and researcher based in Southampton. He completed his doctoral thesis at the University of Southampton in 2015, before working there as a part-time tutor between 2017 and 2020. He is the author of “‘Unconsciously Influenced’: Alan Burns, Ian McEwan, and the Angles, 13 | 2021 Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... Subjects Who Cannot Be Consoled: The Discarded Opening of Albert Angelo and t... 17 Lasting Legacies of Postwar British Experimental Fiction”, MFS: Modern Fiction Studies (Spring 2018). He has also written an essay on the “poetry of the inarticulate” in the early experimental novels of Eva Figes, which appeared in British Avant-Garde Fiction of the 1960s (Edinburgh University Press, 2019). Contact: cfclarke16 [at] gmail.com Angles, 13 | 2021
https://openalex.org/W3209296761
http://www.journalofdairyscience.org/article/S0022030221009693/pdf
English
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Milking system and premilking routines have a strong effect on the microbial community in bulk tank milk
Journal of dairy science
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J. Dairy Sci. 105:123–139 https://doi.org/10.3168/jds.2021-20661 © 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). J. Dairy Sci. 105:123–139 https://doi.org/10.3168/jds.2021-20661 © 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). © 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Associat This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Milking system and premilking routines have a strong effect on the microbial community in bulk tank milk Li Sun,1* Åse Lundh,1 Annika Höjer,2 Gun Bernes,3 David Nilsson,4 Monika Johansson,1 Mårten Hetta,3 Anders H. Gustafsson,5 Karin Hallin Saedén,2 and Johan Dicksved6 1Department of Molecular Sciences, Swedish University of Agricultural Sciences, Box 7015, SE-750 07 Uppsala, Sweden 2Norrmejerier Ek. Förening, Mejerivägen 2, SE-906 22 Umeå, Sweden 3Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden 4Computational Life Science Cluster, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden 5Växa Sverige, Ulls väg 26, SE-750 07 Uppsala, Sweden 6 Li Sun,1* Åse Lundh,1 Annika Höjer,2 Gun Bernes,3 David Nilsson,4 Monika Johansson,1 Mårten Hetta,3 Anders H. Gustafsson,5 Karin Hallin Saedén,2 and Johan Dicksved6 1Department of Molecular Sciences, Swedish University of Agricultural Sciences, Box 7015, SE-750 07 Uppsala, Sweden 2Norrmejerier Ek. Förening, Mejerivägen 2, SE-906 22 Umeå, Sweden 3Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden 4Computational Life Science Cluster, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden 5Växa Sverige, Ulls väg 26, SE-750 07 Uppsala, Sweden 6Department of Animal Nutrition and Management Swedish University of Agricultural Sciences Box 7024 SE 750 07 Uppsala Sweden Received April 25, 2021. Accepted September 7, 2021. *Corresponding author: li.sun@​slu​.se ABSTRACT TBC/mL for AMS and tiestalls, respectively). Among tiestall farms, milk from farms using a chemical agent in connection to teat preparation and a more frequent use of acid to clean the milking equipment had lower TBC in milk, than milk from farms using water for teat preparation and a less frequent use of acid to clean the milking equipment (log 3.68 vs. 4.02 TBC/mL). There were no significant differences in the number of thermoresistant bacteria between farm types. The evaluated factors explained only a small proportion of total variation in the microbiota data, however, despite this, the study highlights the effect of routines associ- ated with teat preparation and cleaning of the milking equipment on raw milk microbiota, irrespective of type of milking system used. In this study, we investigated the variation in the microbial community present in bulk tank milk samples and the potential effect of different farm management factors. Bulk tank milk samples were collected repeat- edly over one year from 42 farms located in northern Sweden. Total and thermoresistant bacteria counts and 16S rRNA gene-based amplicon sequencing were used to characterize microbial community composi- tion. The microbial community was in general hetero- geneous both within and between different farms and the community composition in the bulk tank milk was commonly dominated by Pseudomonas, Acinetobacter, Streptococcus, unclassified Peptostreptococcaceae, and Staphylococcus. Principal component analysis including farm factor variables and microbial taxa data revealed that the microbial community in milk was affected by type of milking system. Milk from farms using an au- tomatic (robot) milking system (AMS) and loose hous- ing showed different microbial community composition compared with milk from tiestall farms. A discriminant analysis model revealed that this difference was depen- dent on several microbial taxa. Among farms using an automatic milking system, there were further differ- ences in the microbial community composition depend- ing on the brand of the milking robot used. On tiestall farms, routines for teat preparation and cleaning of the milking equipment affected the microbial community composition in milk. Total bacteria count (TBC) in milk differed between the farm types, and TBC were higher on AMS than tiestall farms (log 4.05 vs. log 3.79 Key words: milking system, premilking routines, bulk tank milk microbiota, microbial community composition MATERIALS AND METHODS ing heat treatment, some nonstarter lactic acid bacteria (NSLAB; e.g., lactobacilli) have been shown to enter into a viable but nonculturable state in cheese curd (Quigley et al., 2013). The NSLAB are believed to find their way to the cheese from handling and processing of the milk in the dairy plant, but raw farm milk is also suggested to be an important source (Vacheyrou et al., 2011). Study Design and Milk Sampling The study was conducted during the period from February 2016 to February 2017. All dairy farmers de- livering milk to the participating cheese-making plant were asked about their willingness to participate in the study, and 42 farmers agreed to participate. Herd and individual animal data (i.e., breed and milk yield) were obtained from the Swedish cow-recording scheme. Information relating to management practices (e.g., systems used for feed production and feeding facilities, housing and milking systems, and routines for milking and cleaning of the equipment) was collected through a questionnaire and farm visits (Priyashantha et al., 2021). Each farm was visited once during the indoor period (February or March) and once in the outdoor period (July). A recent study on transfer of bacteria from the en- vironment to raw milk found that most environmental bacteria detected in the milk were also present in the barn and milking parlor environment (Vacheyrou et al., 2011). However, technologically important bacteria such as lactobacilli were rarely found in the barn en- vironment, but were identified in the milk and on the teat surface (Vacheyrou et al., 2011). Monsallier et al. (2012) identified teat skin as an important source of bacteria of technological importance in cheese produc- tion, and found that higher counts of Lactobacillus and Enterococcus were associated with a silage-based diet, loose-house systems with straw bedding, and moderate milking hygiene. Hygiene measures in conjunction with milking and effective cleaning of the milking equipment were identified as important factors in determining the balance between bacterial populations in milk in studies by Verdier-Metz et al. (2009) and Michel et al. (2001). In Sweden in general, tank milk is collected by the dairy processor every second day. Upon each milk collection, a representative tank milk sample is rou- tinely collected by a device installed on the truck. MATERIALS AND METHODS This sample is transported refrigerated to the official milk testing laboratory (Eurofins Steins laboratory, Jönköping, Sweden), where analysis of milk quality pa- rameters must be initiated within 24 h. One time each in March, May, July, August, October, and December 2016, and in February 2017, an extra 250-mL bottle of tank milk was sampled manually by the truck driver when collecting milk on participating farms. Whereas the routinely collected milk samples were analyzed for total bacteria count (TBC) and thermoresistant bacte- ria count (TRBC) at the milk testing laboratory, the identical, manually collected tank milk samples were transported at 4°C to the Department of Molecular Sci- ences, Swedish University of Agricultural Sciences for analysis of milk microbiota. Upon arrival, the samples were aliquoted and stored as whole milk at −80°C until analysis. The time from sampling on the farm, to stor- age at −80°C was maximum 30 h. For various practical reasons, a complete set of samples was not available for all farms for the 7 mo. Therefore, the numbers of farms and farm milk samples have been indicated for each analysis in the results section of this paper. ( ) ( ) The rapid intensification in dairy production during the past 20 years has resulted in fewer and larger farms with high levels of mechanization (e.g., in feeding) and automation (e.g., use of robotic milking systems; Clay et al., 2020), but also stricter hygienic criteria. The transition in technology and the shift from tiestalls to loose-house systems have undoubtedly had effects on the composition, microbiota, and technological proper- ties of raw milk. A recent study by Priyashantha et al. (2021) reported a strong influence of dairy farm factors (e.g., type of housing and milking system, dominant breed in herd) on quality traits of the bulk tank milk. However, little knowledge is available on the influence of these farm factors on microbial community structure. Therefore, in this study we hypothesized that variation in bacteria counts and microbial community structure in bulk tank milk from commercial farms is related to farm factors, such as breed, technology, and housing system. The aim of the study was to characterize the variation in milk microbiota within and between farms and to identify major on-farm factors influencing the microbial community structure. MATERIALS AND METHODS Because our overall research focus is related to the link between farm fac- tors and the quality and ripening time of a traditional Swedish long-ripening cheese produced in the region, the abundance of LAB in the raw milk was of special interest. INTRODUCTION The microbial community in raw milk is very diverse, with thousands of different taxa present (Quigley et al., 2013), but for obvious reasons pathogens and spoilage bacteria in milk have attracted most attention. Cold storage of milk, both on-farm and at the dairy plant be- fore processing, permits growth of psychrotrophs such as Pseudomonas (De Jonghe et al., 2011). This increas- es the risk of proteolytic or lipolytic spoilage of dairy products caused by heat-resistant enzymes (Andersson et al., 1981; Stoeckel et al., 2016). Most bacteria and all pathogens are killed during pasteurization of milk, but spore-formers and other heat-resistant bacteria may survive, causing spoilage in different dairy prod- ucts during processing and storage. Lactic acid bacteria (LAB) in the raw milk do not multiply at refrigeration temperatures and are generally not considered to sur- vive pasteurization in high numbers. However, follow- Received April 25, 2021. Accepted September 7, 2021. *Corresponding author: li.sun@​slu​.se 123 124 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Journal of Dairy Science Vol. 105 No. 1, 2022 DNA Extraction of Milk Samples Extraction of microbial DNA was performed using a PowerFood DNA isolation kit (Qiagen AB) according to a customized protocol (Sun et al., 2019). In brief, milk samples (1.8 mL) were thawed at room tempera- ture for 15 min, centrifuged at 13,000 × g for 15 min at 4°C, and then incubated on ice for 5 min. The resulting cell pellets with carefully collected fat layer were resus- pended in 450 μL of MBL buffer (provided with kit). The resuspended mixture was transferred to MicroBead tubes (provided with kit). Cell lysis was conducted by incubating the tubes at 65°C for 10 min, after which they were processed in a Fastprep 24 instrument (MP Biomedicals) at 5.0 speed for 60 s, repeated 2 times with a 5-min pause. The tubes were then centrifuged at 13,000 × g for 15 min at 4°C, followed by incubation on ice for 5 min. The supernatant excluding the fat layer was transferred to a new 2-mL collection tube and the remaining steps were carried out according to the manufacturer’s protocol. The resulting DNA was eluted with 50 μL of buffer EB and stored at −20°C until use. The risk of introducing contamination in the labora- tory, resulting in misinterpretation of data for samples with low microbial biomass, is obvious, as reported by (Dahlberg et al., 2019). It is therefore important to have careful sample preparation procedures and to include relevant controls. In addition to milk samples, negative DNA extraction controls as well as PCR controls were included by using buffer EB instead of milk. The cows on the farms were fed grass/clover silage as main forage. Whole-crop silage was also fed on several farms (n = 10). Forage from different cuts (first, sec- ond, or third cut) or of different types (grass or clover silage, hay, whole-crop silage) were often mixed and used at the same time. More than half of the farmers (n = 23) had solely round bales for all forage. The rest had either tower (n = 10) or bunker silos (n = 9) as their main storage system, often in combination with round bales. Additives for preservation of forage was mainly used in the silos [chemical additives, mainly propionic and formic acid (n = 17 farms) or bacterial inoculants (n = 3 farms)]. Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA access to pasture varied from mainly offering outdoor activity, to pasture comprising a large part of the daily forage intake. All farms had year-round calving without any obvious seasonal pattern. access to pasture varied from mainly offering outdoor activity, to pasture comprising a large part of the daily forage intake. All farms had year-round calving without any obvious seasonal pattern. DNA Extraction of Milk Samples According to farm visits, hay was used as the major forage on 3 farms at some occasion during the year (farm 9, 11, and 28). Forage comprised on average 60% of the diet DM. The rest of the diet consisted of a readymade mixed concentrate, or cereals (mostly barley) with a commercial protein concentrate mix. The quality of the water (analyzed for total number of culturable bacteria, Escherichia coli and coliform bacteria, pH, hardness of water, nitrate or nitrite concentration) used to wash the milking equip- ment was controlled by the dairy cooperative at least once per year. The drinking water to the cows was from the same wells. The most common bedding material was sawdust, which was used on 39 of the 42 farms. Of the remaining 3 farms, one farm used straw, the second peat, and the third used recycled manure solids (farm 23, 13, and 12, respectively). Space per cow was according to the Swedish animal welfare regulations (Jordbruksverket, 2019). According to these regula- tions, dairy cows in the northern part of the country must have access to pasture at least 60 d/yr, which mainly occurs during the period of June to August. This was the case on all the studied farms. However, Illumina Amplicon Library Construction, Sequencing, and Bioinformatic Analysis Total Bacteria and Thermoresistant Bacteria Count in Milk Samples Total bacteria count and TRBC were analyzed in bulk tank milk samples at Eurofins Steins Laboratory (Jönköping, Sweden). The TBC was determined using BactoScan FC (Foss) and TRBC was determined using a culturing method (Wehr et al., 2004). Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA 125 milking system (AMS), 19 used tiestall milking (TIE), and 5 had a milking parlor. All AMS and milking parlor farms had loose housing in which cows were allowed to move freely, with cubicles for resting, whereas all TIE farms with the exception of one (farm 24) had tiestall housing. Farm 24 had loose housing, but the cows were milked in a tiestall. The 18 AMS farms used 2 different brands, with 7 farms using an AMS of brand A and 11 farms of brand B. There are several general differences between the 2 robot brands. Regarding cleaning of the teats, brand A robots have a system whereby the teats are cleaned in washing cups with lukewarm water and then dried by blowing air, whereas brand B robots use 2 parallel rolling brushes to clean the teats. The milk- ing devices and brushes are cleaned between each cow. After milking, 5 of 7 farmers with brand A robots used an iodine-based spray for the teats, as did also about half of those with brand B robots. The rest used either a spray based on lactic acid or no treatment at all. milking system (AMS), 19 used tiestall milking (TIE), and 5 had a milking parlor. All AMS and milking parlor farms had loose housing in which cows were allowed to move freely, with cubicles for resting, whereas all TIE farms with the exception of one (farm 24) had tiestall housing. Farm 24 had loose housing, but the cows were milked in a tiestall. The 18 AMS farms used 2 different brands, with 7 farms using an AMS of brand A and 11 farms of brand B. There are several general differences between the 2 robot brands. Regarding cleaning of the teats, brand A robots have a system whereby the teats are cleaned in washing cups with lukewarm water and then dried by blowing air, whereas brand B robots use 2 parallel rolling brushes to clean the teats. The milk- ing devices and brushes are cleaned between each cow. After milking, 5 of 7 farmers with brand A robots used an iodine-based spray for the teats, as did also about half of those with brand B robots. The rest used either a spray based on lactic acid or no treatment at all. Participating Farms The farms participating in the study were representa- tive for dairy farming in the northern part of Sweden, regarding size, animal material, feeding routines and milking systems. Categorizing the 42 farms according to their milking system, 18 farms used an automatic Journal of Dairy Science Vol. 105 No. 1, 2022 Variation in TBC and Microbial Community Within and Between Farms The TBC in bulk tank milk samples varied from log 3.48 to 5.00 TBC/mL over the sampling period, and varied both between and within farms during the pe- riod. This is illustrated in Figure 1, where results for farms with data for 5 or more of the 7 selected sampling months are presented. The smallest variation between sampling months was observed in tank milk from farm 24, with total bacteria counts varying from log 3.85/ mL to log 3.95/mL. The largest variation was found in milk from farm 40, with bacteria counts varying from log 3.48 to log 4.76 TBC/mL. Illumina Amplicon Library Construction, Sequencing, and Bioinformatic Analysis The SILVA SSU Ref NR 99 132 data set was first trimmed to the corresponding primer region and trained as classify-sklearn taxonomy classi- fier (Pedregosa et al., 2011; Quast et al., 2013; Bokulich et al., 2018). Amplicon sequence variants (ASV) were assigned taxonomy using the resulting classifier. The ASV table was rarefied at 7,139 reads/sample and the core microbiota was computed using the QIIME2 feature-table plugin (Weiss et al., 2017). The weighted UniFrac distance matrix and alpha rarefaction was generated using the QIIME2 diversity plugin (Bolyen et al., 2019). 2-step PCR approach described previously (Sun et al., 2019). The 16S rRNA library was sequenced using the Illumina Miseq platform at SciLifeLab (Uppsala, Swe- den) and Macrogen (Seoul, Korea). The raw sequencing data have been deposited to the Sequence Read Archive at the National Center for Biotechnology Information database (http:​/​/​www​.ncbi​.nlm​.nih​.gov/​sra), under accession number PRJNA715838. Bioinformatic data processing was performed using Quantitative Insights into Microbial Ecology 2 (Core 2019.04; Bolyen et al., 2019). The raw demultiplexed reads were trimmed using Cutadapt to remove primer sequences (Martin, 2011). Any base from the 3′ end which had quality below 30 was trimmed. A read was discarded if it con- tained N base or did not contain primer sequences. The trimmed reads were further processed using DADA2 to de-noise, de-replicate reads, merge pair end reads, and remove chimeras (Callahan et al., 2016), using a trun- cation length of 210 and 160 bp for forward and reverse reads, respectively. A phylogenetic tree was built using FastTree and MAFFT alignment (Katoh et al., 2002; Price et al., 2010). The SILVA SSU Ref NR 99 132 data set was first trimmed to the corresponding primer region and trained as classify-sklearn taxonomy classi- fier (Pedregosa et al., 2011; Quast et al., 2013; Bokulich et al., 2018). Amplicon sequence variants (ASV) were assigned taxonomy using the resulting classifier. The ASV table was rarefied at 7,139 reads/sample and the core microbiota was computed using the QIIME2 feature-table plugin (Weiss et al., 2017). The weighted UniFrac distance matrix and alpha rarefaction was generated using the QIIME2 diversity plugin (Bolyen et al., 2019). Illumina Amplicon Library Construction, Sequencing, and Bioinformatic Analysis The DNA extracted from the milk samples was used to construct a 16S rRNA library with primers 515F and 805R (Hugerth et al., 2014). Negative PCR con- trols and negative DNA extraction controls were both included in the sequencing library. Illumina adaptors and barcode were used for amplification, following a Journal of Dairy Science Vol. 105 No. 1, 2022 126 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Cross-validation score plots were created for visual as- sessment of separation between modeled classes. Num- ber of observations (n) and number of variables (K) were reported for each PCA and OPLS-DA model. Am- plicon sequence variants with a confidence interval >0 and predictive variable importance for the projection (VIP-predictive) >1 were used for model interpretation (Galindo-Prieto, 2017). To evaluate differences in TBC and TRBC between type of dairy farming system, a mixed effects linear model was analyzed with the pack- ages lme4, lmerTest, pbkrtest, and emmeans in R, using milking system, brand of AMS robot, acid wash, and teat preparation within TIE system as fixed effect and farm as random effect (Bates et al., 2014; Halekoh and Højsgaard, 2014; Kuznetsova et al., 2017; Lenth, 2021). Both TBC and TRBC data were log-transformed to better fit the normal distribution, resulting in the unit log bacteria count per milliliter. Differences with P < 0.05 were considered significant. 2-step PCR approach described previously (Sun et al., 2019). The 16S rRNA library was sequenced using the Illumina Miseq platform at SciLifeLab (Uppsala, Swe- den) and Macrogen (Seoul, Korea). The raw sequencing data have been deposited to the Sequence Read Archive at the National Center for Biotechnology Information database (http:​/​/​www​.ncbi​.nlm​.nih​.gov/​sra), under accession number PRJNA715838. Bioinformatic data processing was performed using Quantitative Insights into Microbial Ecology 2 (Core 2019.04; Bolyen et al., 2019). The raw demultiplexed reads were trimmed using Cutadapt to remove primer sequences (Martin, 2011). Any base from the 3′ end which had quality below 30 was trimmed. A read was discarded if it con- tained N base or did not contain primer sequences. The trimmed reads were further processed using DADA2 to de-noise, de-replicate reads, merge pair end reads, and remove chimeras (Callahan et al., 2016), using a trun- cation length of 210 and 160 bp for forward and reverse reads, respectively. A phylogenetic tree was built using FastTree and MAFFT alignment (Katoh et al., 2002; Price et al., 2010). Journal of Dairy Science Vol. 105 No. 1, 2022 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Figure 1. Box plot showing the total number of bacteria (log cells/mL) in bulk tank milk samples from participating farms. Only the data for farms represented by at least 5 milk sampling occasions are shown. The horizontal bars through the boxes show the median (i.e., the 50th percentile) of the distance bacterial distribution. The lower and upper extents of the boxes indicate the 25th and 75th percentiles of the distri- bution, respectively. The upper and lower whiskers indicate the maximum and minimum values, respectively. The detection limit is 1,000 cells/ mL. In the case of farm 11, the variation in total bacteria number was very low between the different samples, explaining the absence of a box. In this case, the lower bar shows the median, and the upper bar is the upper whisker. netobacter, Streptococcus, unclassified Peptostreptococ- caceae, and Staphylococcus. as the fifth principal component (PC) for the larger data set (n = 210) used for analysis of the influence of farm factors and also in some of the orthogonal compo- nents of the OPLS-DA models. , p y Assessment of microbial community stability in milk over time, using weighted UniFrac matrix comparison, revealed variation within individual farms (Figure 2A). Most of the farms showed heterogeneous community composition in milk over the different sampling occa- sions, whereas a few farms had quite stable community structure. As indicated by the homogeneity comparison (Figures 2A and 2B; Supplemental Figure S1, https:​/​ /​doi​.org/​10​.7910/​DVN/​OL0ASF), the microbial com- munity was sometimes similar and sometimes varied substantially within the same farm across different sampling occasions. The dominant genera also varied between farms. For instance, farms 1, 11, and 45 showed little variation across the different milk sampling oc- casions (Figure 2A), but Acinetobacter dominated in milk on all sampling occasions for farm 45, whereas multiple genera instead of a single genus dominated on all sampling occasions for farms 1 and 11 (Figure 2B). In milk from farms showing greater variation between sampling occasions (e.g., farms 18 and 22; Figure 2A), the dominant genera varied to a larger extent (Figure 2B). Although the effect of season was not within the scope of this study, it was evaluated during the initial screening of the data. Data showed that the seasonal effect was not interfering with the relationships studied in the 3 OPLS-DA models. Statistical Analysis The rarefied ASV table and data related to farm management factors were analyzed using SIMCA (Ver- sion 14.0.0.1359, Umetrics, Sartorius) and R (https:​/​ /​r​-project​.org). Principal component analysis (PCA) was performed in SIMCA to analyze the variation in microbial community as influenced by farm factors (see Priyashantha et al., 2021, Supplemental File S1, for the final farm factors that were included in the screen- ing), using standard settings. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) models were cross-validated and the associated measure of predictive performance, Q2Y, was reported for each model. The cross-validation procedure employed an ex- clusion strategy where all observations from the same farm were excluded and predicted in the same round. As an additional measure of statistical significance, the P-value of the CV-ANOVA diagnostic was determined. The 16S rRNA gene sequencing yielded an average of 35,299 (median 26,866) sequences per sample. The sequences were distributed in 3,096 ASV, representing 194 taxonomic families and 464 genera. One particular ASV (68f648), classified as Pseudomonas, was found in negative DNA extractions, negative PCR controls, and milk samples. Because Pseudomonas is commonly identified as part of the natural microbial community in raw milk, it is difficult to distinguish natural occur- rence of this ASV from contamination. To limit the potential risk of this ASV heavily influencing the data analysis, milk samples with relative abundance (RA) > 10% of this ASV were removed from the analysis. In total, 15 bacterial genera were found to represent a core microbiota, present in at least 90% of the bulk tank milk samples (Table 1). The 5 most dominant bacteria overall in tank milk samples were Pseudomonas, Aci- Journal of Dairy Science Vol. 105 No. 1, 2022 127 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Table 1. Mean relative abundance (RA) and SD of the 15 genera that were present in at least 90% of the bulk tank milk samples collected on the participating 42 farms Phylum   Class   Order   Family   Genus RA SD Actinobacteria Actinobacteria Corynebacteriales Corynebacteriaceae Corynebacterium_1 1.98% 1.95%     Micrococcales Micrococcaceae Kocuria 1.57% 4.18% Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides 1.25% 1.10%       Unclassified Bacteroidales (unclassified) 1.51% 1.42% Firmicutes Bacilli Bacillales Staphylococcaceae Staphylococcus 3.05% 6.57%     Lactobacillales Aerococcaceae Aerococcus 2.31% 5.53%       Streptococcaceae Lactococcus 2.09% 6.50%         Streptococcus 4.46% 11.68%   Clostridia Clostridiales Peptostreptococcaceae Peptostreptococcaceae (unclassified) 4.30% 4.12%       Ruminococcaceae Ruminococcaceae_UCG-005 2.62% 2.31%         Firmicutes (unclassified) 1.26% 1.12% Proteobacteria Gammaproteobacteria Alteromonadales Shewanellaceae Shewanella 0.67% 0.67%     Oceanospirillales Halomonadaceae Halomonas 1.37% 1.32%     Pseudomonadales Moraxellaceae Acinetobacter 10.54% 20.22%     Pseudomonadales Pseudomonadaceae Pseudomonas 17.16% 31.14% AMS and farms using tiestall milking. Mixed effects linear model analysis revealed that TBC was higher in milk from the AMS farms (log 4.05 TBC/mL) than in milk from the TIE farms (log 3.79 TBC/mL, P < 0.01; Table 2). There was no significant difference in TRBC between the AMS and TIE farms. Interestingly, the rarefaction curves of observed ASV revealed a higher number in milk from AMS farms than in milk from TIE farms (Figure 4A). Table 1. Mean relative abundance (RA) and SD of the 15 genera that were present in at least 90% of the bulk tank milk samples collected on the participating 42 farms Phylum   Class   Order   Family   Genus RA SD Actinobacteria Actinobacteria Corynebacteriales Corynebacteriaceae Corynebacterium_1 1.98% 1.95%     Micrococcales Micrococcaceae Kocuria 1.57% 4.18% Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides 1.25% 1.10%       Unclassified Bacteroidales (unclassified) 1.51% 1.42% Firmicutes Bacilli Bacillales Staphylococcaceae Staphylococcus 3.05% 6.57%     Lactobacillales Aerococcaceae Aerococcus 2.31% 5.53%       Streptococcaceae Lactococcus 2.09% 6.50%         Streptococcus 4.46% 11.68%   Clostridia Clostridiales Peptostreptococcaceae Peptostreptococcaceae (unclassified) 4.30% 4.12%       Ruminococcaceae Ruminococcaceae_UCG-005 2.62% 2.31%         Firmicutes (unclassified) 1.26% 1.12% Proteobacteria Gammaproteobacteria Alteromonadales Shewanellaceae Shewanella 0.67% 0.67%     Oceanospirillales Halomonadaceae Halomonas 1.37% 1.32%     Pseudomonadales Moraxellaceae Acinetobacter 10.54% 20.22%     Pseudomonadales Pseudomonadaceae Pseudomonas 17.16% 31.14% ( ) An OPLS-DA model (n = 185, K = 227, Q2Y = 0.688, P < 0.001) was created for assessing the differ- ence in ASV between milk from AMS and TIE farms (Supplemental Figure S2, https:​/​/​doi​.org/​10​.7910/​ DVN/​OL0ASF). Of the 41 ASV that were found to differ between the farm types, 15 showed higher RA in milk from TIE farms (Figure 5). The remaining 26 ASV were present in higher RA in milk from AMS farms. Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Instead, the effect of season was clearly observed in other model components, such Differences in Microbial Community Between Farms as Influenced by Farm Management Factors Differences in Microbial Community Associ- ated with Milking System. The influence of different farm factors on the microbial community in bulk tank milk was initially screened using PCA, with number of observations, n = 210, and number of variables, K = 228 (see Supplemental File S1 of Priyashantha et al., 2021). The PCA identified milking system and type of housing as the 2 most influential factors. As mentioned, all AMS and milking parlor farms had loose housing, whereas all TIE farms except farm 24 had tiestall hous- ing. Thus, dairy farms were categorized according to milking system, being aware that these categories also included the effects of confounded factors, of which the individual effects could not be distinguished within this study. Milk from farms using AMS and from farms a using tiestall milking system showed 2 clear clusters in the PCA, separated along PC 2, whereas milk from farms with milking parlors showed a wider distribution in the plot (Figure 3). In subsequent comparisons, we therefore decided to focus on differences between the 2 dominant farm types, distinguished as farms using Journal of Dairy Science Vol. 105 No. 1, 2022 128 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA It is interesting to note that several ASV classified into the same genus or family showed different dominance in milk from either AMS or TIE farms. For instance, within the genus Corynebacterium, ASV cee9ed showed higher RA in milk from TIE farms, whereas ASV bad597 and 0c35e6 showed higher RA in milk from AMS farms. Similarly, Aerococcaceae_d347bc, Psychrobacter_83c81d, Corynebacteriaceae_700739, and Acinetobacter_b994ec were present in higher RA in milk from TIE farms, whereas Aerococcaceae_1bbfc6, Psychrobacter_24a457, Corynebacteriaceae_db47e9, Acinetobacter_189aec, and Acinetobacter_f2f4ab showed higher RA in milk from AMS farms. Among the 15 ASV with higher RA in milk from TIE than AMS farms, Pseudomonas_69d9bd was identified as the most dominant ASV. Lactobacillus_c9822d, which belongs to the NSLAB, showed higher RA in milk from TIE than in milk from AMS farms. The genus Kocuria and family Enterobacteriaceae were represented by 2 ASV each (303143 and be6f97 for Kocuria; 26d8f0 and fc5860 for Enterobacteriaceae). Of the 26 ASV with higher RA in milk from AMS than in milk from TIE farms, 2 ASV (598071 and bd2ebc) were classified to the same genus (Streptococcus) and 3 ASV (3ca58b, 464a52, and 69a95a) were classified to the same family (Peptostreptococcaceae). In addition, Bacilli_38162e, Lactobacillales_c34a7c, Atopostipes_89db65, Carnobacteriaceae_0b711b, Aerococcus_81618b, Facklamia_83534c, and Firmicutes_dacd87 showed Differences in Microbial Community Between AMS Farms Using Different Brands of Robots. Among the AMS farms, the most influential factor for microbial community composition in tank milk was the brand of milking robot, with PCA (n = 95, K = 202) plots indicating clear clusters of milk samples repre- Journal of Dairy Science Vol. 105 No. 1, 2022 129 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA 2 brands of milking robots, but farms with a brand A robot had a higher number of observed ASV than farms with a brand B robot (Figure 4B). 2 brands of milking robots, but farms with a brand A robot had a higher number of observed ASV than farms with a brand B robot (Figure 4B). senting farms with brand A and B robots, respectively, along PC 2 (Figure 6). Analysis of TBC and TRBC levels revealed no significant differences between the nal of Dairy Science Vol. 105 No. 1, 2022 els revealed no significant differences between the with a brand B robot (Figure 4B). igure 2. (A) Boxplots showing the distribution of weighted UniFrac distance matrix data between bulk tank milk samples from indiv s for which results from at least 5 sampling occasions were available. The horizontal bars through the boxes show the median (i.e., the entile) of the distance matrix distribution. The lower and upper extents of the boxes are the 25th and 75th percentiles of the distribu ectively. The lower and upper whiskers in the box plots are the minimum and maximum values of the distribution, respectively. (B) Re ndance (RA, %) of dominant genera in bulk tank milk samples from some of the participating farms; farm ID followed by sampling m era with maximum RA less than 10% were grouped together (minor group). Figure 2. (A) Boxplots showing the distribution of weighted UniFrac distance matrix data between bulk tank milk samples from individual farms for which results from at least 5 sampling occasions were available. The horizontal bars through the boxes show the median (i.e., the 50th percentile) of the distance matrix distribution. The lower and upper extents of the boxes are the 25th and 75th percentiles of the distribution, respectively. The lower and upper whiskers in the box plots are the minimum and maximum values of the distribution, respectively. (B) Relative abundance (RA, %) of dominant genera in bulk tank milk samples from some of the participating farms; farm ID followed by sampling month. Genera with maximum RA less than 10% were grouped together (minor group). Figure 2. (A) Boxplots showing the distribution of weighted UniFrac distance matrix data between bulk tank milk samples from individual farms for which results from at least 5 sampling occasions were available. Journal of Dairy Science Vol. 105 No. 1, 2022 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Mean values of log-transformed total and thermoresistant bacteria counts in bulk tank milk from farms characterized according to their milking systems [i.e., automatic milking system (AMS) and tiestall milking systems (TIE)]1 Table 2. Mean values of log-transformed total and thermoresistant bacteria counts in bulk tank milk from farms characterized according to their milking systems [i.e., automatic milking system (AMS) and tiestall milking systems (TIE)]1 Item Total bacteria count (log) P Thermoresistant bacteria count (log) P Comparison between milking systems   AMS 4.05±0.31 (n = 88, N = 18) 0.005 2.93±0.65 (n = 58, N = 18) 0.231   TIE 3.79±0.33 (n = 84, N = 19) 2.65±0.73 (n = 50, N = 18) Comparison within AMS farms2     Brand A 4.18±0.29 (n = 30, N = 7) 0.102 2.95±0.81 (n = 19, N = 7) 0.821   Brand B 3.98±0.30 (n = 58, N = 11) 2.92±0.57 (n = 39, N = 11) Comparison within TIE farms3     CHEM-OFT 3.68±0.22 (n = 33, N = 8) 0.024 2.65±0.80 (n = 16, N = 7) 0.131   WATER-FEW 4.02±0.41 (n = 18, N = 3) 3.09±0.68 (n = 12, N = 3)   1Within the group of AMS farms, values were compared between farms with different brand of milking system. Within the group of TIE farms, values were compared between farms with different routines related to preparation of teats before milking and cleaning of the milking equipment. n = number of bulk tank milk samples; N = number of farms. 2Within AMS farms, 2 different brands of robots were used (i.e., brand A and brand B). 3Within TIE farms, different routines related to preparation of teats before milking and cleaning of the milking equipment could be distin- guished: CHEM-OFT = farms using a chemical (CHEM) agent for teat preparation before milking as well as a more frequent use (OFT) of acid wash to clean the milking system (confounded factors); WATER-FEW = farms using only water for teat preparation before milking and a less frequent (FEW) use of acid to clean the milking system (confounded factors). Journal of Dairy Science Vol. 105 No. 1, 2022 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA The horizontal bars through the boxes show the median (i.e., the 50th percentile) of the distance matrix distribution. The lower and upper extents of the boxes are the 25th and 75th percentiles of the distribution, respectively. The lower and upper whiskers in the box plots are the minimum and maximum values of the distribution, respectively. (B) Relative abundance (RA, %) of dominant genera in bulk tank milk samples from some of the participating farms; farm ID followed by sampling month. Genera with maximum RA less than 10% were grouped together (minor group). Journal of Dairy Science Vol. 105 No. 1, 2022 130 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Figure 3. Principal component analysis of microbial taxa in bulk tank milk samples collected on farms using automatic milking (AMS), milking parlor, or tiestall milking (TIE) systems. Each dot represents a unique milk sample from an individual farm, colors indicating type of farm as represented by milking system. The milk samples originated from 18, 5, and 19 farms with AMS, milking parlor, or TIE milking systems, respectively. Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA 130 Figure 3. Principal component analysis of microbial taxa in bulk tank milk samples collected on farms using automatic milking (AMS), milking parlor, or tiestall milking (TIE) systems. Each dot represents a unique milk sample from an individual farm, colors indicating type of farm as represented by milking system. The milk samples originated from 18, 5, and 19 farms with AMS, milking parlor, or TIE milking systems, respectively. An OPLS-DA model (n = 95; K = 204; Q2Y = 0.607, P < 0.001) was created to assess the difference between AMS with different brands of milking robots and to identify ASV contributing to the difference (Supple- mental Figure S3, https:​/​/​doi​.org/​10​.7910/​DVN/​ OL0ASF). Among the 18 ASV identified, 17 showed higher RA in milk from farms with a brand A milking robot and only one ASV, Ralstonia_c021db, showed higher RA in milk from farms with a brand B milking robot (Figure 7). Among the ASV present in higher RA Table 2. Mean values of log-transformed total and thermoresistant bacteria counts in bulk tank milk from farms characterized according to their milking systems [i.e., automatic milking system (AMS) and tiestall milking systems (TIE)]1 Table 2. Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA in milk from farms with a brand A robot, Lactococcus and Acinetobacter were the 2 most frequently identified genera, with 2 (cc1f55 and d90ee6) and 5 ASV (189aec, 90c1ff, a35537, 9defed, and 000d0f), respectively. In ad- dition, Streptococcus_5cac9e, Lactobacillales_3ebdbc, Atopostipes_568dfd, and Carnobacteriaceae_0b711b were observed at higher RA in milk from AMS farms using a brand A milking robot, although these LAB only constituted minor populations. in milk from farms with a brand A robot, Lactococcus and Acinetobacter were the 2 most frequently identified genera, with 2 (cc1f55 and d90ee6) and 5 ASV (189aec, 90c1ff, a35537, 9defed, and 000d0f), respectively. In ad- dition, Streptococcus_5cac9e, Lactobacillales_3ebdbc, Atopostipes_568dfd, and Carnobacteriaceae_0b711b were observed at higher RA in milk from AMS farms using a brand A milking robot, although these LAB only constituted minor populations. terms of microbial community structure in tank milk samples in the PCA plot (n = 90, K = 222). However, these 2 factors were clearly confounded, because TIE farms using acid wash less frequently also more com- monly used only water, or used a dry cloth or paper towel, when preparing the teats before milking. In contrast, TIE farms that applied acid wash more fre- quently (every second wash) more commonly reported use of chemical agents for teat preparation. TIE farms with no or less frequent acid washing of their milking equipment (varying from once every second day to once per week or more seldom) and nonchemical-based teat preparation were more associated with the left side of the PCA plot (Figure 8). Differences in Microbial Community Between Tiestall Farms Using Different Routines for Teat Preparation and Cleaning of the Milking Equipment. Within the TIE farms, the variation in management factors was larger than for the AMS farms. For example, some TIE farms reported using a cloth to clean teats before milking, whereas other farms used paper towels. Likewise, some TIE farms reported premilking directly onto the floor, whereas others premilked into a dedicated cup. Some TIE farms did not have automatic removal of the milking clusters as an option in their milking system, whereas others had. Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Figure 4. (A) The rarefaction curves of observed amplicon sequence variants (ASV) in milk, comparing bulk tank milk samples from herds with different milking systems [represented by 95 and 90 bulk tank milk samples originating from 18 farms using automated milking systems (AMS) and 19 farms using tiestall milking (TIE), respectively]; (B) the rarefaction curves of observed ASV in milk, comparing bulk tank milk samples from AMS farms using different brands of AMS (represented by 32 and 63 bulk tank milk samples originating from 7 farms using brand A and 11 farms using brand B, respectively); (C) the rarefaction curves of observed ASV in milk comparing bulk tank milk samples from TIE farms with different routines related to cleaning of the milking equipment (frequency of acid to alkaline wash) and method for teat cleaning (represented by 34 milk samples originating from 8 farms using a chemical agent in connection to teat cleaning and frequent use of acid to clean the milking equipment (CHEM-OFT), and 19 milk samples originating from 3 farms using water only to clean teats before milking and less frequent use of acid to clean the milking equipment (WATER-FEW). Figure 4. (A) The rarefaction curves of observed amplicon sequence variants (ASV) in milk, comparing bulk tank milk samples from herds with different milking systems [represented by 95 and 90 bulk tank milk samples originating from 18 farms using automated milking systems (AMS) and 19 farms using tiestall milking (TIE), respectively]; (B) the rarefaction curves of observed ASV in milk, comparing bulk tank milk samples from AMS farms using different brands of AMS (represented by 32 and 63 bulk tank milk samples originating from 7 farms using brand A and 11 farms using brand B, respectively); (C) the rarefaction curves of observed ASV in milk comparing bulk tank milk samples from TIE farms with different routines related to cleaning of the milking equipment (frequency of acid to alkaline wash) and method for teat cleaning (represented by 34 milk samples originating from 8 farms using a chemical agent in connection to teat cleaning and frequent use of acid to clean the milking equipment (CHEM-OFT), and 19 milk samples originating from 3 farms using water only to clean teats before milking and less frequent use of acid to clean the milking equipment (WATER-FEW). Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Item Total bacteria count (log) P Thermoresistant bacteria count (log) P Comparison between milking systems   AMS 4.05±0.31 (n = 88, N = 18) 0.005 2.93±0.65 (n = 58, N = 18) 0.231   TIE 3.79±0.33 (n = 84, N = 19) 2.65±0.73 (n = 50, N = 18) Comparison within AMS farms2     Brand A 4.18±0.29 (n = 30, N = 7) 0.102 2.95±0.81 (n = 19, N = 7) 0.821   Brand B 3.98±0.30 (n = 58, N = 11) 2.92±0.57 (n = 39, N = 11) Comparison within TIE farms3     CHEM-OFT 3.68±0.22 (n = 33, N = 8) 0.024 2.65±0.80 (n = 16, N = 7) 0.131   WATER-FEW 4.02±0.41 (n = 18, N = 3) 3.09±0.68 (n = 12, N = 3) 1Within the group of AMS farms, values were compared between farms with different brand of milking system values were compared between farms with different routines related to preparation of teats before milking and c 1Within the group of AMS farms, values were compared between farms with different brand of milking system. Within the group of TIE farms, values were compared between farms with different routines related to preparation of teats before milking and cleaning of the milking equipment. n = number of bulk tank milk samples; N = number of farms. 2Within AMS farms, 2 different brands of robots were used (i.e., brand A and brand B). 3Within TIE farms, different routines related to preparation of teats before milking and cleaning of the milking equipment could be distin- guished: CHEM-OFT = farms using a chemical (CHEM) agent for teat preparation before milking as well as a more frequent use (OFT) of acid wash to clean the milking system (confounded factors); WATER-FEW = farms using only water for teat preparation before milking and a less frequent (FEW) use of acid to clean the milking system (confounded factors). 3Within TIE farms, different routines related to preparation of teats before milking and cleaning of the milking equipment could be distin- guished: CHEM-OFT = farms using a chemical (CHEM) agent for teat preparation before milking as well as a more frequent use (OFT) of acid wash to clean the milking system (confounded factors); WATER-FEW = farms using only water for teat preparation before milking and a less frequent (FEW) use of acid to clean the milking system (confounded factors). Journal of Dairy Science Vol. 105 No. 1, 2022 131 Journal of Dairy Science Vol. 105 No. 1, 2022 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Among the management factors evaluated (Priyashantha et al., 2021), the frequency of applying acid or alkaline wash to descale and clean the milking equipment, and the procedure used for teat preparation before milking gave the largest separation of samples in ( ) In subsequent comparisons of farms with tiestall milking, we therefore decided to focus on the differ- ences between the farms that used chemicals during teat preparation and more frequently used acid wash to clean the milking equipment (CHEM-OFT), and the farms that employed water and less frequent acid washing of the milking equipment (WATER-FEW). The average TBC in tank milk from WATER-FEW farms was log 4.02 TBC/mL, compared with log 3.68 TBC/mL in milk from the CHEM-OFT group (Table 2, P < 0.05). Contrary to expectations, there was no significant difference between the 2 groups in numbers Journal of Dairy Science Vol. 105 No. 1, 2022 132 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA DVN/​OL0ASF). Of the 22 ASV identified as the most important taxa in the model, 11 ASV showed higher RA in milk from the CHEM-OFT group, whereas the remaining 11 showed higher RA in milk from the WATER-FEW group (Figure 9). Two ASV, cc1f55 and d90ee6, that had higher RA in WATER-FEW milk were classified into the same genus (Lactococ- cus). Similarly, 2 ASV, 44e039 and fc5860, classified of thermoresistant bacteria in the milk. The number of observed ASV was higher in milk from the CHEM- OFT group than in milk from the WATER-FEW group (Figure 4C). An OPLS-DA model (n = 53, K = 220, Q2Y = 0.784, P < 0.001) was created for assessing differences in ASV between milk from the 2 groups of tiestall farms (Supplemental Figure S4, https:​/​/​doi​.org/​10​.7910/​ of thermoresistant bacteria in the milk. The number of observed ASV was higher in milk from the CHEM- OFT group than in milk from the WATER-FEW group (Figure 4C). An OPLS-DA model (n = 53, K = 220, Q2Y = 0.784, P < 0.001) was created for assessing differences in ASV between milk from the 2 groups of tiestall farms (Supplemental Figure S4, https:​/​/​doi​.org/​10​.7910/​ Figure 5. The relative abundance (RA, %) of amplicon sequence variants (ASV) in bulk tank milk samples from farms using auto king systems (AMS) and tiestall milking systems (TIE), respectively. The vertical bar through the box is the median (i.e., the 50th perc e. The left and right extents of the box are the 25th and 75th percentiles (q3) of the values, respectively, and the left and right whis box plot represent the minimum and maximum values of the distribution. There are 95 and 90 milk samples originated from the 18 a ms that using AMS and TIE respectively. For ASV outside the axis limit: *1: TIE whisker 3.4%; *2 TIE q3 12%, TIE whisker 30%; *3 2% AMS hisker 8 6% TIE q3 3 0% TIE hisker 7 2% *4 AMS hisker 3 5% TIE hisker3 3% *5 AMS hisker 5 1% *6 A Figure 5. The relative abundance (RA, %) of amplicon sequence variants (ASV) in bulk tank milk samples from farms using automated milking systems (AMS) and tiestall milking systems (TIE), respectively. The vertical bar through the box is the median (i.e., the 50th percentile) value. Journal of Dairy Science Vol. 105 No. 1, 2022 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Within the samples from the CHEM-OFT group, Leuconostoc_0c7492 and Weissella_e9f4ea showed higher RA than in milk from WATER-FEW farms, although these 2 LAB were pres- ent at low RA (<1%). as Enterobacteriaceae showed higher RA in milk from the WATER-FEW group. The genera Acinetobacter and Corynebacterium were identified in milk from both groups, with higher RA of Acinetobacter_66a959 and Corynebacterium_1_c2eea2 in milk from the WATER- FEW group and higher RA of Acinetobacter_90c1ff and Corynebacterium_bad597 in milk from the CHEM- OFT group. It is worth noting that one individual farm, farm 42, was responsible for the observed dif- ferences in ASV between farm groups, including the previously mentioned 4 ASV from Lactococcus and Enterobacteriaceae and one additional ASV, Bacilla- ceae_bed463. Lactobacillales_9cbacb, with higher RA in milk from the WATER-FEW group, was also mainly represented by one farm (farm 15). Within the samples from the CHEM-OFT group, Leuconostoc_0c7492 and Weissella_e9f4ea showed higher RA than in milk from WATER-FEW farms, although these 2 LAB were pres- ent at low RA (<1%). collected over the course of one year. The aim was to learn more about the composition and temporal dy- namics of the microbial community and to identify the most important farm factors in shaping microbial community composition in tank milk. The study was conducted in a region of Sweden where the milk is used for production of long-ripening cheese, so we were par- ticularly interested in the abundance of technologically important NSLAB in the raw milk. The core microbiota in the tank milk consisted of 15 genera, of which the 5 most abundant were Pseu- domonas, Acinetobacter, Streptococcus, unclassified Peptostreptococcaceae, and Staphylococcus (Table 1). A recent Norwegian study also identified Pseudomonas, Streptococcus, Acinetobacter, and Staphylococcus as be- ing among the most abundant bacterial genera in tank milk from 45 farms (Skeie et al., 2019). In the Norwe- gian study, tank milk was stored refrigerated for 3 d on participating farms, in our study for 2 d, before milk was collected by the milk truck. Because cold storage of raw milk is known to permit growth of Gram-negative psychrotrophic bacteria, such as Pseudomonas and Aci- netobacter, it was not surprising to find these genera among the most abundant in our study and also in the Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA The left and right extents of the box are the 25th and 75th percentiles (q3) of the values, respectively, and the left and right whiskers of the box plot represent the minimum and maximum values of the distribution. There are 95 and 90 milk samples originated from the 18 and 19 farms that using AMS and TIE respectively. For ASV outside the axis limit: *1: TIE whisker 3.4%; *2 TIE q3 12%, TIE whisker 30%; *3: AMS q3 4.2%, AMS whisker 8.6%, TIE q3 3.0%, TIE whisker 7.2%; *4: AMS whisker 3.5%, TIE whisker3.3%; *5: AMS whisker 5.1%; *6: AMS q3 3.3%, AMS whisker 7.0%, TIE whisker 4.4%; *7: AMS q3 4.5%, AMS whisker 11%. Journal of Dairy Science Vol. 105 No. 1, 2022 133 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Figure 6. Principal component analysis plot of microbial data for bulk tank milk samples collected on farms with automatic milking systems (AMS). Each dot represents a milk sample from an individual farm, colors indicating brand of AMS. Numbers of farms using brand A and B were 7 and 11, respectively, and the number of bulk tank milk samples associated with the 2 brands of robots were 32 and 63, respectively. 133 1 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Figure 6. Principal component analysis plot of microbial data for bulk tank milk samples collected on farms with automatic milking systems (AMS). Each dot represents a milk sample from an individual farm, colors indicating brand of AMS. Numbers of farms using brand A and B were 7 and 11, respectively, and the number of bulk tank milk samples associated with the 2 brands of robots were 32 and 63, respectively. as Enterobacteriaceae showed higher RA in milk from the WATER-FEW group. The genera Acinetobacter and Corynebacterium were identified in milk from both groups, with higher RA of Acinetobacter_66a959 and Corynebacterium_1_c2eea2 in milk from the WATER- FEW group and higher RA of Acinetobacter_90c1ff and Corynebacterium_bad597 in milk from the CHEM- OFT group. It is worth noting that one individual farm, farm 42, was responsible for the observed dif- ferences in ASV between farm groups, including the previously mentioned 4 ASV from Lactococcus and Enterobacteriaceae and one additional ASV, Bacilla- ceae_bed463. Lactobacillales_9cbacb, with higher RA in milk from the WATER-FEW group, was also mainly represented by one farm (farm 15). Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA 134 study by Skeie et al. (2019). However, this is not always the case, because Breitenwieser et al. (2020) found that Staphylococcus, Corynebacterium, Caryophanon, and Streptococcus were the dominant genera in farm milk samples that had been stored at refrigerated tempera- ture for 2 and 3 consecutive days before sampling. Al- though Pseudomonas and Acinetobacter were dominant genera in tank milk in our study, genera other than psychrotrophs were more abundant in milk from some farms (see examples in Figure 2B). pling occasions, whereas numbers in milk from other farms fluctuated over the sampling period and on some farms bacteria counts in milk remained at relatively high levels. In contrast to O’Connell et al. (2015), who reported a seasonal trend in monthly average TBC, with TBC being greatest at the beginning and end of the year, we did not observe any general trend in TBC related to milk sampling month or season. The results also revealed that the temporal dynam- ics of bulk tank milk microbiota (i.e., the variation in microbial community structure) varied between the participating farms (Figure 2A). Some farms showed relatively large variation in microbial community struc- ture despite low TBC (e.g., farms 18 and 22). Farms 1 and 45, on the other hand, both showed high homoge- neity of the milk microbiota, although farm 1 delivered milk with low TBC and farm 45 delivered milk with relatively high TBC on all occasions. Other farms, such as farms 1 and 11, were characterized by low TBC and low variation in microbial community structure over time but showed relatively high alpha diversity (i.e., with no single dominant bacterial genus). Among the farms that showed large variation in community struc- DISCUSSION In this study we explored the microbial community in tank milk from 42 Swedish dairy farms, using samples Journal of Dairy Science Vol. 105 No. 1, 2022 Journal of Dairy Science Vol. 105 No. 1, 2022 Influence of Different Farm Factors on Farm Milk Microbiota ture between the different sampling occasions, there was no systematic pattern in the variation (e.g., no sea- sonal trend). To our knowledge, the temporal variation in microbial community structure in bulk tank milk over longer periods has not been widely investigated. Although the season effect was not in the scope of this paper, it was investigated during the initial, general analysis of the data. We found that the seasonal effect was not interfering with the relationships studied in the 3 OPLS-DA models. Instead, the effect of season was clearly observed in other model components, such as the fifth PC for the larger data set. Also Skeie et al. (2019) reported that microbial composition in tank milk from the same farm varied significantly between sampling occasions. In their study, milk samples from individual farms were collected on 3 occasions, with 2 to 3 wk between each sampling occasion. Overall, the TBC was similar between the 3 samplings from each individual farm, but the milk microbiota varied between the collection days. Several factors, such as changes in feed quality, farm environment, weather conditions, and animal health, might have contributed to these changes (Skeie et al., 2019). ture between the different sampling occasions, there was no systematic pattern in the variation (e.g., no sea- sonal trend). To our knowledge, the temporal variation in microbial community structure in bulk tank milk over longer periods has not been widely investigated. Although the season effect was not in the scope of this paper, it was investigated during the initial, general analysis of the data. We found that the seasonal effect was not interfering with the relationships studied in the 3 OPLS-DA models. Instead, the effect of season was clearly observed in other model components, such as the fifth PC for the larger data set. Also Skeie et al. (2019) reported that microbial composition in tank milk from the same farm varied significantly between sampling occasions. In their study, milk samples from individual farms were collected on 3 occasions, with 2 to 3 wk between each sampling occasion. Overall, the TBC was similar between the 3 samplings from each individual farm, but the milk microbiota varied between the collection days. Several factors, such as changes in feed quality, farm environment, weather conditions, and animal health, might have contributed to these changes (Skeie et al., 2019). Variation in Tank Milk Microbiota Within and Between Farms The TBC in tank milk was generally low, ranging from log 3.48 to 5.00 TBC/mL, indicating high hygiene quality and farms respecting the European limits for TBC in raw milk (i.e., log 5.00 bacteria/mL milk; Eu- ropean Commission, 2004). The TBC were in line with the log 3.6 to 5.28 TBC/mL reported in the Norwegian study cited above (Skeie et al., 2019). However, the TBC profile in milk from the individual farms varied (Figure 1). Some farms showed low counts on all sam- Figure 7. The relative abundance (RA, %) of amplicon sequence variants (ASV, bacterial taxa followed by ASV numbers) in bulk tank milk samples from farms using automatic milking systems (AMS) of brand A and B, respectively. The vertical bars through the boxes are the median (i.e., the 50th percentile) values. The left and right extents of the boxes are the 25th and 75th percentiles of the values, respectively. The left and right whiskers of the boxes are the minimum and maximum values of the distribution, respectively. There are 32 and 63 milk samples originating from 7 and 11 farms using AMS brands A and B, respectively. Figure 7. The relative abundance (RA, %) of amplicon sequence variants (ASV, bacterial taxa followed by ASV numbers) in bulk tank milk samples from farms using automatic milking systems (AMS) of brand A and B, respectively. The vertical bars through the boxes are the median (i.e., the 50th percentile) values. The left and right extents of the boxes are the 25th and 75th percentiles of the values, respectively. The left and right whiskers of the boxes are the minimum and maximum values of the distribution, respectively. There are 32 and 63 milk samples originating from 7 and 11 farms using AMS brands A and B, respectively. 135 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Figure 8. Principal component analysis plot of microbial data in bulk tank milk samples from tiestall farms only. Each point represents a bulk tank milk sample from an individual farm. Routines used to prepare teats before milking were as follows: CHEM = use of chemical agent in teat preparation, DRY = no liquid-based method for teat preparation, WATER = use of water only to prepare teats before milking. Variation in Tank Milk Microbiota Within and Between Farms Frequency of acid wash to clean the milking equipment was as follows: FEW = less frequent use of acid wash (from once every second day to once per week or less frequently); NO = never use acid wash, only use alkaline wash; OFT = frequent use of acid wash (every second wash). Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA 135 Figure 8. Principal component analysis plot of microbial data in bulk tank milk samples from tiestall farms only. Each point represents a bulk tank milk sample from an individual farm. Routines used to prepare teats before milking were as follows: CHEM = use of chemical agent in teat preparation, DRY = no liquid-based method for teat preparation, WATER = use of water only to prepare teats before milking. Frequency of acid wash to clean the milking equipment was as follows: FEW = less frequent use of acid wash (from once every second day to once per week or less frequently); NO = never use acid wash, only use alkaline wash; OFT = frequent use of acid wash (every second wash). Journal of Dairy Science Vol. 105 No. 1, 2022 Influence of Different Farm Factors on Farm Milk Microbiota The OPLS-DA models presented in this study had Q2Y values ranging from 0.607 to 0.784, indicating strong model performance. This was also seen in the cross-validated score plots where the different farm types were found to be nearly nonoverlapping. The strong model performance could partly be attributed to a sufficiently large sample size, enabling reliable load- ing interpretations from significant models. Although several samples originated from each farm, causing a possible redundancy in the data, the cross-validation procedure effectively mitigated this, as all observations from the same farm were excluded and predicted in the same cross-validation round. The PCA identified type of dairy farm, categorized according to milking system, as the most influential factor for the milk microbiota. A clear difference in milk microbiota between AMS and tiestall farms was observed, whereas the milk microbiota from farms with a milking parlor could not be separated from the other 2 systems. However, this clustering was linked to PC 2, Journal of Dairy Science Vol. 105 No. 1, 2022 136 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA 136 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Figure 9. Relative abundance (RA, %) of amplicon sequence variants (ASV, bacterial taxa followed by ASV number) in bulk tank milk samples from tiestall farms grouped according to method for preparation of teats before milking and frequency of acid wash to clean the milk- ing equipment. CHEM-OFT consisted of farms that used chemicals in connection to teat preparation and more frequently (every second wash) applied acid solution to clean the milking equipment (34 milk samples originated from 8 farms); WATER-FEW consisted of farms that used water for teat cleaning and less frequently (from once every second day to once per week) applied acid solution to clean the milking equipment (19 milk samples originated from 3 farms). The vertical bars through the boxes are the 50th percentile values. The left and right extents of the boxes are the 25th and 75th percentiles (q3) of the values, respectively. The left and right whiskers of the boxes are the minimum and maximum values of the distribution, respectively. *1: WATER-FEW q3 28%, WATER-FEW whiskers 57%; *2: WATER-FEW q3 5.4%, WATER-FEW whiskers 13%; *3: WATER-FEW q3 14%, WATER-FEW whiskers 17%. Figure 9. Influence of Different Farm Factors on Farm Milk Microbiota (1999) on factors affecting the number of Bacillus cereus spores in raw milk during the grazing period found that the spore content of milk was strongly associated with de- gree of soil contamination on the teats, but that the number of spores could be reduced by teat cleansing. The influence of teat preparation on microbial com- munity composition in milk was also investigated by Doyle et al. (2016), who concluded that the teat sur- face was the most significant source of contamination of the milk. In agreement with previous studies, within each of the 2 farm types of our study (i.e., AMS and tiestall milking), differences in milk microbiota were seen between farms using different cleaning procedures before milking. Tank milk from the 2 brands of milk- ing robots represented (A, B) showed clear clustering, although only a small proportion of the variation in the data was explained by the model. Five ASV belong- ing to the genus Acinetobacter, but also some LAB, Lactococcus, and Streptococcus, were more abundant in milk from brand A robots. This difference in milk microbiota was most likely attributable to differences in the procedure used for teat preparation before milk- ing. Brand A robots have a system whereby teats are cleaned in washing cups with lukewarm water and then dried by blowing air, whereas brand B robots use 2 parallel rolling brushes to clean the teats. For the farms with tiestall milking routines for teat prepara g q p Interestingly, milk from AMS farms was associated with higher species richness than milk from tiestall farms. Skeie et al. (2019) suggested that differences in herd size could have contributed to observed differences in microbial diversity between milk samples collected from the farms in their study. Considering that the first source of bacterial contamination in milk is the indi- vidual cow, farms with more cows could show greater microbial diversity in the tank milk. There was a clear difference in average herd size between AMS (85 cows) and TIE (30 cows) farms in our study (data not shown; for details see Priyashantha et al., 2021), which could have contributed to the differences in microbial diver- sity between the herd types. Influence of Different Farm Factors on Farm Milk Microbiota Relative abundance (RA, %) of amplicon sequence variants (ASV, bacterial taxa followed by ASV number) in bulk tank milk samples from tiestall farms grouped according to method for preparation of teats before milking and frequency of acid wash to clean the milk- ing equipment. CHEM-OFT consisted of farms that used chemicals in connection to teat preparation and more frequently (every second wash) applied acid solution to clean the milking equipment (34 milk samples originated from 8 farms); WATER-FEW consisted of farms that used water for teat cleaning and less frequently (from once every second day to once per week) applied acid solution to clean the milking equipment (19 milk samples originated from 3 farms). The vertical bars through the boxes are the 50th percentile values. The left and right extents of the boxes are the 25th and 75th percentiles (q3) of the values, respectively. The left and right whiskers of the boxes are the minimum and maximum values of the distribution, respectively. *1: WATER-FEW q3 28%, WATER-FEW whiskers 57%; *2: WATER-FEW q3 5.4%, WATER-FEW whiskers 13%; *3: WATER-FEW q3 14%, WATER-FEW whiskers 17%. which explained only 5% of the variation in the data. Several confounding factors, such as housing and milk- ing system, breed, and number of cows, contributed to the characterization of the 2 major farm types in our study (i.e., AMS and tiestall farms, respectively). The influence from the confounded farm factors could not be distinguished and should not be overlooked. More- over, effects of some individual factors might have been hidden or masked, due to low frequency among the farms. To study the influence of individual factors, a case-study or a controlled trial might give better results than screening of factors, which this study was aiming for. Milk from tiestall farms showed higher RA, espe- cially of Pseudomonas_69d9bd, whereas bacteria from Peptostreptococcaceae and Ruminococcaceae, commonly found in dairy cow feces samples, were more abundant in milk from AMS farms. In agreement with expectations and with findings in a previous review by Hogenboom et al. (2019), we found that levels of TBC were higher in tank milk from AMS farms than in milk from farms with a tiestall milking system (P < 0.01). An increase in TBC is in most cases Journal of Dairy Science Vol. 105 No. Influence of Different Farm Factors on Farm Milk Microbiota In agreement with previous studies, within each of the 2 farm types of our study (i.e., AMS and tiestall milking), differences in milk microbiota were seen between farms using different cleaning procedures before milking. Tank milk from the 2 brands of milk- ing robots represented (A, B) showed clear clustering, although only a small proportion of the variation in the data was explained by the model. Five ASV belong- ing to the genus Acinetobacter, but also some LAB, Lactococcus, and Streptococcus, were more abundant in milk from brand A robots. This difference in milk microbiota was most likely attributable to differences in the procedure used for teat preparation before milk- ing. Brand A robots have a system whereby teats are cleaned in washing cups with lukewarm water and then dried by blowing air, whereas brand B robots use 2 parallel rolling brushes to clean the teats. For the farms with tiestall milking, routines for teat prepara- tion were largely confounded by the frequency of use of acid wash to clean the equipment. Nevertheless, these factors influenced the microbial community in tank associated with shortcomings in the teat preparation process. Teat skin is reported to be the major source of bacteria for milk microbiota, even more important than environmental sources, such as feces, bedding material, and milking equipment (Vacheyrou et al., 2011). In a study examining the microbial communities in raw cow milk and potential transfer from the barn environment to the milk, Vacheyrou et al. (2011) found that coryneforms were commonly associated with teat skin, whereas some cocci, such as Staphylococcus, were very common in the environment, and others, such as Lactococcus and Enterococcus, were found only in the milk. Differences in microbial community composition between teat skin and milk samples, with higher pro- portions of Corynebacterium on teat skin than in milk, were also found in a study by Dahlberg et al. (2020). Skeie et al. (2019) concluded that milking system and on-farm management practices have a great effect on the bacterial composition of milk, with milk from AMS farms showing higher TBC than milk from farms us- ing a milking parlor. They attributed those differences to inferior washing of the teat, lack of a drying step before milking, and challenges with equipment clean- ing in AMS. A study by Christiansson et al. Influence of Different Farm Factors on Farm Milk Microbiota 1, 2022 137 Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA milk, with some bacteria, such as Enterobacteriaceae, Lactococcus, and Bacillaceae, being more abundant in milk from tiestall farms that only used water for teat preparation and less frequently used acid wash to clean their milking equipment. milk, with some bacteria, such as Enterobacteriaceae, Lactococcus, and Bacillaceae, being more abundant in milk from tiestall farms that only used water for teat preparation and less frequently used acid wash to clean their milking equipment. associated with shortcomings in the teat preparation process. Teat skin is reported to be the major source of bacteria for milk microbiota, even more important than environmental sources, such as feces, bedding material, and milking equipment (Vacheyrou et al., 2011). In a study examining the microbial communities in raw cow milk and potential transfer from the barn environment to the milk, Vacheyrou et al. (2011) found that coryneforms were commonly associated with teat skin, whereas some cocci, such as Staphylococcus, were very common in the environment, and others, such as Lactococcus and Enterococcus, were found only in the milk. Differences in microbial community composition between teat skin and milk samples, with higher pro- portions of Corynebacterium on teat skin than in milk, were also found in a study by Dahlberg et al. (2020). Skeie et al. (2019) concluded that milking system and on-farm management practices have a great effect on the bacterial composition of milk, with milk from AMS farms showing higher TBC than milk from farms us- ing a milking parlor. They attributed those differences to inferior washing of the teat, lack of a drying step before milking, and challenges with equipment clean- ing in AMS. A study by Christiansson et al. (1999) on factors affecting the number of Bacillus cereus spores in raw milk during the grazing period found that the spore content of milk was strongly associated with de- gree of soil contamination on the teats, but that the number of spores could be reduced by teat cleansing. The influence of teat preparation on microbial com- munity composition in milk was also investigated by Doyle et al. (2016), who concluded that the teat sur- face was the most significant source of contamination of the milk. CONCLUSIONS Differences in teat preparation before milking most likely contributed to the differences in tank milk micro- biota observed between types of farms (AMS vs. tiestall farms) and within farm types. However, the variation in microbiota data explained by milking system, robot brand, and routines used for teat preparation and clean- ing the milking equipment was limited, and most could not be explained by the models. The routines used on tiestall farms, (i.e., CHEM-OFT and WATER-FEW) displayed a weaker association to the microbial data than milking system (AMS or TIE) or robot brand (A or B). The microbial community in tank milk reflects events taking place in the herd and routines associated with milking and handling of the milk. It is challeng- ing to predict or control this microbial community, but modifying the routines associated with teat preparation and cleaning of the milking equipment, irrespective of type of milking system used, could provide some degree of control. Christiansson, A., J. Bertilsson, and B. Svensson. 1999. Bacillus cereus spores in raw milk: Factors affecting the contamination of milk during the grazing period. J. Dairy Sci. 82:305–314. https:​/​/​doi​ .org/​10​.3168/​jds​.S0022​-0302(99)75237​-9. / / ( ) Clay, N., T. Garnett, and J. Lorimer. 2020. Dairy intensification: Driv- ers, impacts and alternatives. Ambio 49:35–48. https:​/​/​doi​.org/​10​ .1007/​s13280​-019​-01177​-y. Ö / Dahlberg, J., L. Sun, K. Persson Waller, K. Östensson, M. McGuire, S. Agenäs, and J. Dicksved. 2019. Microbiota data from low bio- mass milk samples is markedly affected by laboratory and reagent contamination. PLoS One 14:e0218257. https:​/​/​doi​.org/​10​.1371/​ journal​.pone​.0218257. j Dahlberg, J., J. E. Williams, M. A. McGuire, H. K. Peterson, K. Östensson, S. Agenäs, J. Dicksved, and K. P. Waller. 2020. Mi- crobiota of bovine milk, teat skin, and teat canal: Similarity and variation due to sampling technique and milk fraction. J. Dairy Sci. 103:7322–7330. https:​/​/​doi​.org/​10​.3168/​jds​.2019​-17783. p // g/ /j De Jonghe, V., A. Coorevits, K. Van Hoorde, W. Messens, A. Van Landschoot, P. De Vos, and M. Heyndrickx. 2011. Influence of storage conditions on the growth of Pseudomonas species in refrig- erated raw milk. Appl. Environ. Microbiol. 77:460–470. Doyle, C. J., D. Gleeson, P. W. O’Toole, and P. D. Cotter. 2016. Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study. Appl. Environ. Microbiol. 83:e02694-16. https:​/​/​doi​.org/​10​.1128/​AEM​.02694​-16. // / / European Commission. 2004. Regulation (EC) No. Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Sun et al.: MILKING ROUTINES AFFECT MILK MICROBIOTA Breitenwieser, F., E. V. Doll, T. Clavel, S. Scherer, and M. Wenning. 2020. Complementary use of cultivation and high-throughput am- plicon sequencing reveals high biodiversity within raw milk micro- biota. Front. Microbiol. 11:1557. contribute to the outcomes of this study, including the observed differences linked to farm type, milking equip- ment, and teat cleaning method. Callahan, B. J., P. J. McMurdie, M. J. Rosen, A. W. Han, A. J. A. Johnson, and S. P. Holmes. 2016. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13:581–583. https:​/​/​doi​.org/​10​.1038/​nmeth​.3869. ACKNOWLEDGMENTS This study was conducted with financial support from the Swedish Farmers’ Foundation for Agricultural Research (Stockholm, Sweden), the Kamprad Family Foundation (Växjö, Sweden), the Regional Fund for Agricultural Research in Northern Sweden (Uppsala, Sweden), and the Faculty of Natural Resources and Agriculture, SLU (Uppsala, Sweden). We thank all the dairy farmers who participated in the study. The au- thors have not stated any conflicts of interest. Galindo-Prieto, B. 2017. Novel variable influence on projection (VIP) methods in OPLS, O2PLS, and OnPLS models for single- and multi-block variable selection. Doctoral thesis. Department of Chemistry, Industrial Doctoral School, Umeå University, Sweden. Halekoh, U., and S. Højsgaard. 2014. A Kenward-Roger approxima- tion and parametric bootstrap methods for tests in linear mixed models: The R package pbkrtest. J. Stat. Softw. 59:1–32. Hogenboom, J. A., L. Pellegrino, A. Sandrucci, V. Rosi, and P. D’Incecco. 2019. Invited review: Hygienic quality, composition, and technological performance of raw milk obtained by robotic milking of cows. J. Dairy Sci. 102:7640–7654. https:​/​/​doi​.org/​10​ .3168/​jds​.2018​-16013. / Hugerth, L. W., H. A. Wefer, S. Lundin, H. E. Jakobsson, M. Lind- berg, S. Rodin, L. Engstrand, and A. F. Andersson. 2014. De- gePrime, a program for degenerate primer design for broad-tax- onomic-range PCR in microbial ecology studies. Appl. Environ. Microbiol. 80:5116–5123. Influence of Different Farm Factors on Farm Milk Microbiota Comparing the microbial diversity in milk from AMS farms using different brands of milking robots, milk from farms using brand A ro- bots (teats prepared in washing cups with lukewarm water, dried by blowing air) showed higher species richness than milk from farms using brand B robots (Figure 4B). In comparison with milk from AMS farms, milk from tiestall farms was associated with lower TBC and higher RA of Pseudomonas, where the latter could be an additional explanation for lower species richness. Unlike on AMS farms, on tiestall farms higher species richness was associated with lower TBC (P < 0.05), represented by milk from farms using chemical agents in teat preparation in combination with more frequent use of acid wash. One explanation could be that the higher TBC in milk from farms with less frequent use of acid wash, and no chemical agents in teat prepa- ration, was associated with a few dominant bacterial taxa, including Bacillaceae, Enterobacteriaceae, and Acinetobacter, contributing to lower species richness. However, it is important to note that the differences between tiestall farms using different routines for clean- ing equipment and teat preparation were observed in PC3 and PC5. Moreover, because fewer milk samples were available for comparison within tiestall farms, the findings should be treated with caution and require further validation. Pseudomonas is commonly found in milk, and it was therefore difficult to determine whether the particular ASV Pseudomonas 68f648 observed in this study origi- nated from the milk or was introduced during labora- tory analysis of the milk samples. As a precautionary strategy, we removed samples with high abundance of this ASV (>10% RA), which eliminated its poten- tial contaminating effect on the data set. Moreover, in discriminant analysis this particular ASV did not Journal of Dairy Science Vol. 105 No. 1, 2022 138 CONCLUSIONS 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on prod- ucts of animal origin intended for human consumption. Off. J. Eur. Union L 139:55–205. 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Variability of microbial teat skin flora in relation to farming practices and individual dairy cow characteristics. Dairy Sci. Technol. 92:265–278. https:​/​/​doi​.org/​10​.1007/​s13594​-012​ -0064​-7. Stoeckel, M., M. Lidolt, T. Stressler, L. Fischer, M. Wenning, and J. Hinrichs. 2016. Heat stability of indigenous milk plasmin and pro- teases from Pseudomonas: A challenge in the production of ultra- high temperature milk products. Int. Dairy J. 61:250–261. https:​/​ /​doi​.org/​10​.1016/​j​.idairyj​.2016​.06​.009. Å O’Connell, A., S. McParland, P. L. Ruegg, B. O’Brien, and D. Glee- son. 2015. Seasonal trends in milk quality in Ireland between 2007 and 2011. J. Dairy Sci. 98:3778–3790. https:​/​/​doi​.org/​10​.3168/​jds​ .2014​-9001. / / / Sun, L., J. Dicksved, H. Priyashantha, Å. Lundh, and M. Johansson. 2019. Distribution of bacteria between different milk fractions, investigated using culture-dependent methods and molecular- based and fluorescent microscopy approaches. J. Appl. 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Exome Sequencing Analysis Identifies Rare Variants in ATM and RPL8 That Are Associated With Shorter Telomere Length
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ORIGINAL RESEARCH published: 30 April 2020 doi: 10.3389/fgene.2020.00337 Exome Sequencing Analysis Identifies Rare Variants in ATM and RPL8 That Are Associated With Shorter Telomere Length Ashley van der Spek1,2, Sophie C. Warner3, Linda Broer4, Christopher P. Nelson3,5, Dina Vojinovic1, Shahzad Ahmad1, Pascal P. Arp4, Rutger W. W. Brouwer6, Matthew Denniff3, Mirjam C. G. N. van den Hout6, Jeroen G. J. van Rooij4,7, Robert Kraaij4, Wilfred F. J. van IJcken6, Nilesh J. Samani3,5, M. Arfan Ikram1, André G. Uitterlinden1,4, Veryan Codd3,5, Najaf Amin1,8 and Cornelia M. van Duijn1,8* Ashley van der Spek1,2, Sophie C. Warner3, Linda Broer4, Christopher P. Nelson3,5, Dina Vojinovic1, Shahzad Ahmad1, Pascal P. Arp4, Rutger W. W. Brouwer6, Matthew Denniff3, Mirjam C. G. N. van den Hout6, Jeroen G. J. van Rooij4,7, Robert Kraaij4, Wilfred F. J. van IJcken6, Nilesh J. Samani3,5, M. Arfan Ikram1, André G. Uitterlinden1,4, Veryan Codd3,5, Najaf Amin1,8 and Cornelia M. van Duijn1,8* 1 Department of Epidemiology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands, 2 SkylineDx B.V., Rotterdam, Netherlands, 3 Department of Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom, 4 Department of Internal Medicine, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands, 5 NIHR Leicester Biomedical Research Centre, Glenfield Hospital, Leicester, United Kingdom, 6 Center for Biomics, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands, 7 Department of Neurology, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands, 8 Nuffield Department of Population Health, University of Oxford, Oxford, United Kingdom Reviewed by: Reviewed by: Eleonora Porcu, Université de Lausanne, Switzerland Nabila Bouatia-Naji, Institut National de la Santé et de la Recherche Médicale (INSERM), France Telomeres are important for maintaining genomic stability. Telomere length has been associated with aging, disease, and mortality and is highly heritable (∼82%). In this study, we aimed to identify rare genetic variants associated with telomere length using whole-exome sequence data. We studied 1,303 participants of the Erasmus Rucphen Family (ERF) study, 1,259 of the Rotterdam Study (RS), and 674 of the British Heart Foundation Family Heart Study (BHF-FHS). We conducted two analyses, first we analyzed the family-based ERF study and used the RS and BHF-FHS for replication. Second, we combined the summary data of the three studies in a meta- analysis. Telomere length was measured by quantitative polymerase chain reaction in blood. We identified nine rare variants significantly associated with telomere length (p-value < 1.42 × 10−7, minor allele frequency of 0.2–0.5%) in the ERF study. Eight of these variants (in C11orf65, ACAT1, NPAT, ATM, KDELC2, and EXPH5) were located on chromosome 11q22.3 that contains ATM, a gene involved in telomere maintenance. Although we were unable to replicate the variants in the RS and BHF- FHS (p-value ≥0.21), segregation analysis showed that all variants segregate with shorter telomere length in a family. In the meta-analysis of all studies, a nominally significant association with LTL was observed with a rare variant in RPL8 (p- value = 1.48 × 10−6), which has previously been associated with age. Additionally, a novel rare variant in the known RTEL1 locus showed suggestive evidence for association (p-value = 1.18 × 10−4) with LTL. To conclude, we identified novel rare variants associated with telomere length. Larger samples size are needed to confirm these findings and to identify additional variants. Telomeres are important for maintaining genomic stability. Telomere length has been associated with aging, disease, and mortality and is highly heritable (∼82%). In this study, we aimed to identify rare genetic variants associated with telomere length using whole-exome sequence data. We studied 1,303 participants of the Erasmus Rucphen Family (ERF) study, 1,259 of the Rotterdam Study (RS), and 674 of the British Heart Foundation Family Heart Study (BHF-FHS). We conducted two analyses, first we analyzed the family-based ERF study and used the RS and BHF-FHS for replication. Second, we combined the summary data of the three studies in a meta- analysis. Keywords: telomere, aging, whole exome sequencing, meta-analysis, ATM, RPL8 Reviewed by: Telomere length was measured by quantitative polymerase chain reaction in blood. We identified nine rare variants significantly associated with telomere length (p-value < 1.42 × 10−7, minor allele frequency of 0.2–0.5%) in the ERF study. Eight of these variants (in C11orf65, ACAT1, NPAT, ATM, KDELC2, and EXPH5) were located on chromosome 11q22.3 that contains ATM, a gene involved in telomere maintenance. Although we were unable to replicate the variants in the RS and BHF- FHS (p-value ≥0.21), segregation analysis showed that all variants segregate with shorter telomere length in a family. In the meta-analysis of all studies, a nominally significant association with LTL was observed with a rare variant in RPL8 (p- value = 1.48 × 10−6), which has previously been associated with age. Additionally, a novel rare variant in the known RTEL1 locus showed suggestive evidence for association (p-value = 1.18 × 10−4) with LTL. To conclude, we identified novel rare variants associated with telomere length. Larger samples size are needed to confirm these findings and to identify additional variants. *Correspondence: Cornelia M. van Duijn Cornelia.vanDuijn@ndph.ox.ac.uk *Correspondence: Cornelia M. van Duijn Cornelia.vanDuijn@ndph.ox.ac.uk Specialty section: This article was submitted to Human Genomics, a section of the journal Frontiers in Genetics Received: 17 September 2019 Accepted: 20 March 2020 Published: 30 April 2020 Edited by: Edited by: Serena Sanna, Institute for Genetic and Biomedical Research (IRGB), Italy Edited by: Serena Sanna, Institute for Genetic and Biomedical Research (IRGB), Italy RESULTS LTL is highly heritable with heritability estimates ranging from 34 to 82% (Slagboom et al., 1994; Bischoffet al., 2005; Vasa-Nicotera et al., 2005; Andrew et al., 2006; Broer et al., 2013). Previously, genome-wide association studies (GWASs) in European ancestry studies have identified common variants associated with LTL located in multiple genes, including: TERC (Codd et al., 2010, 2013; Mangino et al., 2012; Pooley et al., 2013), TERT (Codd et al., 2013; Pooley et al., 2013), NAF1 (Codd et al., 2013), OBFC1 (Levy et al., 2010; Mangino et al., 2012; Codd et al., 2013; Pooley et al., 2013), RTEL1 (Codd et al., 2013), CTC1 (Mangino et al., 2012), ZNF676 (Mangino et al., 2012), ZNF208 (Codd et al., 2013), ACYP2 (Codd et al., 2013), DCAF4 (Mangino et al., 2015), and PXK (Pooley et al., 2013). However, these variants explain < 5% of the heritability. The Manhattan plot and the distribution of the test statistic [quantile-quantile (QQ) plot, λ = 1.04] of the WES analysis in the ERF study are presented in Figures 1, 2, respectively. We observed significant association of nine rare variants [Minor Allele Frequency (MAF) between 0.2% and 0.5%] with LTL as shown in Table 2. The significance threshold (p- value < 1.42 × 10−7) was adjusted for multiple testing using Bonferroni correction based on the number of variants in the analysis (0.05/353,075 variants). Each variant was negatively associated with LTL and the estimated effects of the minor allele of these variants were large (−2.18 < standardized β < −1.34), suggesting a significant decrease in LTL for each minor allele. The top eight variants are located in a dense region on chromosome 11q22.3 (position range: 108004687 – 108384666, Figure 3) and appear to be a part of a haplotype that spans the C11orf65, ACAT1, NPAT, ATM, KDELC2, and EXPH5 genes. A haplotype can describe a pair of genes inherited together from one parent on one chromosome, or it can describe all of the genes on a chromosome that were inherited together from a single parent. This haplotype segregates with shorter LTL in a family (Supplementary Figure 1), where it is carried by 14 individuals, 11 of whom were related within 4 generations according to the TABLE 1 | Descriptive statistics of the study populations. RESULTS Telomeres are DNA structures located at the ends of chromosomes and consist of tandem hexanucleotide sequence repeats (TTAGGG) (Blackburn and Gall, 1978). They are important for maintaining genomic stability by preventing DNA degradation and chromosomal fusions (Blackburn, 2000). Telomeres are shortened with each cell division due to the inability of DNA polymerase to fully extend the 3′ end of the DNA strand during replication. When the telomeres reach a critical length, this leads to cellular senescence and ultimately to cell death, making them regulators of the replicative capacity of a cell and markers of biological age (Lindsey et al., 1991; Allsopp et al., 1992). Descriptive statistics of the family-based and population-based studies are provided in Table 1. Mean age at LTL measurement was 49 years (SD = 15.0) in the ERF study and 61% of the study participants were female. The RS participants were older (¯xage = 75 years, SD = 7.7) and 57% of the participants were female, while mean age in the BHF-FHS was 58 years (SD = 8.2) and most study participants were male (26% female). Mean LTL values, measured in each participant using a quantitative polymerase chain reaction (qPCR) based technique as the ratio of telomere repeat length to copy number of the single copy gene 36B4, were higher in the ERF study (¯xLTL = 1.85, SD = 0.35) than in the RS (¯xLTL = 0.94, SD = 0.18) and BHF-FHS (¯xLTL = 1.37, SD = 0.22). After adjusting LTL values for age and sex, mean LTL was comparable between studies (Table 1). Shorter leukocyte telomere length (LTL) has been associated with several age-related diseases including cardiovascular diseases (Brouilette et al., 2003, 2007; Benetos et al., 2004; Fitzpatrick et al., 2007; Willeit et al., 2010a; Haycock et al., 2014), cancer (Hastie et al., 1990; Willeit et al., 2010b, 2011; Bojesen et al., 2013) and dementia (Martin-Ruiz et al., 2006; Grodstein et al., 2008; Honig et al., 2012). LTL has also been associated with mortality (Cawthon et al., 2003; Kimura et al., 2008; Fitzpatrick et al., 2011; Honig et al., 2012; Deelen et al., 2014; Rode et al., 2015; Marioni et al., 2016). However, this association has been inconsistent (Martin-Ruiz et al., 2005; Harris et al., 2006; Njajou et al., 2009; Houben et al., 2011; Strandberg et al., 2011; Needham et al., 2015). Citation: van der Spek A, Warner SC, Broer L, Nelson CP, Vojinovic D, Ahmad S, Arp PP, Brouwer RWW, Denniff M, van den Hout MCGN, van Rooij JGJ, Kraaij R, van IJcken WFJ, Samani NJ, Ikram MA, Uitterlinden AG, Codd V, Amin N and van Duijn CM (2020) Exome Sequencing Analysis Identifies Rare Variants in ATM and RPL8 That Are Associated With Shorter Telomere Length. Front. Genet. 11:337. doi: 10.3389/fgene.2020.00337 Keywords: telomere, aging, whole exome sequencing, meta-analysis, ATM, RPL8 April 2020 | Volume 11 | Article 337 1 Frontiers in Genetics | www.frontiersin.org Exome-Wide Association Study of Telomere Length van der Spek et al. Frontiers in Genetics | www.frontiersin.org RESULTS FIGURE 1 | Manhattan plot of the association analysis with LTL in the ERF study. This plot shows –log10 transformed p-values (y-axis) for all variants present in the association analysis according to their position on each chromosome (x-axis). The red dashed line represents the Bonferroni corrected p-value threshold for significance (p-value < 1.42 × 10−7), while the blue dashed line represents the p-value threshold for suggestive significance (p-value ≤1.42 × 10−4). FIGURE 1 | Manhattan plot of the association analysis with LTL in the ERF study. This plot shows –log10 transformed p-values (y-axis) for all variants present in the association analysis according to their position on each chromosome (x-axis). The red dashed line represents the Bonferroni corrected p-value threshold for significance (p-value < 1.42 × 10−7), while the blue dashed line represents the p-value threshold for suggestive significance (p-value ≤1.42 × 10−4). FIGURE 1 | Manhattan plot of the association analysis with LTL in the ERF study. This plot shows –log10 transformed p-values (y-axis) for all variants present in the association analysis according to their position on each chromosome (x-axis). The red dashed line represents the Bonferroni corrected p-value threshold for significance (p-value < 1.42 × 10−7), while the blue dashed line represents the p-value threshold for suggestive significance (p-value ≤1.42 × 10−4). variants are missense variants: rs79119325 (NPAT, PolyPhen = 1, CADD score = 18.1) and rs12146512 (EXPH5, PolyPhen = 0.624, CADD score = 4.5). The eighth significant variant, rs2234993, is located within an intron of ATM (MAF = 0.5%, β = −1.37, SE = 0.26, p-value = 1.25 × 10−7). The ninth significant variant, rs144114619 (MAF = 0.2%, p-value = 1.29 × 10−7), is a missense variant located on chromosome six in the BTN3A1 gene, which is predicted to be damaging (PolyPhen = 1, CADD score = 12.2) and has the largest effect size (β = −2.18, SE = 0.41). There were six carriers of this variant in the ERF population. Interestingly, four of these carriers are also carriers of the rare variants in the chromosome 11q22.3 region (Supplementary Figure 2). pedigree data (Figure 4). Further, the genetic kinship estimates show that the other three individuals are also related within 3–4 generations. These 8 variants are in strong linkage disequilibrium (pairwise LD: r2 between 0.93 and 1.00, D′ = 1) and show very similar p-values. RESULTS ERF RS BHF-FHS N 1303 1257 674 Mean age (SD) 48.9 (15.0) 74.5 (7.7) 58.0 (8.2) Age range 18.2–95.7 55.0–105.8 36.0–81.0 % female 60.5 57.0 25.8 Mean LTL (SD) 1.85 (0.35) 0.94 (0.18) 1.37 (0.22) LTL range 0.77–3.17 0.31–1.79 0.69–2.14 Adjusted mean 8.85 × 10−18 (0.32) 1.37 × 10−17 (0.18) 1.11 × 10−10 (0.21) LTL (SD)* Adjusted LTL −1.15–1.08 −0.71–0.89 −0.63–0.71 range* ERF = Erasmus Rucphen Family study; RS = Rotterdam Study; BHF-FHS = British Heart Foundation Family Heart Study; N = Number of variants; SD = Standard Deviation, LTL = Leukocyte Telomere Length. *LTL values were adjusted for age and sex and information on residuals is shown. Up until now, no systemic whole exome or genome screen for rare variants has been published, despite the fact that these may explain part of the heritability (Manolio et al., 2009). Rare variants are not well captured by microarrays used for GWAS and remain difficult to impute, despite the recent improvements in imputation panels (McCarthy et al., 2016). Next generation sequencing technologies, such as whole-exome sequencing (WES), are better suited to study rare variants. In this study, we present a dual analysis. First, we conducted a genome- wide WES analysis of LTL in 1,303 individuals of the Dutch Erasmus Rucphen Family (ERF) study to search for rare genetic variants associated with LTL. The advantage of a family-based study is that the segregation of rare variants can be studied. We performed a replication analysis in the Rotterdam Study (RS) and the British Heart Foundation Family Heart Study (BHF-FHS). Next, we pooled the data together and conducted a meta-analysis of the association results of all three cohorts. TABLE 1 | Descriptive statistics of the study populations. ERF = Erasmus Rucphen Family study; RS = Rotterdam Study; BHF-FHS = British Heart Foundation Family Heart Study; N = Number of variants; SD = Standard Deviation, LTL = Leukocyte Telomere Length. *LTL values were adjusted for age and sex and information on residuals is shown. ERF = Erasmus Rucphen Family study; RS = Rotterdam Study; BHF-FHS = British Heart Foundation Family Heart Study; N = Number of variants; SD = Standard Deviation, LTL = Leukocyte Telomere Length. *LTL values were adjusted for age and sex and information on residuals is shown. April 2020 | Volume 11 | Article 337 2 Exome-Wide Association Study of Telomere Length van der Spek et al. RESULTS The top variant rs185270276 is located in an intron of the C11orf65 gene (MAF = 0.5%, β = −1.34, SE = 0.25, p-value = 7.99 × 10−8). The next six variants significantly associated with LTL (MAF = 0.5%, β = −1.38, SE = 0.26, p-value = 1.21 × 10−7) are located in the ACAT1, NPAT, ATM/C11orf65, KDELC2, and EXPH5 genes. Two of these six FIGURE 2 | Quantile-quantile plot of the association analysis with LTL in the ERF study. The QQ-plot shows the observed test statistics (y-axis) plotted against the expected values of the test statistics (x-axis) (X2-distribution). The red line shows the distribution under the null hypothesis. For replication analysis, we used WES data from two independent cohorts of European ancestry, the RS and the BHF- FHS. Results of the replication analysis are shown in Table 3, together with the results of the meta-analysis of summary statistics from all three cohorts for these variants. Six out of nine rare variants significantly associated with LTL in the ERF study (located in ACAT1, NPAT, ATM/C11orf65, EXPH5, and BTN3A1) were present in the RS and/or the BHF-FHS but were not significantly (p-value ≥0.21) associated with LTL (p-value < 0.025, 0.05/2 independent tests). The direction of effect of most variants was similar in the ERF study and the RS, while the BHF-FHS showed opposite direction of effect for most variants. Three variants, located in C11orf65, KDELC2, and ATM, were not present in the RS and BHF-FHS data. As these are unique to an isolated population, we were unable to confirm or reject their association with LTL in the replication cohorts. Finally, to increase the statistical power, we performed an inverse-variance weighted meta-analysis of the association results from all three cohorts using METAL software. Variants were included in the meta-analysis if they were present in at least two out of three cohorts and had a minimum minor allele count of five in one or more cohorts, resulting in a multiple testing corrected significance threshold of 3.02 × 10−7 (0.05/165,311 variants). The top results of the meta-analysis (p-value < 3.02 × 10−4) are FIGURE 2 | Quantile-quantile plot of the association analysis with LTL in the ERF study. The QQ-plot shows the observed test statistics (y-axis) plotted against the expected values of the test statistics (x-axis) (X2-distribution). The red line shows the distribution under the null hypothesis. RESULTS April 2020 | Volume 11 | Article 337 Frontiers in Genetics | www.frontiersin.org 3 Exome-Wide Association Study of Telomere Length van der Spek et al. TABLE 2 | Significant variants from the association analysis in the ERF study. REF/ PolyPhen PhastCons CADD rsID Gene Chr Position* MAF ALT GVS function** score** score** score** β SE p-value rs185270276 C11orf65 11 108263828 0.005 T/C Intron Unknown 0 6.51 −1.34 0.25 7.99 × 10−8 rs12365364 ACAT1 11 108004687 0.005 G/A Intron Unknown 0 3.28 −1.38 0.26 1.21 × 10−7 rs79119325 NPAT 11 108032614 0.005 C/T Missense 1 0.998 18.13 −1.38 0.26 1.21 × 10−7 rs3092910 ATM,C11orf65 11 108180917 0.005 T/C Intron, synonymous Unknown 0.997 9.87 −1.38 0.26 1.21 × 10−7 rs3218711 ATM,C11orf65 11 108236264 0.005 C/G 3-prime-UTR, intron Unknown 0.002 5.56 −1.38 0.26 1.21 × 10−7 rs11212668 KDELC2 11 108352576 0.005 T/C Intron Unknown 0 1.44 −1.38 0.26 1.21 × 10−7 rs12146512 EXPH5 11 108384666 0.005 T/C Missense 0.624 0.011 4.47 −1.38 0.26 1.21 × 10−7 rs2234993 ATM 11 108129599 0.005 C/G Intron Unknown 0 4.25 −1.37 0.26 1.25 × 10−7 rs144114619 BTN3A1 6 26408145 0.002 T/A Missense 1 0.002 12.24 −2.18 0.41 1.29 × 10−7 Chr = Chromosome; MAF = Minor Allele Frequency; REF = Reference allele; ALT = Alternative allele; CADD = Combined Annotation Dependent Depletion; β = effect of the minor allele; SE = Standard Error. *Position according to Hg19. **Seattleseq Annotation Database 138. TABLE 2 | Significant variants from the association analysis in the ERF study. Chr = Chromosome; MAF = Minor Allele Frequency; REF = Reference allele; ALT = Alternative allele; CADD = Combined Annotation Dependent Depletion; β = effect of the minor allele; SE = Standard Error. *Position according to Hg19. **Seattleseq Annotation Database 138. FIGURE 3 | Regional association plot for the top hits on chromosome 11. The plot was constructed using LocusZoom (http://locuszoom.org/). The –log10 transformed p-values are plotted on the y-axis. The x-axis shows the position of the variants (dots) on chromosome 11 and the genes in this region. The most significant variant (rs185270276) is shown in purple and the color of the dots indicates the extend of linkage disequilibrium between the variant and the top variant. FIGURE 3 | Regional association plot for the top hits on chromosome 11. The plot was constructed using LocusZoom (http://locuszoom.org/). The –log10 transformed p-values are plotted on the y-axis. Frontiers in Genetics | www.frontiersin.org RESULTS The x-axis shows the position of the variants (dots) on chromosome 11 and the genes in this region. The most significant variant (rs185270276) is shown in purple and the color of the dots indicates the extend of linkage disequilibrium between the variant and the top variant. available in Table 4 and in Supplementary Table 1, which also contains cohort specific information. The Manhattan and QQ- plots are provided in Supplementary Figures 3, 4, respectively. The λ of 0.97 suggests the power has been low. Although there were no variants genome-wide significantly associated with LTL in the meta-analysis after adjusting for multiple testing, many of the top findings show a consistent effect across cohorts. The variant most significantly associated with LTL was a highly conserved synonymous variant (PhastCons score = 0.999) located in the RPL8 gene on chromosome 8 (p-value = 1.48 × 10−6), which is predicted to be deleterious (CADD score = 15.11). Additionally, we used the meta-analysis results to perform a look-up of variants in loci identified by previous European ancestry GWASs (Supplementary Table 2). There were 153 variants present in these loci and we found suggestive evidence (p-valuemeta < 3.02 × 10−4) for a positive association of a rare variant with LTL (rs181080831, β = 0.74, SE = 0.19, p-value = 1.18 × 10−4) in the known RTEL1 locus. April 2020 | Volume 11 | Article 337 Frontiers in Genetics | www.frontiersin.org 4 Exome-Wide Association Study of Telomere Length van der Spek et al. FIGURE 4 | Segregation plot of the rare variants on chromosome 11q22.3 in the ERF study. The carriers of the rare variants located on chromosome 11q22.3 are depicted in red. The T/S ratio is added below the individuals of whom whole-exome sequencing data was available. Squares are males and rounds are females. Deceased individuals are denoted by a line through that specific individual. One person is twice in the pedigree, this is shown with the dotted line. FIGURE 4 | Segregation plot of the rare variants on chromosome 11q22.3 in the ERF study. The carriers of the rare variants located on chromosome 11q22.3 are depicted in red. The T/S ratio is added below the individuals of whom whole-exome sequencing data was available. Squares are males and rounds are females. Deceased individuals are denoted by a line through that specific individual. One person is twice in the pedigree, this is shown with the dotted line. DISCUSSION Interestingly, we identified three unique rare variants in the ATM (Ataxia Telangiectasia Mutated) gene associated with LTL in the ERF study. ATM is the homolog of the Tel1 gene in yeast (Greenwell et al., 1995) and has been implicated in important telomere maintenance processes (Pandita, 2002; Lee et al., 2015; Tong et al., 2015). The ATM protein kinase is a master controller of cell cycle checkpoint signaling pathways required for cell response to DNA damage and for genomic stability (https: //www.ncbi.nlm.nih.gov/gene/?term=472). Additionally, ATM kinase is necessary for telomere elongation (Lee et al., 2015; Tong et al., 2015). ATM is involved in the genetic disorder ataxia telangiectasia (AT), which is characterized by cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, and a predisposition to cancer (Savitsky et al., 1995). Cells of AT patients also show telomeric fusions and have accelerated telomere shortening with increasing age (Metcalfe et al., 1996). Furthermore, ATM was significantly associated with In the family-based ERF study, we identified nine rare variants (MAF between 0.2% and 0.5%) associated with LTL by performing a WES association analysis. The eight most significantly associated variants are located in a region on chromosome 11q22.3 and segregate together with shorter LTL in one family. This region contains the ATM locus that has previously been shown to be involved in telomere maintenance and genomic stability and is thus an obvious candidate gene. In the meta-analysis of discovery and replication cohorts, we identified another rare missense variant in the RPL8 gene strongly associated with LTL. Although we were not able to replicate either of the associations, both ATM and RPL8 have been previously found to be strong predictors of telomere length (ATM) and chronological age (ATM and RPL8). April 2020 | Volume 11 | Article 337 Frontiers in Genetics | www.frontiersin.org 5 Exome-Wide Association Study of Telomere Length van der Spek et al. chronological age in a meta-analysis of gene expression profiles, showing lower transcript abundance in older individuals (Peters et al., 2015). Although genetic variants in ATM have been associated with various cancers (Barrett et al., 2011; Helgason et al., 2015; Ransohoffet al., 2017; Scelo et al., 2017), only one genetic variant in ATM, rs227080, was genome-wide significantly associated with LTL in a Singaporean Chinese population (Dorajoo et al., 2019). DISCUSSION This variant was not significantly associated with LTL in the ERF study, the Rotterdam Study or the BHF-FHS, implicating it was not driving the association observed in the current study. TABLE 3 | Replication results of the association analysis. rsID Gene ERF (N = 1,303) RS (N = 1,257) BHF-FHS (N = 674) Meta-analysis (N = 3,234) MAF β SE p-value MAF β SE p-value MAF β SE p-value Direction* β SE p-value rs185270276 C11orf65 0.005 −1.34 0.25 7.99 × 10−8 – – – – – – – – – – – – rs12365364 ACAT1 0.005 −1.38 0.26 1.21 × 10−7 0.006 −0.20 0.25 0.43 0.010 0.30 0.26 0.25 --+ −0.40 0.15 7.84 × 10−3 rs79119325 NPAT 0.005 −1.38 0.26 1.21 × 10−7 0.006 −0.24 0.28 0.39 0.010 0.30 0.26 0.25 --+ −0.43 0.16 5.91 × 10−3 rs3092910 ATM,C11orf65 0.005 −1.38 0.26 1.21 × 10−7 0.007 −0.18 0.27 0.50 0.010 0.30 0.26 0.25 --+ −0.41 0.16 8.67 × 10−3 rs3218711 ATM,C11orf65 0.005 −1.38 0.26 1.21 × 10−7 0.005 −0.24 0.28 0.39 0.010 0.30 0.26 0.25 --+ −0.43 0.16 5.93 × 10−3 rs11212668 KDELC2 0.005 −1.38 0.26 1.21 × 10−7 – – – – – – – – – – – – rs12146512 EXPH5 0.005 −1.38 0.26 1.21 × 10−7 – – – – 0.016 0.27 0.21 0.21 -?+ −0.38 0.17 2.61 × 10−2 rs2234993 ATM 0.005 −1.37 0.26 1.25 × 10−7 – – – – – – – – – – – – rs144114619 BTN3A1 0.002 −2.18 0.41 1.29 × 10−7 0.004 0.008 0.29 0.98 0.003 −0.43 0.48 0.38 -+- −0.63 0.22 3.73 × 10−3 ERF Erasmus Rucphen Family study; RS Rotterdam Study; BHF FHS British Heart Foundation Family Heart Study; N sample size; MAF Minor Allele Frequency; β Effect of the minor allele; SE Standard Segregation analysis of the variants within the chromosome 11q22.3 region in the ERF study showed that the variants segregate with shorter LTL in one family. We did not detect a specific disease that segregates in this family, which may be explained by the relatively young age of most of the carriers. In the replication analysis, we were not able to confirm the association of the nine variants associated with LTL in the RS and BHF-FHS, due to lack of association (six variants) or absence of the variant in the replication cohorts (three variants). Frontiers in Genetics | www.frontiersin.org DISCUSSION However, it is possible this signal comes from variants that are family-specific and thus may not be transferable to the general population. Likewise, it may also be that the whole haplotype has an effect on telomere length, an haplotype that is most likely unique to the isolated ERF population. At this locus, the ATM gene is currently the most likely causative gene. Lastly, we performed a meta-analysis of the three cohorts. The top variant of the meta-analysis is located in RPL8 (Ribosomal Protein L8). This is interesting as RPL8, together with six other RPL genes, was negatively associated with chronological age (Peters et al., 2015). In this transcriptomic study, Peters et al. identified 1,497 genes whose expression level changes associated with age; this gene list includes RPL8 and ATM. We additionally found suggestive evidence for an association of a novel rare variant in the known RTEL1 locus. It would be interesting to perform a WES or whole-genome sequencing meta-analysis with larger sample size to increase the statistical power. p The advantage of our study is that we used a family-based setting that allowed us to show segregation of the variants located on chromosome 11q22.3 in a family. The ERF study population has shown a low immigration rate and a high level of inbreeding, which has increased the frequency of many rare alleles (Pardo et al., 2005). Another advantage is that all three studies used the standardized qPCR method to quantify LTL. However, there are also several limitations to our study. The first limitation is that our findings in the ERF study are not easy to generalize to the general population as these findings may be family-specific. However, we identified genes that are known to be related to telomere processes and thus are plausible candidate genes. The second limitation of our study may be that we used blood measurements of telomere length instead of tissue specific measurements, although previous studies have shown that mean telomere length in blood and other tissues are highly correlated (Okuda et al., 2002; Wilson et al., 2008) and, therefore, we expect this did not influence our findings. The third limitation is the difference in LTL distribution between the three studies, which may be explained by the different age distributions in the studies. April 2020 | Volume 11 | Article 337 6 Exome-Wide Association Study of Telomere Length van der Spek et al. DISCUSSION TABLE 4 | Suggestive findings of the meta-analysis (p-value ≤3.02 × 10−4). REF/ PolyPhen PhastCons CADD rsID Gene Chr Position* ALT GVS function** score** score** score** β SE p-value Direction† 8:146017422 RPL8 8 146017422 G/A Synonymous Unknown 0.999 15.11 1.93 0.40 1.48 × 10−6 +++ rs77919685 LATS2 13 21563311 G/A Missense 0.002 0 7.001 0.44 0.09 2.23 × 10−6 +++ rs56041036 ZFPM1 16 88599023 A/G Synonymous-near- splice Unknown 0.998 10.77 −0.20 0.04 7.44 × 10−6 --- rs199779997 MGA 15 42058958 A/C Missense 0.001 0.866 9.45 1.77 0.40 8.60 × 10−6 +++ rs4895944 VNN2 6 133070995 G/T Missense, non-coding-exon 0.038 0.003 16.69 −1.29 0.29 8.99 × 10−6 --? rs7735563 RAPGEF6 5 130764936 T/C Intron Unknown 1 19.08 −0.77 0.18 1.53 × 10−5 --- rs189691392 SF3B5 6 144416667 G/A 5-prime-UTR Unknown 0 7.119 −1.82 0.42 1.77 × 10−5 -?- rs138765444 ZKSCAN4 6 28219377 A/G Synonymous, intron Unknown 0.036 9.324 −1.27 0.30 2.59 × 10−5 --- rs1783091 none 21 33964605 T/C Intergenic Unknown 0.004 0.154 0.22 0.05 2.67 × 10−5 +?+ rs2170177 DOCK3 3 51349887 C/A Intron Unknown 0 0.044 −0.36 0.09 5.06 × 10−5 --- rs5930 LDLR 19 11224265 A/G Synonymous Unknown 0.001 0.579 −0.10 0.03 5.73 × 10−5 --- rs55648406 TUB 11 8060566 G/A Missense, intron 0.086 1 15.76 −0.62 0.16 5.85 × 10−5 --- rs140456008 SLC35G2 3 136574420 A/G Missense 0.941 1 10.84 1.89 0.47 6.67 × 10−5 ++? DISCUSSION rs139380413 COL8A1 3 99513830 G/A Missense 0.071 0.966 11.23 −1.26 0.32 7.88 × 10−5 --- rs11656725 LRRC48 17 17900726 C/T Intron Unknown 0 0.471 0.38 0.10 8.25 × 10−5 +?+ rs7193541 RFWD3 16 74664743 T/C Missense 0.008 0.485 9.1 −0.10 0.03 8.28 × 10−5 --- rs17222435 SLC28A1 15 85488335 C/T Intron Unknown 0 3.598 0.69 0.18 9.07 × 10−5 +++ rs117223521 none 8 38964715 T/C Upstream-gene Unknown 0 1.194 −1.36 0.35 1.01 × 10−4 -?- rs11700220 MTG2 20 60770931 A/G Missense 1 1 21.6 0.47 0.12 1.03 × 10−4 +++ rs187466877 GPN1 2 27862872 A/G Intron Unknown 0 1.47 0.85 0.22 1.05 × 10−4 +?+ rs56188826 MARK1 1 220791870 C/T Synonymous Unknown 0.123 6.414 0.41 0.11 1.14 × 10−4 +?+ rs1872592 PIF1 15 65113493 G/A Intron Unknown 0 0.005 −0.10 0.02 1.15 × 10−4 --- rs73056605 CLEC4C 12 7894056 G/A Missense 0.037 0 0.005 0.11 0.03 1.18 × 10−4 +++ rs181080831 RTEL1, RTEL1- TNFRSF6B 20 62326874 G/A Intron, non-coding-exon, synonymous Unknown 0 4.04 0.74 0.19 1.18 × 10−4 +++ rs13014800 CENPA 2 27015118 G/A Intron Unknown 0.163 11.67 −0.13 0.03 1.23 × 10−4 -?- rs143463783 TRIM27 6 28889741 G/A Synonymous Unknown 1 9.216 −1.22 0.32 1.24 × 10−4 -?- rs374215951 PIEZO1 16 88788318 G/A Synonymous Unknown 0.21 0.893 −2.57 0.67 1.31 × 10−4 -?- rs181215404 EPPK1 8 144941659 G/A Synonymous Unknown 0.011 4.835 0.94 0.25 1.32 × 10−4 +?+ rs377359525 FTCD 21 47572869 A/G Missense 1 1 16.44 1.68 0.44 1.33 × 10−4 +++ rs10936599 MYNN 3 169492101 C/T Synonymous, non-coding-exon, 5-prime-UTR Unknown 1 10.1 −0.11 0.03 1.38 × 10−4 --- rs74730846 STXBP5L 3 120924764 C/T Intron-near-splice Unknown 0.629 5.818 −0.19 0.05 1.39 × 10−4 -?- rs137853096 HSD17B4 5 118788316 G/A Missense, 5-prime-UTR 1 1 24 −0.73 0.19 1.67 × 10−4 --? DISCUSSION rs41284136 IFIT3 10 91087805 G/C 5-prime-UTR Unknown 0 7.796 0.40 0.11 1.76 × 10−4 +++ rs58106741 SLC4A1AP 2 27886820 G/T Synonymous Unknown 0 5.939 0.70 0.19 1.80 × 10−4 +?+ rs58068845 UTP6 17 30200363 G/A Intron Unknown 0 4.223 0.35 0.09 1.90 × 10−4 +++ rs200602887 GREB1 2 11751072 G/C Synonymous Unknown 0.986 10.5 −1.02 0.27 2.01 × 10−4 --- rs141180155 LRP2 2 170127559 G/A Synonymous, intron Unknown 0 13.06 −0.36 0.10 2.08 × 10−4 --- rs7837242 LONRF1 8 12600622 C/T Intron Unknown 0.001 4.941 −0.18 0.05 2.08 × 10−4 -?- rs115018606 C2orf16 2 27799773 A/C Missense 0.972 0.002 5.869 0.41 0.11 2.12 × 10−4 +?+ rs151309008 REXO2 11 114310345 C/T Missense 0.437 1 17.76 −0.64 0.17 2.12 × 10−4 --- rs143759519 PYGL 14 51382637 G/A Missense 1 0.975 34 0.55 0.15 2.13 × 10−4 +++ rs10936600 LRRC34 3 169514585 A/T Missense 1 0.001 12.07 −0.11 0.03 2.14 × 10−4 --- rs117178504 DYNC2H1 11 103153788 C/A Synonymous Unknown 0.996 8.615 0.38 0.10 2.18 × 10−4 +++ rs367644268 COA5 2 99224742 C/T Intron Unknown 0.015 8.626 −1.42 0.39 2.28 × 10−4 --? rs146979490 GPN1 2 27864089 A/G Intron Unknown 0.025 11.52 0.51 0.14 2.43 × 10−4 +++ rs146033252 MTA3, OXER1 2 42991127 G/A Missense, intron 0.084 0.023 8.404 0.71 0.19 2.48 × 10−4 +++ (Continued) TABLE 4 | Suggestive findings of the meta-analysis (p-value ≤3.02 × 10−4). April 2020 | Volume 11 | Article 337 Frontiers in Genetics | www.frontiersin.org 7 Exome-Wide Association Study of Telomere Length van der Spek et al. DISCUSSION TABLE 4 | Continued REF/ PolyPhen PhastCons CADD rsID Gene Chr Position* ALT GVS function** score** score** score** β SE p-value Direction† rs141280036 PADI4 1 17634718 A/G Missense 0.992 0.881 13.72 0.79 0.22 2.51 × 10−4 +++ rs79400176 DZANK1 20 18414309 C/T Missense 0.129 0.994 2.801 −0.36 0.10 2.55 × 10−4 --- rs116604207 RBM5 3 50147061 G/A Synonymous, non-coding-exon Unknown 0.453 11.12 0.79 0.22 2.62 × 10−4 +++ rs41307740 LBR 1 225601614 C/A Intron Unknown 0.004 5.956 −0.65 0.18 2.76 × 10−4 -?- rs369623673 ELAC2 17 12899160 C/T Intron Unknown 0 6 0.93 0.26 2.77 × 10−4 +?+ rs9997727 C4orf50 4 5969113 G/A Intron Unknown 0 0.613 −0.10 0.03 2.85 × 10−4 --- rs150538926 PDZD2 5 32037369 C/T Synonymous Unknown 0 0.482 0.40 0.11 2.87 × 10−4 +++ rs55868421 TRIM5 11 5688948 A/G Intron-near-splice, intron Unknown 0 5.512 0.44 0.12 2.94 × 10−4 +++ rs323895 ACY1, ABHD14A- ACY1 3 52021316 A/G Intron Unknown 0 1.818 −0.36 0.10 3.00 × 10−4 --- rs7188880 RFWD3 16 74664810 A/T Synonymous Unknown 1 10.78 −0.09 0.02 3.01 × 10−4 --- Chr = Chromosome; REF = Reference allele; ALT = Alternative allele; CADD = Combined Annotation Dependent Depletion; β = effect of the minor allele; SE = Standard Error. *Position according to Hg19. **Seattleseq Annotation Database 138. †Order of cohorts in direction column: ERF, RS, BHF-FHS; Direction of effect represented by −(negative association) + (positive association) or ? (not available). Mean age is lowest in the ERF study (49 years), highest in the RS (75 years) and mean age in the BHF-FHS is in between (58 years). As LTL decreases significantly with age (Lindsey et al., 1991; Slagboom et al., 1994; Blackburn, 2001) and is associated with mortality (Cawthon et al., 2003; Kimura et al., 2008; Fitzpatrick et al., 2011; Honig et al., 2012; Deelen et al., 2014; Rode et al., 2015; Marioni et al., 2016), the variation in LTL becomes less with the aging populations. Mean LTL values were comparable between studies after adjusting LTL values for age and sex. To further standardize across the three cohorts, a z-transformation of the LTL values was performed resulting in comparable distributions with mean of zero and standard deviation of one. Nevertheless, these differences between studies, together with the small sample size of the BHF-FHS cohort, could potentially explain the lack of replication. DISCUSSION The fourth limitation is that we were unable to calculate the effect of the variants on telomere length in base pairs because of the quantification method of LTL in our study together with the z-transformation that was applied to the LTL values to standardize across the cohorts. recent genetically isolated community in the Southwest of the Netherlands, studied as part of the Genetic Research in Isolated Population program (Pardo et al., 2005). All ERF participants are descendants of 22 founder couples who had at least six children baptized in the community church in the 18th century, or their spouses. Baseline data collection, including blood withdrawal, took place between 2002 and 2005. As the ERF population shows a low rate of immigration and a high level of inbreeding, the frequency of several rare alleles is increased in this population (Pardo et al., 2005). The ERF study was approved by the Medical Ethics Commitee of the Erasmus Medical Center (MC), Rotterdam, Netherlands. All participants provided written informed consents and all investigations were carried out in accordance with the Declaration of Helsinki. The replication cohorts included participants from the Rotterdam Study (RS) and the British Heart Foundation Family Heart Study (BHF-FHS). The RS is a prospective cohort study ongoing since 1990 in the well-defined Ommoord district in Rotterdam, The Netherlands. The original RS cohort (RS-I) included 7,983 individuals of 55 years of age or over. At baseline, participants were interviewed at home and had an extensive set of examinations, which were repeated every 3–4 years (Ikram et al., 2017). The Rotterdam Study has been approved by the Medical Ethics Committee of the Erasmus MC and by the Dutch Ministry of Health, Welfare and Sport. The Rotterdam Study has been entered into the Netherlands National Trial Register (NTR; www.trialregister.nl) and into the WHO International Clinical Trials Registry Platform (ICTRP; www.who.int/ictrp/ network/primary/en/) under shared catalogue number NTR6831. All participants provided written informed consent to participate in the study and to have their information obtained from treating physicians. To conclude, this first study using WES data to search for rare genetic variants associated with LTL has identified interesting variants and genes associated with shorter LTL. Eight out of nine rare variants associated with LTL are located on chromosome 11q22.3 and all variants segregate within an ERF family. DISCUSSION As we were not able to replicate findings, future studies should further investigate this region and the other genes identified in this study to confirm their involvement in telomere length regulation. Telomere Length g Mean LTL values in the ERF study and the BHF-FHS study were measured using a qPCR method in all samples (Cawthon, 2002). The measurements were performed in Leicester, United Kingdom, and details of the measurements were previously described (Codd et al., 2010, 2013). In summary, mean LTL was measured in leukocytes and expressed as the ratio (T/S ratio) of telomere repeat length (T) to the copy number of a single-copy gene, 36B4 (S). Samples were quantified relative to a calibrator sample used on each run (DNA from the K562 cell line) (Codd et al., 2010). In the ERF study, we calculated the pairwise LD (r2 and D′) between the top eight variants on chromosome 11 that were significantly associated with LTL using the –ld command of PLINK 1.9 software (Chang et al., 2015) (www.cog- genomics.org/plink/1.9/). Additionally, we performed an inverse- variance weighted meta-analysis using METAL software (Willer et al., 2010). In the analysis using data of the family-based ERF study, we corrected the significance thresholds for multiple testing using Bonferroni correction, resulting in a significance threshold of 1.42 × 10−7 (0.05/353,075). In the replication analysis, the multiple testing corrected p-value thresholds was 0.025 (0.05/2 independent tests). In the meta-analysis, we adjusted for the number of variants tested, resulting in a significance threshold of 3.02 × 10−7 (0.05/165,311). In the RS, mean LTL values were also measured using a qPCR assay based on the method described by Cawthon (2002) with minor modifications. For each sample the telomere and 36B4 assay were run in separate wells but in the same 384 wells PCR plate. Each reaction contained 5 ng DNA, 1 uM of each of the telomere primers (tel1b-forward: GGTT TGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT, tel2b- reverse: GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTA CCCT) or 250 nM of the 36B4 primers (36B4u-forward: CAG CAAGTGGGAAGGTGTAATCC, 36B4d-reverse: CCCATTCT ATCATCAACGGGTACAA) and 1x Quantifast SYBR green PCR Mastermix (Qiagen). The reactions for both assays were performed in duplicate for each sample in a 7900HT machine (Applied Biosystems). Ct values and PCR efficiencies were calculated per plate using the MINER algorithm (Zhao and Fernald, 2005). Duplicate Ct values that had a Coefficient of Variance (CV) of more than 1% were excluded from further analysis. Using the average Ct value per sample and the average PCR efficiency per plate the samples were quantified using the formula Q = 1/(1 + PCR eff)ˆCt. DATA AVAILABILITY STATEMENT The datasets analyzed for each individual cohort can be requested by contacting the responsible Principal Investigator. Because of restrictions based on privacy regulations and informed consent of the participants, data cannot be made freely available in a public repository. For the Rotterdam Study data, requests should be directed toward the management team of the Rotterdam Study (secretariat.epi@erasmusmc.nl), which has a protocol for approving data requests. Exome Sequencing The exomes of 1,336 ERF participants were sequenced at the Erasmus Center for Biomics of the department of Cell Biology, Erasmus MC, The Netherlands. The exomes of a randomly selected subset of 2,628 individuals from the RS-I population were sequenced at the Human Genotyping facility of the Department of Internal Medicine, Erasmus MC, The Netherlands. Details of the methods and quality control for ERF and RS are described elsewhere (Amin et al., 2016; van Rooij et al., 2017). In total, 1,303 ERF participants and 1,257 RS participants had both exome sequence and LTL data available and were included in this analysis. A subset of the BHF-FHS, comprising of 674 unrelated individuals of Caucasian ancestry who had previously undergone exome sequencing as part of the Leicester Myocardial Infarction Study (Khera et al., 2017) and had LTL data available (Codd et al., 2010) were included in this analysis. Telomere Length The relative telomere length was calculated by dividing the Q of the telomere assay by the Q of the 36B4 assay. To validate the assay 96 random samples were run twice and the CV of that experiment was 4.5%. ETHICS STATEMENT The studies involving human participants were reviewed and approved by their respective Medical Ethics Boards and all investigations were carried out in accordance with the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study. Statistical Analyses Statistical Analyses For each individual cohort quantitative trait association analysis was performed using Rare Variant tests (RVtests) software, which supports the analysis of related individuals (Zhan et al., 2016). Association analysis was performed using a score test, assuming an additive model, suitable for analysis with related and unrelated individuals. We applied a z-transformation of LTL values for the three cohorts separately to standardize values across cohorts. All analyses were adjusted for age, sex and batch effects (if needed). Furthermore, we adjusted for familial relationships in ERF using the kinship matrix estimated from the genotyped data, while in the RS we corrected for the first four principal components as the fourth principal component was significantly associated with LTL. Only variants with a minor allele count ≥5 were included. Study Populations The British Heart Foundation Family Heart study recruited families with at least two siblings diagnosed with premature (<66 years) coronary artery disease (CAD) within the Our discovery population consisted of participants from the family-based Erasmus Rucphen Family (ERF) study. The ERF study comprises approximately 3,000 inhabitants of a April 2020 | Volume 11 | Article 337 Frontiers in Genetics | www.frontiersin.org 8 Exome-Wide Association Study of Telomere Length van der Spek et al. United Kingdom between 1998 and 2003. Full details are provided elsewhere (Samani et al., 2005, 2007). United Kingdom between 1998 and 2003. Full details are provided elsewhere (Samani et al., 2005, 2007). REFERENCES Blackburn, E. H. (2000). Telomere states and cell fates. Nature 408, 53–56. doi: 10.1038/35040500 Allsopp, R. C., Vaziri, H., Patterson, C., Goldstein, S., Younglai, E. V., Futcher, A. B., et al. (1992). Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 89, 10114–10118. doi: 10.1073/pnas.89.21.10114 Blackburn, E. H. (2001). Switching and signaling at the telomere. Cell 106, 661–673. doi: 10.1016/s0092-8674(01)00492-5 Blackburn, E. H., and Gall, J. G. (1978). A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. J. Mol. Biol. 120, 33–53. doi: 10.1016/0022-2836(78)90294-2 Amin, N., Jovanova, O., Adams, H. H., Dehghan, A., Kavousi, M., Vernooij, M. W., et al. (2016). Exome-sequencing in a large population-based study reveals a rare Asn396Ser variant in the LIPG gene associated with depressive symptoms. Mol. Psychiatry 22, 537–543. doi: 10.1038/mp.2016.101 Biol. 120, 33–53. doi: 10.1016/0022-2836(78)90294-2 Bojesen, S. E., Pooley, K. A., Johnatty, S. E., Beesley, J., Michailidou, K., Tyrer, J. P., et al. (2013). Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer. Nat. Genet. 45, 371–384. 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Biol. 23, 842–846. doi: 10.1161/01.atv.0000067426. 96344.32 Benetos, A., Gardner, J. P., Zureik, M., Labat, C., Xiaobin, L., Adamopoulos, C., et al. (2004). Short telomeres are associated with increased carotid atherosclerosis in hypertensive subjects. Hypertension 43, 182–185. doi: 10. 1161/01.hyp.0000113081.42868.f4 Brouilette, S. W., Moore, J. S., Mcmahon, A. D., Thompson, J. R., Ford, I., Shepherd, J., et al. (2007). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene. 2020.00337/full#supplementary-material ACKNOWLEDGMENTS ERF study: we are grateful to all study participants and their relatives, general practitioners, and neurologists for their contributions and to P. Veraart for her help in genealogy, J. Vergeer for the supervision of the laboratory work and P. Snijders for his help in data collection. Rotterdam Study: we thank Mr. Pascal Arp, Ms. Mila Jhamai, and Mr. Marijn Verkerk for their help in creating the RS-Exome Sequencing database. We are grateful to the study participants, the stafffrom the Rotterdam Study and the participating general practitioners and pharmacists. AUTHOR CONTRIBUTIONS AS, NA, and CD designed the study and wrote the manuscript. AS, SW, CN, and SA performed the analyses. LB, PA, RB, MD, MH, JR, RK, and NA were involved in the data collection and April 2020 | Volume 11 | Article 337 Frontiers in Genetics | www.frontiersin.org 9 Exome-Wide Association Study of Telomere Length van der Spek et al. www.bbmri.nl; project number CP2010-41). The Rotterdam Study was funded by Erasmus Medical Center and Erasmus University, Rotterdam, Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. The BHF-FHS study was funded by the British Heart Foundation (BHF). Exome sequencing of the Leicester Myocardial Infarction Study was supported by grant 5U54HG003067 from the National Institutes of Health. CN and NS were supported by the BHF. NS is a NIHR Senior Investigator. provided the data. WI, NS, MI, AU, and CD were involved in the supervision of individual cohorts. AS, SW, CN, DV, NS, VC, NA, and CD interpreted the results. All authors critically reviewed and approved the manuscript. provided the data. WI, NS, MI, AU, and CD were involved in the supervision of individual cohorts. AS, SW, CN, DV, NS, VC, NA, and CD interpreted the results. All authors critically reviewed and approved the manuscript. FUNDING The ERF study as a part of EUROSPAN (European Special Populations Research Network) was supported by European Commission FP6 STRP grant number 018947 (LSHG-CT- 2006-01947) and also received funding from the European Community’s Seventh Framework Programme (FP7/2007- 2013)/grant agreement HEALTH-F4-2007-201413 by the European Commission under the programme “Quality of Life and Management of the Living Resources” of 5th Framework Programme (No. QLG2-CT-2002-01254). High-throughput analysis of the ERF data was supported by joint grant from Netherlands Organization for Scientific Research and the Russian Foundation for Basic Research (NWO-RFBR 047.017.043). Exome sequencing analysis in ERF was supported by the ZonMw grant (project 91111025). AS and CD have used exchange grants from Personalized pREvention of Chronic DIseases consortium (PRECeDI) (H2020-MSCA-RISE-2014). The generation and management of the exome sequencing data for the Rotterdam Study was executed by the Human Genotyping Facility of the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, the Netherlands. The Exome Sequencing data set was funded by the Netherlands Genomics Initiative (NGI)/Netherlands Organisation for Scientific Research (NWO) sponsored Netherlands Consortium for Healthy Aging (NCHA; project nr. 050-060-810), by the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, and by the and by a Complementation Project of the Biobanking and Biomolecular Research Infrastructure Netherlands (BBMRI-NL; REFERENCES B., Hindorff, L. A., Hunter, D. J., et al. (2009). Finding the missing heritability of complex diseases. Nature 461, 747–753. Deelen, J., Beekman, M., Codd, V., Trompet, S., Broer, L., Hagg, S., et al. 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Cell Rep. 13, 1633–1646. doi: 10.1016/j.celrep.2015.10.041 Frontiers in Genetics | www.frontiersin.org Copyright © 2020 van der Spek, Warner, Broer, Nelson, Vojinovic, Ahmad, Arp, Brouwer, Denniff, van den Hout, van Rooij, Kraaij, van IJcken, Samani, Ikram, Uitterlinden, Codd, Amin and van Duijn. 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. April 2020 | Volume 11 | Article 337 Conflict of Interest: AS is an employee of the company SkylineDx. Conflict of Interest: AS is an employee of the company SkylineDx. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. van Rooij, J. G. J., Jhamai, M., Arp, P. P., Nouwens, S. C. A., Verkerk, M., Hofman, A., et al. (2017). Population-specific genetic variation in large sequencing data sets: why more data is still better. Eur. J. Hum. Genet. 25, 1173–1175. doi: 10.1038/ejhg.2017.110 Copyright © 2020 van der Spek, Warner, Broer, Nelson, Vojinovic, Ahmad, Arp, Brouwer, Denniff, van den Hout, van Rooij, Kraaij, van IJcken, Samani, Ikram, Uitterlinden, Codd, Amin and van Duijn. 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. Copyright © 2020 van der Spek, Warner, Broer, Nelson, Vojinovic, Ahmad, Arp, Brouwer, Denniff, van den Hout, van Rooij, Kraaij, van IJcken, Samani, Ikram, Uitterlinden, Codd, Amin and van Duijn. 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. Vasa-Nicotera, M., Brouilette, S., Mangino, M., Thompson, J. R., Braund, P., Clemitson, J. R., et al. (2005). Mapping of a major locus that determines telomere length in humans. Am. J. Hum. Genet. 76, 147–151. doi: 10.1086/ 426734 Willeit, P., Willeit, J., Brandstatter, A., Ehrlenbach, S., Mayr, A., Gasperi, A., et al. (2010a). Cellular aging reflected by leukocyte telomere length predicts advanced atherosclerosis and cardiovascular disease risk. Arterioscler. Thromb. Vasc. Biol. 30, 1649–1656. doi: 10.1161/atvbaha.110.205492 April 2020 | Volume 11 | Article 337 Frontiers in Genetics | www.frontiersin.org 12
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A new measurement on <sup>56</sup>Fe(n,inl) using GAINS@GELINA
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Alexandru Negret1,∗, Catalin Borcea1, Marian Boromiza1, François Claeys2, Philippe Dessagne2, Cristiano Fontana3, Greg Henning2, Nasser Kalantar-Nayestanaki4, Myroslav Kavatsyuk4, Maelle Kerveno2, Markus Nyman5, Adina Olacel1, Andreea Oprea3, Carlos Paradela3, and Arjan Plompen3 1Horia Hulubei National Institute for Physics and Nuclear Engineering, M˘agurele, Romania 2Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg, France 3European Commission, Joint Research Centre, Geel, Belgium 4University of Groningen, The Netherlands 5University of Helsinki, Finland 1Horia Hulubei National Institute for Physics and Nuclear Engineering, M˘agurele, Romania 2Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg, France 3European Commission, Joint Research Centre, Geel, Belgium 4University of Groningen, The Netherlands 5University of Helsinki, Finland 1Horia Hulubei National Institute for Physics and Nuclear Engineering, M˘agurele, Romania 2Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg, France 3European Commission, Joint Research Centre, Geel, Belgium 4University of Groningen, The Netherlands 5University of Helsinki, Finland Abstract. The extended dataset of 56Fe(n,n’γ) cross sections measured by our group more than a decade ago at GELINA (Geel Linear Accelerator) was used in many recent evaluations like ENDF, JEFF and CIELO. De- spite the special measures we took to ensure reliability and accuracy, concerns were raised by various groups with regard to several features of this dataset (absolute normalization and/or shape) and therefore the 56Fe(n,inl) cross section is still under the evaluation by the International Nuclear Data Evaluation Network (INDEN). Con- sequently, a new experiment is now under preparation aiming to take advantage of the numerous experimental improvements of the GAINS (Gamma Array for Inelastic Neutron Scattering) setup implemented over the years. While γ spectroscopy combined with the time-of-flight method will remain the main technique involved, several other experimental details will differ substantially. Figure 1. Result of the CIELO evaluation compared with the re- sult of our previous measurement and the result of an experiment performed by R. Nelson et al. [8] at Los Alamos National Labo- ratory that was rescaled. Figure taken from Ref. [9]. ∗e-mail: negret@nipne.ro EPJ Web of Conferences , 01034 (2023) 284 ND2022 EPJ Web of Conferences , 01034 (2023) 284 ND2022 https://doi.org/10.1051/epjconf/202328401034 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http ://creativecommons.org/licenses/by/4.0/). s P Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4.0/). A new measurement on 56Fe(n,inl) using GAINS@GELINA Alexandru Negret1,∗, Catalin Borcea1, Marian Boromiza1, François Claeys2, Philippe Dessagne2, Cristiano Fontana3, Greg Henning2, Nasser Kalantar-Nayestanaki4, Myroslav Kavatsyuk4, Maelle Kerveno2, Markus Nyman5, Adina Olacel1, Andreea Oprea3, Carlos Paradela3, and Arjan Plompen3 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http ://creativecommons.org/licenses/by/4.0/). s 1 Introduction Neutron leakage in a 70-cm diameter sphere with a Cf source. Discrepancies were linked to the inelastic cross section of 56Fe. Figure taken from Ref. [11], page 11. of emission angles allows a precise integration of the γ- ray angular distribution as explained in Section 4.1.1 from Ref. [13]. The neutron beam is monitored by a fission chamber with multiple layers of 235U. The primary experimental results consist of the γ- production cross sections for the most important transi- tions from the nucleus of interest. In most cases the excep- tional γ-energy resolution specific of the HPGe detectors allows very good separation of the main transitions. The γ- production cross sections are reported as absolute values, but one should keep in mind that the beam is monitored using a fission chamber and, therefore, they are calculated relative to the standard 235U cross section [14]. The neu- tron energy resolution of the setup is influenced by the time resolution of the HPGe detectors (≈10 ns) and the length of the flight path (≈100 m), resulting in ≈3 keV at En=1 MeV. Consequently, the resonant structures of the strongest γ-production cross section are visible in many cases (see fig. 3). Whenever statistics is insufficient and the statistical uncertainties are too high (i.e. larger than 2-3% for the strong transitions), one can combine several time-of-flight channels and sacrifice neutron energy reso- lution in order to increase the statistics per channel. This procedure is generally avoided at small neutron energies where the resonant structures of the cross section are visi- ble (see Fig. 3) but can be safely applied at higher energies. Figure 2. Neutron leakage in a 70-cm diameter sphere with a Cf source. Discrepancies were linked to the inelastic cross section of 56Fe. Figure taken from Ref. [11], page 11. elastic data for En=1-8 MeV. Consequently, the new eval- uation of 56Fe(n,inl) cross sections performed within the INDEN project rejects, at least partially, our data [11]. Until now we did not discover any experimental reason to question the result published in Ref. [3]. However, fol- lowing the same arguments as the INDEN evaluators, we consider that a new measurement of the 56Fe(n,inl) cross sections using the GAINS setup at GELINA is required. 1 Introduction Iron represents, arguably, the most important material used in the structure of any nuclear facility. Therefore, a pre- cise knowledge of all cross sections of neutron-induced reactions on iron isotopes is essential for the design of nuclear installations. Iron has four stable isotopes: 54Fe [natural abundancy 5.85(11)%], 56Fe [91.75(11)%], 57Fe [2.12(3)%], and 58Fe [0.28(1)%] [1]. Over the last decade our group published the neutron inelastic cross sections of 54Fe [2], 56Fe [3] and 57Fe [4] for incident energies from ≈70 keV to ≈18 MeV. The mea- surement on the major isotope 56Fe was performed in 2007 using the GAINS (Gamma Array for Inelastic Neutron Scattering) spectrometer at the GELINA (Geel Linear Ac- celerator) neutron source and published a few years later in Ref. [3]. Out of these three experiments it was the only one using a natural target. It allowed the direct measure- ment of 20 γ-production cross sections and, using the eval- uated level scheme [5], the computation of 10 level cross sections and of the total inelastic cross section. Figure 1. Result of the CIELO evaluation compared with the re- sult of our previous measurement and the result of an experiment performed by R. Nelson et al. [8] at Los Alamos National Labo- ratory that was rescaled. Figure taken from Ref. [9]. The results of the CIELO evaluation on iron iso- topes, also adopted by ENDF/B-VIII.0, were published in Ref. [9]. It overlaps well with our experimental result for a large energy range. Fig. 1, taken from Ref. [9], displays this result. Two major evaluation projects were implemented after the publications of our results on the inelstic cross sections of 56Fe: CIELO (Collaborative International Evaluated Li- brary Organisation) [6] followed by INDEN (International Nuclear Data Evaluation Network) [7]. In both cases the neutron inelastic cross section of 56Fe were considered es- sential quantities for the development of a new generation of nuclear facilities and were re-evaluated. Nevertheless, within the next important evaluation project, INDEN, it was noted that the 56Fe evaluation from ENDF/B-VIII.0 does not perform well when it is bench- marked against certain integral measurements [10]. In par- ticular, the discrepancies observed in an iron sphere leak- age spectrum shown in Fig. 2 were traced back to the in- https://doi.org/10.1051/epjconf/202328401034 EPJ Web of Conferences , 01034 (2023) 284 EPJ Web of Conferences , 01034 (2023) 284 ND2022 Figure 2. 1 Introduction In the present paper we emphasize the differences and simi- larities between the current setup and the one previously used arguing that the upgrades already implemented or that are now under consideration are significant and will probably allow us to identify any possible hidden issue of our previous measurement. 56Fe(n,n' 846.8 keV) (b) En (keV) 900 1 2 0 1000 1100 Figure 3. 846.8-keV γ-production cross section from 56Fe ob- tained in our previous measurement. A limited energy range is displayed to show the resonant structures visible due to the good resolution. 56Fe(n,n' 846.8 keV) (b) En (keV) 900 1 2 0 1000 1100 2 Experimental setup and technique The experimental setup consists of the neutron source and time-of-flight facility GELINA and the HPGe array GAINS operated by the European Commission’s Joint Re- search Centre in Geel, Belgium. These experimental facil- ities were used together for more than 15 years generating a significant quantity of high precision neutron inelastic data [12]. Figure 3. 846.8-keV γ-production cross section from 56Fe ob- tained in our previous measurement. A limited energy range is displayed to show the resonant structures visible due to the good resolution. GELINA consists of a high intensity electron acceler- ator (140 MeV) operated in a pulsed mode at 400 Hz, a depleted uranium target and several flight paths. GAINS is an array of 12 high volume HPGe detectors currently in- stalled at the end of a 100-m flight path of GELINA. The detectors are placed at backward angles of 110◦, 125◦and 150◦with respect to the incoming neutron beam. They point to a sample that usually has a thickness of 1-3 mm and a diameter larger than the neutron beam that is colli- mated to 61 mm. The total efficiency of GAINS is around 2% for 1.3-MeV γ rays. A more detailed description of GAINS, including a picture of the current setup can be found in Ref. [12]. Several corrections are applied using Monte Carlo sim- ulations, including the multiple-scattering correction and the correction for the detection efficiency from the ex- tended sample. The final step in data analysis employs an external nu- clear structure database - namely the ENSDF (Evaluated Nuclear structure Data File) [15] - to generate transition probabilities from the γ-production cross section (i.e. cor- recting for the internal conversion when necessary) and further, using the branching ratios, to generate the total inelastic and the level cross sections. These are accurate only over a limited energy range depending on which γ rays from the reaction were observed. In this context it should be noted that the neutron inelastic scattering is a rather non-selective process, populating most of the acces- sible levels of the target nucleus. The experimental method makes use of the very good time resolution of GELINA (≈2 ns) and the long flight path to determine very accurately the neutron energy. The γ rays emitted following inelastic scattering of neutrons are detected by the HPGe detectors. 2 Experimental setup and technique The special choice 2 EPJ Web of Conferences , 01034 (2023) 284 ND2022 https://doi.org/10.1051/epjconf/202328401034 More details on the experimental setup and the data analysis technique are available in Ref. [16]. built using conventional electronics and was delivered to the card only when at least one of the two input channels showed an event in the time range of interest. The system resulted in a counting rate of only 10-15 events/second in each detector. However, recently it became unstable most probably because of the ageing of the electronics. 4 Conclusions The sample used in the previous measurement was composed of three natFe disks, each of them with a thick- ness of 1 mm, a diameter of 80 mm and a purity of 99.5%. For the future measurement an enriched sample (99.77(1)% of 56Fe), procured from the Oak Ridge na- tional Laboratory, will be used. It has a thickness of 1 mm and a diameter of 70 mm. The main conclusion of this report is that a new measure- ment of the neutron inelastic cross section of 56Fe is being proposed using the GAINS spectrometer at GELINA, 15 years after a similar experiment was performed. The main effort is to decouple as much as possible the two measure- ments: although the same general method will be used, many other experimental details will be changed including the target, the data acquisition and possibly the normaliza- tion method. Over the years GAINS was continuously upgraded. An overview of these upgrades (and of the experimental program conducted there) is given in Ref. [12]. Practically all HPGe detectors used in 2007 were replaced or at least reconditioned. Moreover, four extra detectors were added to the system at the scattering angle of 125◦allowing a double calculation (though less precise in terms of angular integration) of the γ-production cross sections. A new sup- porting frame and a new nitrogen filling system are now in place. As a more general remark, we note an important speci- ficity of the nuclear data field: if in general in the scientific environment, and particularly in case of basic research the emphasis is always on world premieres, in case of nuclear data experiments the redundancy and the repetition of cer- tain measurements is an essential tool in our quest for re- liability and precision. The data acquisition system processing the signals from the HPGe detectors of GAINS was based for the last 15 years on Acqiris digitizers running at a sampling rate of 420 MHz and having 12 bits. They can accommodate 2 input channels per card with a common trigger (so, for the 8 detectors used in the previous measurement, 4 cards were used in two dedicated crates). The trigger signal was • A double-normalization method is proposed. Compared to the measurement performed in 2007 when a flight path of 200 m was used, the newly proposed experiment will use a new flight path, GAINS being now installed at 100 m from the neutron source. The accel- erator is now operated at 400 Hz instead of 800 Hz and the whole flight path area was refurbished. Consequently the instantaneous flux on the sample is now higher and its shape may be slightly different. In general these dif- ferences are not essential, but they result in a somewhat different neutron flux which constitutes an advantage for the new proposal. • A new data acquisition system will be implemented, • A double-normalization method is proposed. 3 Main differences between the previous and the newly-proposed measurement Therefore, an upgrade of the data acquisition is cur- rently being implemented in collaboration with University of Groningen. The new system developed there is based on Struck SIS3316-250-14 SADC cards with 16 channels, 250 MHz sampling rate and 14 bits. They benefit from a GBE readout and will be operated using internal trigger- ing with an external gate. The fact that all GAINS de- tectors will be connected to the same module will open the possibility for coincidence measurements eliminating the issue of time synchronization among different mod- ules. Although the counting rate in our experiments is rather low and, therefore, the number of coincident events is also small, this may at least allow various checks for cases when overlapping γ transitions are present in the spectra. In the previous section we gave a short overview of the ex- perimental technique and the main facilities used to mea- sure neutron inelastic cross sections. Most of these fea- tures remain unchanged as they represent the basis of our method: a combination of the γ spectroscopy using HPGe detectors with the time-of-flight technique. However, the newly-proposed measurement of the neutron inelastic cross section of 56Fe will benefit from a number of upgrades that make it significantly different from the one reported in Ref. [3]. The most important changes are the following: • A new flight path is used, • A different sample is irradiated, • GAINS was upgraded, Finally, an additional normalization method was pro- posed. As already discussed, the current setup uses a fis- sion chamber and, therefore, the cross sections are scaled to the 235U(n,f) standard [14]. As this is a powerful and reliable technique, it will be kept during the proposed ex- periment. However, we propose to perform a double check simply by measuring a second sample together with the 56Fe target. As this implies additional corrections and complicates some of the corrections already used, it will probably not be used for the entire duration of data taking. 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Pirovano, A. J. M. Plompen, and C. Rouki, Physical Review C93, 024610 (2016). [7] https://www-nds.iaea.org/INDEN/ [8] R.O. Nelson, N. Fotiades, M. Devlin, J. A. Beckerand, P.E. Garrett, and W. Younes, in Proceedings of the In- ternational Conference on Nuclear Data for Science and Technology - ND2004, September 26 - October 1, 2004, Santa Fe, USA, edited by R. C. Haight,M. B. Chadwick, T. Kawano, and P. Talou, AIP Conf. Proc. No. 769 (AIP, New York, 2005), p. 838. [18] A. Olacel, F. Belloni, C. Borcea, M. Boromiza, P. Dessagne, G. Henning, M. Kerveno, A. Negret, M. Ny- man, E. Pirovano, and A. J. M. Acknowledgements This work is supported by a grant of the Ministry of Research, Innovation and Digitalization, CNCS- UEFISCDI, project number PN-III-P4-PCE-2021-0490, within PNCDI III. 3 EPJ Web of Conferences , 01034 (2023) 284 ND2022 https://doi.org/10.1051/epjconf/202328401034 Mughabghab, Q. Jing, G. Zhigang, L. Tingjin, L. Han- lin, R. Xichao, L. Leal, B.V. Carlson, T. Kawano, M. Sin, S.P. Simakov, and K. Guber, Nuclear Data Sheets 148 214-253 (2018) References Plompen, Physical Re- view C96, 014621 (2017). [9] M. Herman, A. Trkov, R. Capote, G.P.A. Nobre, D.A. Brown, R. Arcilla, Y. Danon, A. Plompen, S.F. 4
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Inhibiting efferocytosis reverses macrophage-mediated immunosuppression in the leukemia microenvironment
Frontiers in immunology
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OPEN ACCESS EDITED BY Philip Cohen, Temple University, United States REVIEWED BY Eugenio Antonio Carrera Silva, Academia Nacional de Medicina, Argentina Raymond B. Birge, Rutgers, The State University of New Jersey, United States *CORRESPONDENCE Alisa B. Lee-Sherick alisa.b.lee@gmail.com SPECIALTY SECTION This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology RECEIVED 17 January 2023 ACCEPTED 20 February 2023 PUBLISHED 07 March 2023 Joselyn Cruz Cruz 1, Kristen C. Allison 1, Lauren S. Page 1, Alexis J. Jenkins 1, Xiaodong Wang 2, H. Shelton Earp 3, Stephen V. Frye 2, Douglas K. Graham 4, Michael R. Verneris 1 and Alisa B. Lee-Sherick 1* 1Division of Pediatric Hematology, Oncology, and Bone Marrow Transplant, University of Colorado, Aurora, CO, United States, 2Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 3Lineberger Comprehensive Cancer Center, Departments of Medicine and Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 4Department of Pediatrics, Emory University, Atlanta, GA, United States Background: Previous studies show that the spleen and bone marrow can serve as leukemia microenvironments in which macrophages play a significant role in immune evasion and chemoresistance. We hypothesized that the macrophage driven tolerogenic process of efferocytosis is a major contributor to the immunosuppressive leukemia microenvironment and that this was driven by aberrant phosphatidylserine expression from cell turnover and cell membrane dysregulation. CITATION Cruz Cruz J, Allison KC, Page LS, Jenkins AJ, Wang X, Earp HS, Frye SV, Graham DK, Verneris MR and Lee- Sherick AB (2023) Inhibiting efferocytosis reverses macrophage-mediated immunosuppression in the leukemia microenvironment. Front. Immunol. 14:1146721. doi: 10.3389/fimmu.2023.1146721 Methods: Since MerTK is the prototypic efferocytosis receptor, we assessed whether the MerTK inhibitor MRX2843, which is currently in clinical trials, would reverse immune evasion and enhance immune-mediated clearance of leukemia cells. COPYRIGHT © 2023 Cruz Cruz, Allison, Page, Jenkins, Wang, Earp, Frye, Graham, Verneris and Lee- Sherick. 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. TYPE Original Research PUBLISHED 07 March 2023 DOI 10.3389/fimmu.2023.1146721 TYPE Original Research PUBLISHED 07 March 2023 DOI 10.3389/fimmu.2023.1146721 OPEN ACCESS Results: We found that inhibition of MerTK decreased leukemia-associated macrophage expression of M2 markers PD-L1, PD-L2, Tim-3, CD163 and Arginase-1 compared to vehicle-treated controls. Additionally, MerTK inhibition led to M1 macrophage repolarization including elevated CD86 and HLA-DR expression, and increased production of T cell activating cytokines, including IFN-b, IL-18, and IL-1b through activation of NF-kB. Collectively, this macrophage repolarization had downstream effects on T cells within the leukemia microenvironment, including decreased PD-1+Tim-3+ and LAG3+ checkpoint expression, and increased CD69+CD107a+ expression. 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. Discussion: These results demonstrate that MerTK inhibition using MRX2843 altered the leukemia microenvironment from tumor-permissive toward immune responsiveness to leukemia and culminated in improved immune-mediated clearance of AML. MERTK, Mertk inhibitors, tumor-associated macrophages, efferocytosis, acute myeloid leukemia, leukemia associated macrophages, leukemia microenvironment, tumor micro environment (TME) 01 Frontiers in Immunology frontiersin.org Cruz Cruz et al. Cruz Cruz et al. 10.3389/fimmu.2023.1146721 Murine AML models C57BL/6J, Mertk-null (B6;129-Mertktm1Grl/J), and TCRa-/- (B6.129S2-Tcratm1Mom/J) mice were purchased from Jackson Laboratory and bred in-house. Mertk-null mice were backcrossed with C57BL/6J mice for ≥12 generations (Mertk–/–). Mertk genotype was verified prior to use; control mice were MerTK-wildtype littermates (MertkWT) with identical backcrossing. LysM-Cre (Lyz2tm1(cre/ERT2)Grtn/J) mice were obtained from Jackson Laboratory and crossed with MerTK-loxp mice (gift from Carla Rothlin); after genotype verification, mice were treated with tamoxifen as previously described (20). Mice were inoculated with AML cells via tail vein at 6-8 weeks of life. For in vivo studies, MRX2843 was dissolved in phosphate buffered saline (PBS). Beginning three days after leukemia inoculation, MRX2843 (60mg/kg) or PBS was administered daily via oral gavage. Once mice developed advanced leukemia (>20% weight loss, hind-limb paralysis, inactivity) they were euthanized, and duration of survival was recorded. Alternatively, three weeks after starting MRX2843, mice were euthanized; peripheral blood, BM and spleen were harvested for analysis. Animal experiments were conducted in accordance with University of Colorado IACUC regulatory standards. Previously, we identified MerTK as a cell intrinsic therapeutic target, given that its expression on leukemia cells confers a survival advantage and chemoresistance in vitro and in vivo (16–19). These discoveries led to the development of type 1 MerTK small molecule inhibitors, including MRX2843, which is currently being tested in early phase clinical trials (NCT03510104, NCT04872478, NCT04762199) for its inhibitory effects on MerTK and FLT3. Using MRX2843 to block efferocytosis in syngeneic murine models of acute myeloid leukemia (AML) and using human samples in vitro, we demonstrate that MRX2843 skews M2-like macrophages toward M1 polarization. The resultant altered leukemia microenvironment led to activation of T cells, which lack MerTK. The combined effect on LAMs and the surrounding leukemia microenvironment led to AML clearance in vivo. These results were independent of MerTK inhibition on AML cells, given that the AML cell lines used in these experiments expressed little to no MerTK. Thus, targeting LAM-associated MerTK, using MRX2843 could have a novel use: repolarizing the leukemia microenvironment through blocking efferocytosis. Cell culture Efferocytosis is a tolerogenic process where macrophages phagocytose apoptotic cells, mediated by the binding of phosphatidylserine (Ptdser) on apoptotic cells to MerTK (or other efferocytic receptors) on macrophages via bridging molecules such as Gas6 and Pros1. Efferocytosis inhibits T cell activation and limits tissue damage in the resolution of inflammation/injury by removing self- antigen and secretion of T cell suppressive cytokines. However, Ptdser surface expression – which is usually tightly regulated (i.e., retained on the inner plasma membrane) in healthy cells – becomes externalized during apoptosis or with rapid cell turnover, such as in tumorigenesis (1). In fact, several studies have demonstrated that viable non-apoptotic cancer cells express high levels of PtdSer due to dysregulation of calcium-dependent flippase activity (2–4). Specifically, high levels of PtdSer are often expressed on acute leukemia cells (4, 5). Murine AML cell line harboring a t(11;19) KMT2A-MLLT1 translocation (“MLL-ENL”), and primary murine AML harboring a t(11;19) KMT2A-MLLT3 translocation (“MLL-AF9”) were gifts from Deb DeRyckere. C1498 and Kasumi-1 were purchased from ATCC. Kasumi-1 cell identity was confirmed using short tandem repeat microsatellite loci analysis. Cell lines were maintained in RPMI medium plus 10% FBS and penicillin/streptomycin (cRPMI). MLL-AF9 cells were thawed and used immediately; they did not survive in vitro culture. For in vitro studies, MRX2843 was dissolved in dimethyl sulfoxide (DMSO; Sigma); vehicle controls were administered the equivalent volume of DMSO. It has been proposed that efferocytosis in the solid tumor microenvironment skews tumor-associated macrophages (TAMs) toward a M2-like polarization and subverts T cell responses, aiding in tumor growth (6–9) and metastatic spread (10). Tumor- permissive macrophages have been identified within solid tumors and more recently in the spleen and bone marrow (BM) – creating a leukemia tumor microenvironment (11, 12). These leukemia- associated macrophages (LAMs) are phenotypically similar to wound healing (M2) macrophages and have been implicated in immune evasion, chemotherapy resistance, and extramedullary spread of leukemia (12–15). Due to the basal overexpression of PtdSer on leukemia cells, in addition to the significant cell turnover caused by rapid and ineffective hematopoiesis of leukemic blasts, the leukemia microenvironment contains abundant efferocytosis signals. Given that LAMs express the prototypic efferocytic receptor MerTK, we sought to evaluate whether targeting MerTK-dependent efferocytosis by LAMs would diminish leukemia growth through skewing of the leukemia microenvironment using a novel clinical grade small molecule tyrosine kinase, MRX2843. Cytokine analysis TC treated plates for three to seven days in cRPMI supplemented with 25ng/mL GM-CSF and 5ng/mL M-CSF, after which non- adherent cells were discarded. PtdSer expression on AML cell lines was induced via UV light exposure (15 minutes), followed by incubation at 37° for two to four hours, and then opsonized with 250nM recombinant mGAS6 (R&D Systems). Macrophages were pretreated with 300nM MRX2843 or vehicle for one hour in 24 well plates before AML cells were added (1.0×106 cells/well). After 48 hours, non-adherent AML cells in the supernatant were removed; macrophages were washed with PBS thrice to remove residual AML cells. TC treated plates for three to seven days in cRPMI supplemented with 25ng/mL GM-CSF and 5ng/mL M-CSF, after which non- adherent cells were discarded. PtdSer expression on AML cell lines was induced via UV light exposure (15 minutes), followed by incubation at 37° for two to four hours, and then opsonized with 250nM recombinant mGAS6 (R&D Systems). Macrophages were pretreated with 300nM MRX2843 or vehicle for one hour in 24 well plates before AML cells were added (1.0×106 cells/well). After 48 hours, non-adherent AML cells in the supernatant were removed; macrophages were washed with PBS thrice to remove residual AML cells. Peripheral serum collected from in vivo assays was analyzed for cytokine concentrations with two independent ProcartaPlex immunoassays using the Luminex MAGPIX Instrument System (Luminex). Inhibition of MerTK in leukemia-associated macrophages improves survival in leukemic mice To evaluate MerTK inhibition on leukemia-associated macrophages (LAMs) in vivo without the confounding factor of cell intrinsic effects, we utilized murine AML cell lines and primary murine AML which had very little or no MerTK expression compared to splenic macrophages (Figure 1A). Flow cytometry Harvested cells were treated with Fixation/Permeabilization Kit reagent (BD Biosciences). Immune cell characterization was quantitated on a Gallios (Beckman Coulter) flow cytometer and analyzed using Kaluza software, or a Cytec Aurora (Cytec) spectral flow cytometer and analyzed using FCS Express software (DeNovo Software). Antibodies listed in Supplemental Table 1 were used according to manufacturer recommendations. Leukemia was allowed to establish in syngeneic immunocompetent C57BL/6 mice for three days, and then treatment with MRX2843, a highly specific small molecule MerTK tyrosine kinase inhibitor (MerTKI), was initiated via daily oral gavage at 60mg/kg – a dose that is known to inhibit MerTK in vivo, and has minimal toxic effects (21). Mice were monitored daily and euthanized once they developed symptoms of leukemia. In mice inoculated with MLL-ENL cells, treatment with the MerTKI was associated with a significant median survival prolongation compared to vehicle (40 vs 17 days, p<0.0001) (Figure 1B). These results were validated in identical survival studies using C1498 AML (a cell line with very rapid leukemia progression) and MLL-AF9 AML (a primary mouse AML). In mice with C1498 AML, MRX2843-treated mice survived longer compared to vehicle- treated mice (28 vs 20 days; p<0.0001) (Supplemental Figure 1A). Similarly in mice with MLL-AF9 AML, mice treated with MRX2843 demonstrated prolongation of survival (41 vs 29 days, p<0.0001) (Figure 1C). Macrophage and AML co-cultures Bone marrow derived macrophages (BMDM) were derived from isolated monocytes from the marrow of C57BL/6 mice using EasySep Mouse CD11b Positive Selection Kit II (Stemcell Technologies). Alternatively, human monocytes were isolated from discarded de-identified blood donation leukopaks from the Children’s Hospital Colorado Blood Donor Center, after written informed consent in accordance with the Declaration of Helsinki. Leukopak peripheral blood mononuclear cells (PBMCs), obtained using Lymphoprep (Stemcell Technologies), underwent CD14+ isolation using MojoSort™Human CD14 selection kit (Biolegend). Monocytes were matured to macrophages on non- Frontiers in Immunology 02 frontiersin.org Cruz Cruz et al. 10.3389/fimmu.2023.1146721 10.3389/fimmu.2023.1146721 Immunoblot analysis Whole cell lysates were prepared after two to six hours of co- culture, and proteins were resolved as previously described (18), probed with antibodies in Supplemental Table 1 according to manufacturer recommendations and visualized by horseradish peroxidase chemiluminescence (Perkin-Elmer). Statistics Statistical analyses were performed using GraphPad Prism software (v6.05). One-way ANOVA corrected with Bonferroni’s multiple comparisons test was used for experiments with three treatment groups. Student’s t-test was used when two samples were analyzed. Results indicate the mean values and were considered significant when p<0.05. Mixed leukocyte reaction Flat-bottom 96-well plates (Corning) were coated with 10 µg/ mL anti-CD3e (clone OKT3; Thermo Fisher Scientific) in sterile PBS overnight at 4°C and then washed to remove unbound antibody. T cells were isolated from PBMCs using EasySep™ Human T Cell negative selection kit (Stemcell Technologies) and then cultured on the anti-CD3e coated plates (2x105 cells/well) in cRPMI media. Primed T cells were collected after 48 hours and added to macrophage/leukemia co-cultures in equal proportions (1:1:1 ratio) for 48 hours at 37˚C. Frontiers in Immunology Real-time quantitative RT-PCR We hypothesized that the extended survival associated with MRX2843 treatment was specific to MerTK-expressing macrophages in vivo. Previous research has demonstrated that MerTK is not expressed in murine (22, 23) or human T cell subsets (24–26), however one study demonstrated that hyper- activated T cells can express MerTK in highly over-activated conditions after prolonged stimulation with anti-CD3/CD28 beads (27). Therefore, we assessed MerTK expression on CD4+ and CD8+ T cells in leukemic mice treated with vehicle or After 12 hours of macrophage and AML cell co-culture, RNA was isolated from harvested macrophages using RNeasy Plus Mini Kit (Qiagen). Real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis was performed using TaqMan Universal PCR Master Mix with primers in Supplemental Table 1. Threshold cycle values were normalized to the GAPDH RNA internal control, and analysis was performed by comparing MRX2843 to vehicle values. Frontiers in Immunology 03 frontiersin.org 10.3389/fimmu.2023.1146721 Cruz Cruz et al. D A B E F G I H J K C FIGURE 1 Inhibition of MerTK on leukemia-associated macrophages increases survival in syngeneic murine models of AML. (A) MerTK expression in murine AML cell lines was determined by immunoblot. Splenocytes from a Mertk wild-type (WT splenocytes) and Mertk-null (Mertk-/- splenocytes) mouse were used as positive and negative controls, respectively. Actin is shown as a loading control. (B, C) Syngeneic AML cells were inoculated into C57BL/6 mice by tail vein injection. After three days, mice began daily treatment with MerTK inhibitor MRX2843 (60mg/kg; red line) or an equivalent volume of vehicle (saline; black line). (B) Mice inoculated with 1x105 MLL-ENL cells (vehicle n =21, MRX2843 n =18; three independent replicates). (C) Mice inoculated with 1x105 MLL-AF9 cells (vehicle n = 10, MRX2943 n = 9; two independent replicates). (D) Peripheral blood T cells collected from mice 7 days and 14 days after starting treatment was analyzed by flow cytometry for MerTK expression on CD3+ T cells. (E) 1x105 MLL-ENL AML cells were inoculated into Mertk-/- mice (blue line) or littermate wild-type controls (MerTKWT; black line) by tail vein injection (MerTKWT n =7, Mertk-/- n =8; two independent replicates). (F) MLL-ENL AML cells were inoculated into mice by tail vein injection after genotype confirmation of MerTK knock-down in dual MerTK and LysM expressing macrophages (i.e. Mertk-loxp+/+ Cre+/- or +/+, n = 19). Control littermate mice (Mertk-loxp-/- Cre-/-, n = 19) did not have MerTK knockdown (two independent replicates). Real-time quantitative RT-PCR (B–F) Weight loss and health score were monitored as surrogates for disease burden; Kaplan-Meier survival analysis measured leukemia-free survival. Analyzed using log-rank test, comparing MerTK inhibited (MRX2843, Mertk-/-, Mertk-loxp+/+ Cre+) to controls (vehicle, MerTKWT, Mertk-loxp-/- Cre-/-). (G) Diagram of the murine model used for flow cytometric evaluation in this manuscript. (H-K) C57BL/6 mice inoculated with MLL-ENL AML and treated daily with MerTK inhibitor MRX2843 (red triangles) or vehicle (saline; black circles); mice not inoculated with leukemia were used as a control (open gray circles). (H) Peripheral blood total viable nucleated white blood cell count was quantified by trypan blue exclusion, and (I) percentages of granulocytes, monocytes, B cells, T cells, and NK cells were quantified by flow cytometric analysis. (J) Total viable nucleated cell counts of the spleen and bone marrow were quantified by trypan blue exclusion. (K) Harvested cells were assessed by flow cytometric analysis, and leukemia-associated macrophages (LAMs) within the spleen and marrow were quantified. Analyzed using 1-way ANOVA. (*p<0.05, **p<0.01, NS, not significant). D A B E F G I H C D A B B C C B D E F E F G H H K J J K FIGURE 1 Inhibition of MerTK on leukemia-associated macrophages increases survival in syngeneic murine models of AML. (A) MerTK expression in murine AML cell lines was determined by immunoblot. Splenocytes from a Mertk wild-type (WT splenocytes) and Mertk-null (Mertk-/- splenocytes) mouse were used as positive and negative controls, respectively. Actin is shown as a loading control. (B, C) Syngeneic AML cells were inoculated into C57BL/6 mice by tail vein injection. After three days, mice began daily treatment with MerTK inhibitor MRX2843 (60mg/kg; red line) or an equivalent volume of vehicle (saline; black line). (B) Mice inoculated with 1x105 MLL-ENL cells (vehicle n =21, MRX2843 n =18; three independent replicates). (C) Mice inoculated with 1x105 MLL-AF9 cells (vehicle n = 10, MRX2943 n = 9; two independent replicates). (D) Peripheral blood T cells collected from mice 7 days and 14 days after starting treatment was analyzed by flow cytometry for MerTK expression on CD3+ T cells. (E) 1x105 MLL-ENL AML cells were inoculated into Mertk-/- mice (blue line) or littermate wild-type controls (MerTKWT; black line) by tail vein injection (MerTKWT n =7, Mertk-/- n =8; two independent replicates). Frontiers in Immunology MerTK inhibition skews LAM polarization We previously reported an effect of MerTK inhibition on PD-L1 and PD-L2 expression in acute lymphoblastic leukemia (ALL) (21), however, given the vast differences between these diseases and the microenvironments they create within the spleen and BM, we evaluated LAM PD-L1 and PD-L2 expression in mice inoculated with MLL-ENL AML treated with MRX2843 (or vehicle), and non- leukemic mice (control). The leukemia microenvironment contained significantly more PD-L1 (spleen: p<0.01; BM: p<0.01) and PD-L2 (spleen: p<0.001; BM: p<0.01) expressing LAMs in vehicle-treated mice compared to MRX2843-treated mice (Figures 2A, B; Table 1; Supplemental Figure 2A). When evaluating the median fluorescence intensity of PD-L1 and PD-L2 expression on LAMs, we observed an up-field shift in the PD-L1 curve indicating globally increased expression on all LAMs (Supplemental Figure 2B), whereas PD-L2 upregulation occurred in a discrete subpopulation of LAMs (Supplemental Figure 2C). Similar PD-L1 and PD-L2 changes were observed in mice with C1498 AML treated with MRX2843 or vehicle (Supplemental Figures 2D, E). We next sought to evaluate whether the survival advantage using MRX2843 correlated with altered white blood cell counts (Figure 1G). We examined peripheral blood cell populations, including total nucleated cell count by trypan blue exclusion, and peripheral white blood cell populations using flow cytometry. There was no difference in the number of total peripheral blood nucleated cells (TNC), or percentages of granulocytes, monocytes, B cells and NK cells (Figure 1H). In MRX2843-treated mice, the percentage of peripheral blood CD3+ T cells (13%) was higher than vehicle- treated mice (6%, p<0.05) (Figures 1I, J). Prior studies using MRX2843 in preclinical models demonstrate no alterations in other blood cell indices (white blood cell count, red blood cell count, or platelet count) (21). To evaluate whether human macrophages are affected similarly in response to MRX2843, donor PBMC-derived macrophages were co- cultured with Kasumi-1 human AML cells for 48 hours. Prior to co- culture, donor macrophages expressed abundant MerTK (Supplemental Figure 2F). Of note, UV-exposed AML cells were positive for PtdSer expression (Annexin-V staining, Supplemental Figure 2G), but demonstrated very little irreversible apoptosis (i.e., propidium iodide uptake). After 48 hours of co-culture, there was broad variation of PD- L1 and PD-L2 expression on vehicle-treated donor macrophages, however MRX2843-treatment significantly decreased expression of PD-L1 by 23% (p<0.01) and PD-L2 by 31% (p<0.05) (Figure 2C). Real-time quantitative RT-PCR (F) MLL-ENL AML cells were inoculated into mice by tail vein injection after genotype confirmation of MerTK knock-down in dual MerTK and LysM expressing macrophages (i.e. Mertk-loxp+/+ Cre+/- or +/+, n = 19). Control littermate mice (Mertk-loxp-/- Cre-/-, n = 19) did not have MerTK knockdown (two independent replicates). (B–F) Weight loss and health score were monitored as surrogates for disease burden; Kaplan-Meier survival analysis measured leukemia-free survival. Analyzed using log-rank test, comparing MerTK inhibited (MRX2843, Mertk-/-, Mertk-loxp+/+ Cre+) to controls (vehicle, MerTKWT, Mertk-loxp-/- Cre-/-). (G) Diagram of the murine model used for flow cytometric evaluation in this manuscript. (H-K) C57BL/6 mice inoculated with MLL-ENL AML and treated daily with MerTK inhibitor MRX2843 (red triangles) or vehicle (saline; black circles); mice not inoculated with leukemia were used as a control (open gray circles). (H) Peripheral blood total viable nucleated white blood cell count was quantified by trypan blue exclusion, and (I) percentages of granulocytes, monocytes, B cells, T cells, and NK cells were quantified by flow cytometric analysis. (J) Total viable nucleated cell counts of the spleen and bone marrow were quantified by trypan blue exclusion. (K) Harvested cells were assessed by flow cytometric analysis, and leukemia-associated macrophages (LAMs) within the spleen and marrow were quantified. Analyzed using 1-way ANOVA. (*p<0.05, **p<0.01, NS, not significant). MRX2843 (and vehicle-treated non-leukemic mice as a control), and did not observe appreciable T cell MerTK expression (Figure 1D). Therefore, we determined that any effects of MRX2843 on T cell number or function were likely through alteration of the leukemia microenvironment, rather than attributable to direct cell intrinsic effects on T cells. To further validate that this survival advantage could be attributable to MerTK inhibition on macrophages in the leukemia Frontiers in Immunology 04 frontiersin.org Cruz Cruz et al. 10.3389/fimmu.2023.1146721 a significant decrease of Gr-1+ monocytes, though there was no difference within the marrow (Table 1; Supplemental Figure 1D). microenvironment, we inoculated MLL-ENL or C1498 cells into mice harboring a homozygous Mertk-knockout mutation (Mertk–/–) or their Mertk-wildtype littermates (MertkWT), and leukemia-free survival was monitored. Extension of survival was even more striking in these studies. None of the Mertk–/– mice inoculated with MLL-ENL AML developed leukemia, whereas MertkWT littermates had a median survival of 25 days (p<0.001, Figure 1E). Real-time quantitative RT-PCR To ensure that the observed effects were due to blockade of MerTK in macrophages rather than an off target effect of MerTK knockout in non-immune cell subsets, or due to concomitant Tyro3 deletion which has recently been described in Mertk–/– mice (28), we performed similar survival studies in conditional knockout model of Mertk-loxp+/+ Cre+ mice, in which MerTK is knocked-down in only dual MerTK and LysM expressing macrophages. Mertk-loxp+/+ Cre+ mice inoculated with MLL-ENL AML demonstrated extension of survival of all but one mouse, compared to a median survival of 35 days in Mertk-loxp-/- Cre-/- mice (p<0.001) (Figure 1F). In the more rapidly progressive C1498 model, median survival of Mertk-/- mice was 40 days, versus only 22 days in littermate control MertkWT mice (p<0.01, Supplemental Figure 1B). Collectively, these results demonstrated that MerTK inhibition of LAMs was sufficient to improve leukemia- free survival in vivo. Frontiers in Immunology MerTK inhibition skews LAM polarization We also evaluated the effect of MRX2843 on myeloid populations in the spleen and BM of mice inoculated with MLL- ENL AML and treated with MRX2843 or vehicle (Figure 1G). Vehicle-treated non-leukemic mice were used for comparison. The total number of nucleated cells in the spleen and BM were similar between the three groups (Figure 1J). Within the myeloid cell lineage, MerTK is commonly expressed on mature macrophages, including leukemia-associated macrophages (LAMs; CD11b+CD11c+Gr-1-), but not on monocytes (CD11b+CD11c-Gr- 1-SideScatterlo), granulocytes (CD11b+CD11c-Gr-1+SideScatterhi), or myeloid-derived suppressor cells/myeloid progenitors (MDSCs; CD11b+CD11c-Gr-1intermediateSideScatterhi) (19). Gr-1+ monocytes (CD11b+CD11c-Gr-1+SideScatterlo) have variable MerTK expression. These myeloid populations (Supplemental Figure 1B) were analyzed for alterations with MRX2843 treatment. LAMs were the only population that were significantly decreased with MRX2843 treatment within both the spleen (p<0.05) and marrow (p<0.01) (Figure 1K; Table 1). There was no significant difference in the number of Gr-1- monocytes, granulocytes, or MDSCs/myeloid progenitors between treatment groups (Table 1; Supplemental Figures 1C, E, F). Within the spleen, MRX2843 treatment yielded Additional co-inhibitory receptors that portend a M2-like phenotype were analyzed on human macrophages co-cultured with AML cells. Macrophage expression of B7-H3 was minorly decreased (p<0.05) and B7-H4 was unchanged when MerTK was inhibited (Figure 2D). However, there was a striking 47% decrease in Tim-3 expression on macrophages co-cultured with AML cells and treated with MRX2843, compared to vehicle treatment (p<0.01) (Figure 2D). Similarly, M2-like markers CD163 (decreased 37%, p<0.05) and Arginase-1 (decreased 27%, p<0.05) were significantly downregulated in MRX2843-treated macrophages compared to vehicle treatment (Figure 2E). Furthermore, M1 macrophage activation was observed via a 111% increased cell surface expression of CD86 (p<0.05) (Figure 2F) and increased mean fluorescence intensity of HLA-DR (p<0.05) (Figure 2G). Upregulation of cell surface PD-L1 and PD-L2 has been associated with exposure to IFN-g (29–32), therefore we assessed serum IFN-g concentrations in MRX2843 or vehicle-treated mice by Luminex. Conversely, we observed a trend of slightly higher serum IFN-g concentrations from the MRX2843-treated mice compared to vehicle-treated mice (vehicle: 0.9pg/ml; MRX2843: 1.9pg/ml, p=0.1) though overall serum IFN-g was very low for all groups (Figure 2H). Frontiers in Immunology 05 frontiersin.org Cruz Cruz et al. 10.3389/fimmu.2023.1146721 TABLE 1 Cell counts with MerTK inhibition. MerTK inhibition skews LAM polarization Spleen Marrow No leukemia AML + vehicle AML + MRX2843 No leukemia AML + vehicle AML + MRX2843 Total nucleated cells (x106/dL) 12.2 ± 16.3 18.1 ± 13.7 10.0 ± 8.5 8.6 ± 6.0 10.6 ± 7.9 4.4 ± 3.0 Myeloid (x105 cells/dL) LAMs 1.4 ± 0.5 1.9 ± 0.8 0.9 ± 0.6 0.7 ± 0.4 1.3 ± 0.7 0.4 ± 0.1 MDSCs/ Myeloid Precursors 0.3 ± 0.1 0.3 ± 0.5 0.1 ± 0.1 1.4 ± 0.9 0.9 ± 0.6 0.8 ± 0.7 Gr-1- Monocytes 14.3 ± 0.5 1.6 ± 1.4 1.0 ± 0.6 0.9 ± 0.4 0.7 ± 0.4 0.6 ± 0.4 Granulocytes 1.2 ± 0.5 1.7 ± 1.3 0.7 ± 0.3 29.8 ± 14.3 12.9 ± 10.1 21.6 ± 15.7 Gr-1+ Monocytes 1.1 ± 0.5 2.0 ± 1.5 0.7 ± 0.4 9.9 ± 3.0 6.9 ± 3.7 8.9 ± 6.4 LAMs (x104 cells/dL) PD-L1+ 6.9 ± 2.6 11.9 ± 4.6 4.9 ± 3.1 4.0 ± 2.8 9.8 ± 4.7 2.2 ± 1.3 PD-L2+ 0.7 ± 0.4 3.6 ± 1.8 0.6 ± 0.4 0.5 ± 0.3 3.1 ± 2.0 0.2 ± 0.1 T cells (x104 cells/dL) CD4+ 9.3 ± 2.3 8.6 ± 3.9 8.2 ± 3.3 4.4 ± 2.9 4.8 ± 2.7 3.8 ± 2.2 CD8+ 9.7 ± 4.0 10.4 ± 2.0 9.3 ± 4.4 10.1 ± 7.0 7.5 ± 5.0 7.5 ± 6.3 CD4+ Tim-3+ PD-1+ 0.2 ± 0.2 2.7 ± 1.6 0.2 ± 1.8 0.7 ± 0.6 3.9 ± 2.8 0.7 ± 0.3 CD8+ Tim-3+ PD-1+ 0.3 ± 0.2 1.2 ± 0.8 0.2 ± 1.4 0.6 ± 0.4 6.0 ± 6.8 0.4 ± 0.6 Total viable cell numbers within the spleen and marrow of mice from each treatment groups. Numbers represent means ± standard deviations. LAMs, Leukemia associated macrophages; MDSCs, myeloid derived suppressor cells. Given the previous association between IL-4/STAT6 signaling, M2-like polarization and upregulation of PD-L1, PD-L2 and Tim-3 (29, 33–35), we assessed the murine serum samples for IL-4. Serum IL- 4 concentrations were evaluated, but demonstrated very low mean concentrations (<1pg/ml) in all treatment groups (not shown). Based on our previous leukemia cell line data demonstrating a correlation between STAT6 and MerTK phosphorylation (17), we assessed protein lysates from bone marrow derived macrophages (BMDMs) cultured with (or without) AML cells by immunoblot. MRX2843-treated BMDM demonstrated consistently diminished STAT6 phosphorylation compared to vehicle (Figure 2I), providing a possible mechanism for the observed alterations in polarization. MerTK inhibition skews LAM polarization SOCS1 and SOC3 have previously been associated with MerTK immunomodulation (36) through inhibition of JAK/STAT, however we did not detect a change in SOCS mRNA levels between treatment groups (Supplemental Figure 2H). compared to vehicle-treated controls (171.6pg/ml, p<0.05) (Figure 3A). We also observed a trend toward higher serum concentrations of IL-6, a cancer-promoting cytokine, in some vehicle-treated mice (379.1pg/ml) compared to MRX2843-treated mice (45.4pg/ml, p=0.3) (Supplemental Figure 3A). The following cytokines/chemokines demonstrated no significant difference between MRX2843-treated mice and controls: IL-10, IL-12p70, IL-27, MCP-3 (Supplemental Figure 3B). Additionally, serum levels of IL-1b and IL-2 were evaluated, however, the mean concentration of these cytokines in the serum were <1pg/ml in all treatment groups (not shown). To evaluate cytokine production that may be produced locally in the leukemia microenvironment, but not be detected peripherally in the serum by Luminex, cultured BMDMs were co-cultured with AML cells and treated with MRX2843 or vehicle. After 12 hours, harvested BMDMs were evaluated via RT-qPCR. Compared to vehicle-treated controls, IFN-b transcripts were increased 2.1-fold in MRX2843-treated BMDMs (p<0.05), and IL-1b mRNA was increased 2.6-fold in MRX2843-treated samples (p<0.05) (Figure 3B). There was no significant difference between vehicle and MRX2843-treated groups for INF-a or TGF-b. IL-10 and IL-12 mRNA levels were not consistently detectable in these samples (not shown) despite testing of two distinct commercially available validated primer pairs each. Additionally, co-cultured macrophages did not demonstrate detectable amounts of IFN-g mRNA (not shown), Frontiers in Immunology MerTK inhibition leads to production of cytokines known to activate T cells Three weeks after inoculation, spleens and marrow were harvested and assessed by flow cytometric analysis. (A) Quantification of PD-L1 expressing LAMs in the spleen and marrow. (B) Quantification of PD-L2 expressing LAMs in the spleen and marrow. (C–G) Macrophages derived from human peripheral blood mononuclear cells (PBMC) co-cultured with Kasumi-1 AML cells and MRX2843 or vehicle were analyzed by flow cytometry. Percentage of cultured macrophages expressing (C) PD-L1 and PD-L2, and (D) B7-H3, B7-H4 and Tim-3, (E) Arginase-1, CD163, and (F) CD86. (G) Flow cytometric quantification of HLA-DR mean fluorescence intensity of co-cultured macrophages. (H) Serum from mice described in Figure 1G was subjected to Luminex for interferon-gamma (IFN-g). (I) Murine bone marrow derived macrophages (BMDMs) were co-cultured with MLL-ENL AML cells and macrophage whole cell lysates were analyzed by immunoblot. Representative immunoblot showing phosphorylated STAT6 (p-STAT6) and total STAT6. Actin was used as a loading control. (A, B, H) were analyzed using 1-way ANOVA. (B–G) were analyzed using student’s t-test (*p<0.05, **p<0.01, ***p<0.001, NS, not significant). FIGURE 2 MerTK inhibition skews macrophage polarization in the leukemia microenvironment. (A, B) As described in Figure 1G, C57BL/6 mice were inoculated with syngeneic MLL-ENL AML and treated daily with MerTK inhibitor MRX2843 (red triangle) or vehicle (saline; black circle); mice not inoculated with leukemia are shown as controls (gray open circle). Three weeks after inoculation, spleens and marrow were harvested and assessed by flow cytometric analysis. (A) Quantification of PD-L1 expressing LAMs in the spleen and marrow. (B) Quantification of PD-L2 expressing LAMs in the spleen and marrow. (C–G) Macrophages derived from human peripheral blood mononuclear cells (PBMC) co-cultured with Kasumi-1 AML cells and MRX2843 or vehicle were analyzed by flow cytometry. Percentage of cultured macrophages expressing (C) PD-L1 and PD-L2, and (D) B7-H3, B7-H4 and Tim-3, (E) Arginase-1, CD163, and (F) CD86. (G) Flow cytometric quantification of HLA-DR mean fluorescence intensity of co-cultured macrophages. (H) Serum from mice described in Figure 1G was subjected to Luminex for interferon-gamma (IFN-g). (I) Murine bone marrow derived macrophages (BMDMs) were co-cultured with MLL-ENL AML cells and macrophage whole cell lysates were analyzed by immunoblot. Representative immunoblot showing phosphorylated STAT6 (p-STAT6) and total STAT6. Actin was used as a loading control. (A, B, H) were analyzed using 1-way ANOVA. (B–G) were analyzed using student’s t-test (*p<0.05, **p<0.01, ***p<0.001, NS, not significant). MerTK inhibition leads to production of cytokines known to activate T cells To further assess the immunological implications of efferocytosis/MerTK inhibition with MRX2943, we evaluated the impact of LAM MerTK signaling using multiplex cytokine Luminex panel. IL-18, indicative of M1 macrophage activation, was significantly increased in MerTKI-treated mice (352.7pg/ml) Frontiers in Immunology 06 frontiersin.org Cruz Cruz et al. 10.3389/fimmu.2023.1146721 suggesting that the trend seen in vivo in murine serum samples were not directly derived from LAMs but from other downstream effector cells in the leukemic microenvironment as consequence MerTK inhibition alters NF-kB activation Given the increased levels of IL-18 and IL-1b with MRX2843- D A B E F G I H C FIGURE 2 MerTK inhibition skews macrophage polarization in the leukemia microenvironment. (A, B) As described in Figure 1G, C57BL/6 mice were inoculated with syngeneic MLL-ENL AML and treated daily with MerTK inhibitor MRX2843 (red triangle) or vehicle (saline; black circle); mice not inoculated with leukemia are shown as controls (gray open circle). Three weeks after inoculation, spleens and marrow were harvested and assessed by flow cytometric analysis. (A) Quantification of PD-L1 expressing LAMs in the spleen and marrow. (B) Quantification of PD-L2 expressing LAMs in the spleen and marrow. (C–G) Macrophages derived from human peripheral blood mononuclear cells (PBMC) co-cultured with Kasumi-1 AML cells and MRX2843 or vehicle were analyzed by flow cytometry. Percentage of cultured macrophages expressing (C) PD-L1 and PD-L2, and (D) B7-H3, B7-H4 and Tim-3, (E) Arginase-1, CD163, and (F) CD86. (G) Flow cytometric quantification of HLA-DR mean fluorescence intensity of co-cultured macrophages. (H) Serum from mice described in Figure 1G was subjected to Luminex for interferon-gamma (IFN-g). (I) Murine bone marrow derived macrophages (BMDMs) were co-cultured with MLL-ENL AML cells and macrophage whole cell lysates were analyzed by immunoblot. Representative immunoblot showing phosphorylated STAT6 (p-STAT6) and total STAT6. Actin was used as a loading control. (A, B, H) were analyzed using 1-way ANOVA. (B–G) were analyzed using student’s t-test (*p<0.05, **p<0.01, ***p<0.001, NS, not significant). B A B A B A C C D D D E F G I H E G E E G I H H FIGURE 2 MerTK inhibition skews macrophage polarization in the leukemia microenvironment. (A, B) As described in Figure 1G, C57BL/6 mice were inoculated with syngeneic MLL-ENL AML and treated daily with MerTK inhibitor MRX2843 (red triangle) or vehicle (saline; black circle); mice not inoculated with leukemia are shown as controls (gray open circle). MerTK inhibition alters NF-kB activation (D) RT-PCR was performed as described in (B) Gene expression fold- change of the p105 (precursor to p50) and RelA (p65) components of NF-kB relative to vehicle-treated samples was calculated. Analyzed using Student’s t-test. (E) Representative immunoblot demonstrating phosphorylated p65/RelA and total p65/RelA; Actin was used as a loading control. (*p<0.05, NS, not significant). BMDMs co-cultured with AML cells. Increased NLRP3 was observed by immunoblot analysis when BMDMs were co- cultured with AML cells and MRX2843, compared to vehicle- treatment (Figure 3C). Previously, inhibition of MerTK signaling has been associated with increased NF-kB signaling (36), possibly through differential expression of the p50 subunit (37). We assessed mRNA levels of p105 (p50 precursor) and RelA (p65) in BMDM co- cultured with AML cells and MRX2843 or vehicle, however there was no difference in transcripts (Figure 3D). However, by immunoblot analysis of BMDMs, p65 phosphorylation was increased after MRX2843 treatment compared to vehicle (Figure 3E). Therefore, in this model of AML, MRX2843- mediated immune activation was associated with NF-kB phosphorylation, rather than altered ratios of p50:p65 transcription. TCRa-/- mice (20 days) had equivalent median survival (Figure 4A). As show in Figure 1B, leukemic C57BL/6 mice with a wild-type TCR alpha chain treated with MRX2843 demonstrated a statistically prolonged survival compared to vehicle treatment. MRX2843 treatment extended survival in TCRa-/- leukemic mice (33 days; p<0.0001) compared to vehicle treatment; however, survival was significantly diminished compared to C57BL/6 MRX2843-treated mice (p<0.05), demonstrating that immune-mediated clearance of AML with MRX2843 treatment is optimal when a/b T cells are present. MerTK inhibition alters NF-kB activation suggesting that the trend seen in vivo in murine serum samples were not directly derived from LAMs but from other downstream effector cells in the leukemic microenvironment as consequence of MerTK inhibition in LAMs. suggesting that the trend seen in vivo in murine serum samples were not directly derived from LAMs but from other downstream effector cells in the leukemic microenvironment as consequence of MerTK inhibition in LAMs. Given the increased levels of IL-18 and IL-1b with MRX2843- treatment, we evaluated for components of the inflammasome in Frontiers in Immunology frontiersin.org 07 Cruz Cruz et al. 10.3389/fimmu.2023.1146721 D A B E C FIGURE 3 Inhibition of MerTK results in M1 cytokine production through activation of NF-kB. (A) Serum from mice described in Figure 1G was subjected to Luminex for interleukin-18 (IL-18); analyzed using 1-way ANOVA. (B) BMDM were co-cultured with MLL-ENL cells for 12 hours in the presence of MRX2843 or vehicle, then mRNA was harvested from adherent macrophages. Quantitative real time-PCR was performed, normalized to GAPDH expression, and then gene expression fold-change relative to vehicle-treated samples was calculated. Analyzed using Student’s t-test. (C) As described in Figure 2I, whole cell lysates from BMDM were analyzed by immunoblot. Representative immunoblot demonstrating NLRP3, a component of the inflammasome, is shown; Actin is used as a loading control. (D) RT-PCR was performed as described in (B) Gene expression fold- change of the p105 (precursor to p50) and RelA (p65) components of NF-kB relative to vehicle-treated samples was calculated. Analyzed using Student’s t-test. (E) Representative immunoblot demonstrating phosphorylated p65/RelA and total p65/RelA; Actin was used as a loading control. (*p<0.05, NS, not significant). A C B B E D E D C FIGURE 3 Inhibition of MerTK results in M1 cytokine production through activation of NF-kB. (A) Serum from mice described in Figure 1G was subjected to Luminex for interleukin-18 (IL-18); analyzed using 1-way ANOVA. (B) BMDM were co-cultured with MLL-ENL cells for 12 hours in the presence of MRX2843 or vehicle, then mRNA was harvested from adherent macrophages. Quantitative real time-PCR was performed, normalized to GAPDH expression, and then gene expression fold-change relative to vehicle-treated samples was calculated. Analyzed using Student’s t-test. (C) As described in Figure 2I, whole cell lysates from BMDM were analyzed by immunoblot. Representative immunoblot demonstrating NLRP3, a component of the inflammasome, is shown; Actin is used as a loading control. Frontiers in Immunology Inhibition of MerTK on LAMs activates T cells (B) Three weeks after inoculation, harvested spleen and marrow from MRX2843 or vehicle-treated mice were analyzed by flow cytometry. Quantification of CD4+ and CD8+ T cells was analyzed using 1-way ANOVA. (*p<0.05, ****p<0.0001, NS, not significant). Inhibition of MerTK on LAMs activates T cells To further explore this observation, we evaluated for alteration of CD4+ and CD8+ T cell infiltration into the leukemia microenvironment. However, the total number of CD4+ and CD8+ T cells in the spleen and BM were not different between treatment groups (Table 1; Figure 4B). To assess whether the prolonged survival observed with MerTK inhibition in the leukemia microenvironment is dependent on T cell function, we performed survival studies in leukemic mice that lack the T cell receptor alpha chain (TCRa-/-) rendering them deficient in a/b T cells. Vehicle-treated leukemic C57BL/6 mice with a wild-type TCR alpha chain (17 days) and To assess the downstream effects that LAM MerTK inhibition had on T cell function in the leukemia microenvironment, we assessed checkpoint receptor expression predictive of T cell hypofunction and/or exhaustion. There were significantly less Frontiers in Immunology 08 frontiersin.org Cruz Cruz et al. 10.3389/fimmu.2023.1146721 A B FIGURE 4 MerTK Inhibition on LAMs effects T cells in the leukemia microenvironment. As described in Figure 1G, mice were inoculated with AML and treated with MRX2843 or vehicle. (A) Cohorts of mice lacking the a chain of the T cell receptor (TCRa-/-) were inoculated with AML and treated equivalently to those described in Figure 1B. Symptom-free survival was recorded; Kaplan-Meier curve of TCRa-/- mice treated with MRX2843 (dashed red line; n = 17) or vehicle (dashed black line, n = 9) with the superimposed wild-type C57BL/6 mice described in Figure 1B treated with MRX2843 (solid red line) and vehicle (solid black line). (B) Three weeks after inoculation, harvested spleen and marrow from MRX2843 or vehicle-treated mice were analyzed by flow cytometry. Quantification of CD4+ and CD8+ T cells was analyzed using 1-way ANOVA. (*p<0.05, ****p<0.0001, NS, not significant). A A B B FIGURE 4 MerTK Inhibition on LAMs effects T cells in the leukemia microenvironment. As described in Figure 1G, mice were inoculated with AML and treated with MRX2843 or vehicle. (A) Cohorts of mice lacking the a chain of the T cell receptor (TCRa-/-) were inoculated with AML and treated equivalently to those described in Figure 1B. Symptom-free survival was recorded; Kaplan-Meier curve of TCRa-/- mice treated with MRX2843 (dashed red line; n = 17) or vehicle (dashed black line, n = 9) with the superimposed wild-type C57BL/6 mice described in Figure 1B treated with MRX2843 (solid red line) and vehicle (solid black line). Frontiers in Immunology Discussion PD-1+Tim-3+ expressing CD4+ cells in the spleen (p<0.001) and BM (p<0.01) of MRX2843-treated mice compared to vehicle- treated mice (Figure 5A; Table 1). The same pattern of fewer PD- 1+Tim-3+ expressing CD8+ cells in the spleen (p<0.01) and BM (p<0.05) was observed in MRX2843-treated mice compared to vehicle-treated mice (Figure 5A; Table 1). MerTK inhibition has been evaluated in various tumor models for its cancer cell intrinsic properties (16–19, 38, 39), directing the development and testing of MerTK-targeted small molecule inhibitors. Here, we mechanistically explored the action of MRX2843, a clinically available MerTKI, on macrophage polarization in the leukemia microenvironment (Figure 6). Recent research highlights the profound immunosuppressive effects of intratumoral myeloid cells such as TAMs/LAMs, including the ability to render chimeric antigen receptor T cells and other immunotherapy ineffective (12, 40–42). Given that PtdSer, the ligand for efferocytosis, is overexpressed on cancer cells due to high cell turnover and poor cell membrane maintenance, we posited that efferocytosis is one of the primary causes of macrophage-mediated immunosuppression within the leukemia microenvironment. Therefore, targeting efferocytosis through MerTK may help reverse immunosuppression in the leukemia microenvironment. Here we tested the activity of MRX2843, an orally-active small molecule as immunotherapy with rapid on/off activity of competitive inhibitor, in the AML tumor microenvironment. To validate these findings in human T cells, we assessed CD4+ and CD8+ T cells co-cultured with AML-exposed macrophages in the presence of MRX2843 or vehicle. In MRX2843-treated samples, we observed a 12% mean reduction of PD-1+Tim-3+ expressing CD4+ T cells (p<0.05), and a 14% reduction in CD8+ PD-1+Tim-3+ cells compared to vehicle-treatment (p=0.07) (Figure 5B). In these co-cultures, we also assessed expression of checkpoint receptor LAG3 (Figure 5C), which was reduced by 29% on CD4+ T cells (p<0.05) and by 19% in CD8+ T cells in MRX2843-treated co- cultures compared to vehicle (p<0.05). To assess the impact of altered macrophage polarization on CD8+ T cell activation, co-expression of activation marker CD69 and degranulation marker CD107a was assessed (Figure 5D). In MRX2843-treated mixed lymphocyte co-cultures, there was a 23% increase in CD69+CD107a+ co-expression compared to vehicle- treated co-cultures (p<0.05). Frontiers in Immunology frontiersin.org 09 Cruz Cruz et al. 10.3389/fimmu.2023.1146721 D A B C FIGURE 5 MerTK inhibition boosts T cells activation. (A) As described in Figure 1G, harvested spleen and marrow were analyzed by flow cytometry. Quantified CD4+ and CD8+ T cells co-expressing PD-1 and Tim-3 were analyzed using 1-way ANOVA. 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. Discussion (B) Mixed human leukocyte co-cultures containing macrophages treated with MRX2843 or vehicle, AML cells and T cells were analyzed by flow cytometry. Percentage of cultured CD4+ and CD8+ T cells expressing (B) PD-1 and Tim-3, (C) LAG3, and (D) CD69 and CD107a. Analyzed using Student’s t-test. (*p<0.05, **p<0.01, ***p<0.001). A A D C B B FIGURE 5 MerTK inhibition boosts T cells activation. (A) As described in Figure 1G, harvested spleen and marrow were analyzed by flow cytometry. Quantified CD4+ and CD8+ T cells co-expressing PD-1 and Tim-3 were analyzed using 1-way ANOVA. (B) Mixed human leukocyte co-cultures containing macrophages treated with MRX2843 or vehicle, AML cells and T cells were analyzed by flow cytometry. Percentage of cultured CD4+ and CD8+ T cells expressing (B) PD-1 and Tim-3, (C) LAG3, and (D) CD69 and CD107a. Analyzed using Student’s t-test. (*p<0.05, **p<0.01, ***p<0.001). CD163, and Arginase-1. Additionally, we observed that MRX2843 treatment increased expression of M1 markers CD86 and HLA-DR, and the production of IFN-b, IL-1b and IL-18, cytokines known to stimulate activation of T cells. Collectively, these observations Targeting efferocytic macrophages within the spleen and marrow leukemia microenvironments, we demonstrated that MRX2843 decreased macrophage immunosuppressive features including a reduction in M2-like markers PD-L1, PD-L2, Tim-3, FIGURE 6 Inhibition of Efferocytosis through MerTK in AML. Graphical depiction of the findings in these studies, including the activities of leukemia-associated macrophages when MerTK engages in efferocytosis in AML (left), and when efferocytosis through MerTK it is inhibited in AML (right). FIGURE 6 Inhibition of Efferocytosis through MerTK in AML. Graphical depiction of the findings in these studies, including the activities of leukemia-associated macrophages when MerTK engages in efferocytosis in AML (left), and when efferocytosis through MerTK it is inhibited in AML (right). 10 10 Frontiers in Immunology frontiersin.org Cruz Cruz et al. 10.3389/fimmu.2023.1146721 Author contributions demonstrate macrophage repolarization from M2-like tumor- permissive phenotype toward a M1 anti-tumor activation, and prolongation of leukemia-free survival. JC, KA, LP, AJ, MV, and AL-S designed and performed experiments and analyzed data. HE, SF, XW, and DG analyzed data. JC, KA, LP, AJ, HE, SF, XW, MV, and AL-S wrote the manuscript. All authors contributed to the article and approved the submitted version. The mechanism of this repolarization is likely associated with the inhibition of MerTK downstream signaling. STAT6 is known to augment coinhibitory receptor ligand expression and M2 polarization (29, 43, 44). We previously reported that in leukemia cells, MerTK inhibition using short-hairpin RNA and MerTKIs decreased STAT6 phosphorylation (17, 18). Here, we found that MRX2843 treatment similarly decreased STAT6 phosphorylation in leukemia-exposed macrophages, providing a plausible mechanism by which MerTK inhibition leads to macrophage repolarization from cancer-promoting toward a classically activated phenotype. Additionally, production of T cell and NK cell activating cytokines (45) likely occurred due to inflammasome assembly as a result of NF-kB activation. Conflict of interest SF and XW have filed a patent application describing MRX2843. HE, DG, XW, and SF are stockholders in Meryx, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest Taken together, these studies demonstrate how inhibition of efferocytosis could be harnessed to alter the microenvironment from tumor-permissive to immune responsive in leukemia. Furthermore, given that a wide variety of cancers are known to express MerTK, including nearly 80-90% of AML patient samples (17), MerTK inhibitors could potentially have a dual cell intrinsic effect on AML cells and antitumor immune response through LAMs. Future work will focus on how effectively MerTK inhibitors can serve these dual purposes. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Acknowledgments We also thank the University of Colorado Cancer Center Flow Cytometry Core Facilities (P30CA046934) for expert technical assistance. Funding This work was supported by the National Institutes of Health (5K08CA222699, ABLS), V Foundation Cancer Research Grants, American Society of Hematology Scholar Award, Hyundai Hope on Wheels Young Investigator grant, the Pablove Foundation. In this study, we demonstrated that LAM repolarization associated with MRX2843 treatment had additional influence on the leukemia microenvironment. MRX2843 altered T cell checkpoint receptor expression and activation markers. However, we conclude that this effect was likely indirect given that that vast majority of T cells did not express MerTK. Though MRX2843 treatment of LAMs alone (in the absence of a/b T cells) was sufficient to prolong survival in leukemic mice, it is notable that when host T cells were present, MRX2843 had a more potent effect on survival prolongation. 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Tetrapod swim techniques interpreted from swim trace fossils from the Lower Triassic Baranów Formation, Holy Cross Mountains, central Poland
Paläontologische Zeitschrift
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PalZ (2021) 95:167–177 https://doi.org/10.1007/s12542-019-00510-w PalZ (2021) 95:167–177 https://doi.org/10.1007/s12542-019-00510-w RESEARCH PAPER Abstract Swimming tetrapods may leave their traces under water if their digits or limbs stir the bottom sediment. Resulting trace fos- sils are evidence of a swim behavior. Tetrapods swim techniques depend on the functional morphology of the swimmers. Examination of swim trace fossils may reveal the swim techniques employed and swimmers’ functional morphologies behind the behaviors. The present paper analyzes swim trace fossils of tetrapods from the fluvial Lower Triassic Baranów Formation in the Holy Cross Mountains (central Poland). The examination focuses on swim techniques. An attempt is made to correlate the inferred technique with functional morphology of the swimmer. It is concluded that the two types of swim traces occur in the Baranów Formation. These record two different swim techniques—paddling with limbs and body/tail undulation. The distinct types of swim trace fossils point to two types of swimmers: fully terrestrial archosaurs paddling with their limbs and amphibious tetrapods swimming with undulatory movements—likely utilizing their laterally flattened tails. Keywords  Early triassic · Swimming tetrapods · Trace fossils · Swimming technique · Webbed foot · Archosaurs · Amphibians rly triassic · Swimming tetrapods · Trace fossils · Swimming technique · Webbed foot · Archosaurs Keywords  Early triassic · Swimming tetrapods · Trace fossils · Swimming technique · Webbed foot · Archosaurs · Amphibians Tetrapod swim techniques interpreted from swim trace fossils from the Lower Triassic Baranów Formation, Holy Cross Mountains, central Poland Grzegorz Sadlok1 · Krzysztof Pawełczyk1 Received: 18 June 2019 / Accepted: 28 December 2019 / Published online: 29 January 2020 © The Author(s) 2020 * Grzegorz Sadlok gregsadlok@gmail.com 1 Faculty of Natural Sciences, Institute of Earth Sciences, University of Silesia in Katowice, Będzińska 60, PL‑43‑200 Sosnowiec, Poland Handling Editor: Mike Reich. Geological settings The ichnites were photographed, measured and their interpretative drawings were made. The applied terminol- ogy follows mostly McAllister (1989); it uses terms like footmark and footmarks to refer to swim traces. Associated sequence of footmarks is a traceway in McAllister (1989) terminology; however, we use more general and widely known term trackway to address such a sequence. The strata constituting the Baranów Formation are Lower Triassic deposits representing braided fluvial system (Kuleta and Nawrocki 2000; Kuleta et al. 2005; Bujok et al. 2008). The formation outcrops at the northern mar- gin of the Holy Cross Mountains—north of city of Kielce (central Poland, Fig. 1a–c). The formation comprises two main lithofacies: reddish mudstones and sandstones—representing floodplain and channel subenvironments, respectively (see Kuleta et al. 2005, see fig. 2 therein and Fig. 1 herein). The thicknesses and proportions of the lithofacies are variable—mudstone intervals become thicker in the upper part of the Baranów Formation. Sandstones associated with mud-dominated intervals tend to form thinner beds (Kuleta et al. 2005; Bujok 2007). The contacts between mudstones and sand- stones (sandstones-over-mudstones) are sharp to erosive (Fig. 1e–g). Locally, inclined sharp to erosive contacts are observed within sandstone packages (Fig. 1e). Thicker sandstone-dominated intervals display upward thinning trends (Fig. 1e)—an indication of channel evolution (Walker and Douglas 1984). The thickest sandstone inter- vals show remnants of trough cross bedding (Fig. 1g)—a record of in-channel bed forms migration. Locally, sur- faces with pebble-rich lags (mudclasts) and scour-and-fill gutters (cf. Shukla et al. 2006) are also observed (Fig. 3a). A rich trace fossil assemblage (root traces invertebrate The formation comprises two main lithofacies: reddish mudstones and sandstones—representing floodplain and channel subenvironments, respectively (see Kuleta et al. 2005, see fig. 2 therein and Fig. 1 herein). The thicknesses and proportions of the lithofacies are variable—mudstone intervals become thicker in the upper part of the Baranów Formation. Sandstones associated with mud-dominated intervals tend to form thinner beds (Kuleta et al. 2005; Bujok 2007). The contacts between mudstones and sand- stones (sandstones-over-mudstones) are sharp to erosive (Fig. 1e–g). Locally, inclined sharp to erosive contacts are observed within sandstone packages (Fig. 1e). Thicker sandstone-dominated intervals display upward thinning trends (Fig. 1e)—an indication of channel evolution (Walker and Douglas 1984). The thickest sandstone inter- vals show remnants of trough cross bedding (Fig. 1g)—a record of in-channel bed forms migration. Locally, sur- faces with pebble-rich lags (mudclasts) and scour-and-fill gutters (cf. Shukla et al. Introduction of interest for ichnologists who analyze and classify such fossilized traces of locomotion as repichnia (sensu Vallon et al. 2016). Swim trace fossils are meaningful and impor- tant piece of information on swim behaviors (e.g., Sarjeant 1975). Terrestrial tetrapods may be occasional swimmers and swim if they need to cross a lake or river—for example, during migrations (see, e.g., Yeager 1991) or exceptionally—dur- ing the hunt-escape situation (see, e.g., Sweeney et al. 1971; Nelson and Mech 1984; Jordan et al. 2010). Other tetra- pods have amphibious life styles—they stay close to, or in the water and swimming is very much a part of their daily behavioral repertoire (see, e.g., Somers and Purves 1996; Barbraud and Weimerskirch 2001; Seebacher et al. 2003; Willson et al. 2006). In any of those cases, tetrapods may stir the muddy sediment on the bottom with their limbs/digits and produce swim traces. Traces of swim behavior may pass into the fossil record. If they do so, they become a matter The functional morphology puts physical constrains on the way the vertebrates propel themselves in water. Fish, amphibians, amphibious reptiles (e.g., crocodiles), terres- trial and marine mammals swim in various ways (see, e.g., Webb 1988; Fish 1996). The details of swim behavior can be preserved in and deciphered from trace fossil morphology. The Lower Triassic Baranów Formation from the Holy Cross Mountains (central Poland) is a fluvial formation. Its sediments are rich in trace fossils, including tetrapod swim traces (Kuleta et al. 2005; Bujok et al. 2008). Despite the abundance of those swim ichnites, the detailed analysis of behaviors and trackmakers these traces represent has been lacking. The present paper aims to fill that void and exam- ines the swim techniques and potential identities of the swimmers. Handling Editor: Mike Reich. Handling Editor: Mike Reich. 1 Faculty of Natural Sciences, Institute of Earth Sciences, University of Silesia in Katowice, Będzińska 60, PL‑43‑200 Sosnowiec, Poland :(0123 1 23456789) 3 168 G. Sadlok, K. Pawełczyk Parallel ridges These trackways are preserved on lower surfaces of cen- timeter- to decimeter-thick sandstone beds (convex hyp- ichnia), herein referred to as specimens WNoZ/S/7/62 and WNoZ/S/7/258. Specimen WNoZ/S/7/62  There are four imprints (I–IV) vis- ible on the specimen WNoZ/S/7/62 (Fig. 2a); measurements of length, width and relief are provided in Table 1. Two dis- tinct morphologies are present (Fig. 2a–f). Footmarks I and II are sets of ridges. These two are almost identical in terms of their morphology and symmetry (Fig. 2b, c). These are described in details below. Tracks marked as III and IV are indistinct imprints, wider than longer (Fig. 2a). Footmarks I and II are sets of long ridges (Fig. 2a–c). Each set comprises two narrow ridges (21 cm long and 2–5 cm width) and one shorter and slightly wider (18–19 cm long and 5–6 cm width). The ridges are parallel (Fig. 2a–c). The sets appear compact as the ridges contact each other (Fig. 2a–c). The outlines of the footmarks are sharp. The ridges taper on one end (Fig. 2b–c). Longitudinal striations are present on the sides of the ridges (inset in Fig. 2b). There are irregular depressions associated with the ridges (Fig. 2b, c). Simple horizontal to subhorizontal invertebrate trace fossils (small burrows, cf. Planolites and shallow burrows/ superficial furrows, cf. Helminthoidichnites) occur within the depressions and on the surface of sediment around the sets of ridges (Fig. 2a–e). The invertebrate trace fossils are cut, in places, by the sets of ridges (Fig. 2d). Distance between footmarks I and II—measured along the direction of their elongation—is approximately 50 cm (Fig. 2a, f). The distance measured perpendicular to that direction is approximately 19 cm (Fig. 2a, f). Desiccation cracks are cut (postdated) by the set of parallel ridges forming the imprint I (Fig. 2d). A rich trace fossil assemblage (root traces, invertebrate and vertebrate ichnites; Figs. 1, 2 and 3) occurs in the for- mation (see, e.g., Kuleta et al. 2005; Bujok 2007; Bujok et al. 2008). Regarding the composition and preservation of trace fossils—e.g., the high abundance of tetrapod swim traces, the strata from Baranów resemble those from the lower Triassic Moenkopi Formation (Utah, USA; Thom- son and Droser 2015). The trace fossils are mostly associ- ated with lower surfaces of the sandstone beds (Fig. 1h–i). Geological settings 2006) are also observed (Fig. 3a). A rich trace fossil assemblage (root traces, invertebrate and vertebrate ichnites; Figs. 1, 2 and 3) occurs in the for- mation (see, e.g., Kuleta et al. 2005; Bujok 2007; Bujok et al. 2008). Regarding the composition and preservation of trace fossils—e.g., the high abundance of tetrapod swim traces, the strata from Baranów resemble those from the lower Triassic Moenkopi Formation (Utah, USA; Thom- son and Droser 2015). The trace fossils are mostly associ- ated with lower surfaces of the sandstone beds (Fig. 1h–i). Material The present study is based on material (Figs. 2 and 3) from Baranów quarry where the Baranów Formation outcrops (Fig. 1). The quarry is an active mining site and the stud- ied specimens were found ex situ, on excavated blocks of sandstone. The location of findings and their presumed stratigraphic proveniences are shown in Fig. 1d. The stud- ied material includes two large specimens photographed during our field work in autumn of 2018 (Fig. 3) and mate- rial stored in the Museum of Institute of Earth Sciences of the University of Silesia, in Sosnowiec (Fig. 2, museum labels: WNoZ/S/7/62 and 258). Specimen WNoZ/S/7/258  There are three imprints (I– III) visible on the specimen WNoZ/S/7/258 (Fig. 2g); measurements of length, width and relief are provided in Table 1. Footmarks I–II are sets of parallel ridges (three per 1 3 Triassic tetrapod trace fossils from central Poland 169 100 km 3 2 1 - Field specimen 1 and 2 - WNoZ/S/7/62 - WNoZ/S/7/258 Poland Warsaw Cracow Lower Triassic Paleozoic a City/Town Mudstone Sandstone Sandstone Thinning beds Mudstone Sandstone 1 m 1 m 1 m Sandstone Track 10 cm 1 cm D D D H H Casts of desiccation cracks es1 es es Trough cross bedding d e f g h es2 G 4 m M1 S2 S3 M2 S1, S2, S3 - sandstones M1, M2, M3 - mudstones Mudstone i h - S-M sequence base N Kielce S1 S1 1 2 3 i - Specimen shown in i - Specimen shown in h h Kielce Holy Cross Mountains 10 km 5 km b b c i  Location of Baranów quarry in the Holy Cross Mountains and mentological summary of Baranów Formation. Material a  Poland with e of the Holy Cross Mountains; b The Holy Cross Mountains ocation of major city—Kielce; c A map showing road S7 lead- om Kielce to the Baranów quarry; d A view of Baranów quarry ew toward west), main sandstone–mudstone sequences (S–M nces) as seen in the central part of the quarry and collection/ g points and stratigraphic provenience of the studied specimens imen shown in h has been photographed to the north from the main view); e Erosive contact between thick sandstone packag mudstone (es1) and inclined erosive contact within the san package (es2); f Erosive contact between thinner sandstone be mudstone (es); g  Thick sandstone interval with trough cros ding (erosive contact with mudstones; es); h A sole of sandston with casts of desiccation cracks and tetrapod track (field photog i An example of sandstone sole with multiple trails/shallow bu of Helminthoidichnites (H), Diplopodichnus (D) and Gordi (field photograph) 100 km Poland Warsaw Cracow Lower Triassic Paleozoic a City/Town N Kielce Kielce Holy Cross Mountains 10 km 5 km b b c d e Track 10 cm Casts of desiccation cracks h Fig. 1   Location of Baranów quarry in the Holy Cross Mountains and sedimentological summary of Baranów Formation. Material ecimen WNoZ/S/7/62 with footmarks (I–II) and indistinct foot- ks III–IV; b Enlargement of footmark II, the inset shows longi- nal striations on the side of the footmark II (dotted lines outline essions within the footmark); c Enlargement of footmark I (dot- lines outline depressions within the footmark); d  Enlargement etail from c, imprint cuts shallow horizontal to subhorizontal rtebrate trace fossils (itf) and desiccation crack; e  Enlargement etail from c, shows an irregular depression within the imprint preserved shallow horizontal to subhorizontal invertebrate trace fossils (itf) within the depression; f An interpretative d of imprints seen in a; Note: basic measurements are provid dashed lines represent axes of imprints; g Specimen WNoZ/S with footprints (I–II) and a track (III); Note: crescent depr associated with footmark II and track III; h  Enlargement of from g, shows crescent depression associated with footmark its cross-cutting relationship with invertebrate horizontal trac (itf); i An interpretative drawing of imprints seen in g; Note measurements are provided 10 cm 1 cm a b c b c d 1 cm d e 1 cm e 1 cm 1 cm g g Swim traces 5 cm g f d h i Secondary striation h f i Fig. 2   Sets of parallel ridges—swim traces of paddling technique. a Specimen WNoZ/S/7/62 with footmarks (I–II) and indistinct foot- marks III–IV; b Enlargement of footmark II, the inset shows longi- tudinal striations on the side of the footmark II (dotted lines outline depressions within the footmark); c Enlargement of footmark I (dot- ted lines outline depressions within the footmark); d  Enlargement of detail from c, imprint cuts shallow horizontal to subhorizontal invertebrate trace fossils (itf) and desiccation crack; e  Enlargement of detail from c, shows an irregular depression within the imprint and preserved shallow horizontal to subhorizontal invertebrate Fig. 2   Sets of parallel ridges—swim traces of paddling technique. Material a  Poland with outline of the Holy Cross Mountains; b The Holy Cross Mountains and location of major city—Kielce; c A map showing road S7 lead- ing from Kielce to the Baranów quarry; d A view of Baranów quarry (a view toward west), main sandstone–mudstone sequences (S–M sequences) as seen in the central part of the quarry and collection/ finding points and stratigraphic provenience of the studied specimens (specimen shown in h has been photographed to the north from the main view); e Erosive contact between thick sandstone package and mudstone (es1) and inclined erosive contact within the sandstone package (es2); f Erosive contact between thinner sandstone bed and mudstone (es); g  Thick sandstone interval with trough cross bed- ding (erosive contact with mudstones; es); h A sole of sandstone bed with casts of desiccation cracks and tetrapod track (field photograph); i An example of sandstone sole with multiple trails/shallow burrows of Helminthoidichnites (H), Diplopodichnus (D) and Gordia (G) (field photograph) main view); e Erosive contact between thick sandstone package and mudstone (es1) and inclined erosive contact within the sandstone package (es2); f Erosive contact between thinner sandstone bed and mudstone (es); g  Thick sandstone interval with trough cross bed- ding (erosive contact with mudstones; es); h A sole of sandstone bed with casts of desiccation cracks and tetrapod track (field photograph); i An example of sandstone sole with multiple trails/shallow burrows of Helminthoidichnites (H), Diplopodichnus (D) and Gordia (G) (field photograph) 1 3 1 3 170 G. Sadlok, K. Pawełczyk Swim traces 5 cm 10 cm 1 cm a b c g f b c d h i Secondary striation h 1 cm d e 1 cm e 1 cm 1 cm 2   Sets of parallel ridges—swim traces of paddling technique. Material a Specimen WNoZ/S/7/62 with footmarks (I–II) and indistinct foot- marks III–IV; b Enlargement of footmark II, the inset shows longi- tudinal striations on the side of the footmark II (dotted lines outline depressions within the footmark); c Enlargement of footmark I (dot- ted lines outline depressions within the footmark); d  Enlargement of detail from c, imprint cuts shallow horizontal to subhorizontal invertebrate trace fossils (itf) and desiccation crack; e  Enlargement of detail from c, shows an irregular depression within the imprint and preserved shallow horizontal to subhorizontal invertebrate trace fossils (itf) within the depression; f An interpretative drawing of imprints seen in a; Note: basic measurements are provided, the dashed lines represent axes of imprints; g Specimen WNoZ/S/7/258 with footprints (I–II) and a track (III); Note: crescent depressions associated with footmark II and track III; h  Enlargement of detail from g, shows crescent depression associated with footmark II and its cross-cutting relationship with invertebrate horizontal trace fossil (itf); i An interpretative drawing of imprints seen in g; Note: basic measurements are provided footmark) and lie in one line (one after the other). Footmark I is incomplete, it terminates at the slab edge. Footmark II is close to the slab edge. It appears to be nearly complete as compared with footmark I. The distance between footmarks is close to the slab edge. It appears to be nearly complete as compared with footmark I. The distance between footmarks 1 3 3 171 Triassic tetrapod trace fossils from central Poland Bed sole view Pebbles' casts Gutter cast? U-shaped burrows Pebbles' casts Bed sole view Swim traces Swim traces Swim traces Swim traces? a b c e d e b 10 cm 10 cm 10 cm 10 cm 10 cm current? F f g 1 2 3 i h d lateral repetition and cross-cutting lateral repetition and cross-cutting Well defined end Well defined end Poorly defined end Sine-shaped ridges—swim traces of undulatory technique. Material andstone bed fragment (sole view) with sine-shaped ridges traces), fr—footprints rows; the ridges are associated with ped burrows and pebble-rich horizon (both postdate the ridges); rged part seen in a; c A sandstone bed fragment (sole view) ine-shaped ridges (swim traces); d  Enlarged part seen in c; rged part seen in a; f An interpretative drawing of swim traces a (dotted line shows the sine-like shapes of imprints; arrow shows plausible swim direction, not to scale); h  An interpre drawing of e (meaning of dotted line as in f; arrow shows plau swim direction, not to scale); i Schematic drawing showing the backstroke movement and the resulting relief of the trace (ridge trace fossil; a rowing foot with interdigital web is used on the tration); the direction of swim is interpreted from changes in re lower ends of ridges are thought to be entry spots of limb stirrin sediment; solid arrow shows plausible swimmer’s move dire Bed sole view Pebbles' casts Gutter cast? U-shaped burrows Pebbles' casts Bed sole view Swim traces Swim traces Swim traces a b c d e b 10 cm 10 cm 10 cm 10 cm d lateral repetition and cross-cutting Well defined end Well defined end Poorly defined end U-shaped burrows Pebbles' casts Swim traces Swim traces b 10 cm 10 cm d lateral repetition and cross-cutting Well defined end Well defined end Poorly defined end a a b d c 10 cm current? F f g 1 2 3 i h lateral repetition and cross-cutting Well defined end Swim traces? e 10 cm current? F f g 1 2 3 i h lateral repetition and cross-cutting Well defined end Swim traces? e 10 cm h e current? g i lateral repetition and cross-cutting lateral repetition and cross-cutting g Fig. 3   Sine-shaped ridges—swim traces of undulatory technique. Material a  A sandstone bed fragment (sole view) with sine-shaped ridges (swim traces), fr—footprints rows; the ridges are associated with U-shaped burrows and pebble-rich horizon (both postdate the ridges); b Enlarged part seen in a; c A sandstone bed fragment (sole view) with sine-shaped ridges (swim traces); d  Enlarged part seen in c; e Enlarged part seen in a; f An interpretative drawing of swim traces from a (dotted line shows the sine-like shapes of imprints; arrow shows plausible swim direction, not to scale); g  An interpretative drawing of imprints seen in c (meaning of dotted line as in f; arrow shows plausible swim direction, not to scale); h  An interpretative drawing of e (meaning of dotted line as in f; arrow shows plausible swim direction, not to scale); i Schematic drawing showing the limb backstroke movement and the resulting relief of the trace (ridge in the trace fossil; a rowing foot with interdigital web is used on the illus- tration); the direction of swim is interpreted from changes in relief— lower ends of ridges are thought to be entry spots of limb stirring the sediment; solid arrow shows plausible swimmer’s move direction; dotted arrows show backstroke direction shows plausible swim direction, not to scale); h  An interpretative drawing of e (meaning of dotted line as in f; arrow shows plausible swim direction, not to scale); i Schematic drawing showing the limb backstroke movement and the resulting relief of the trace (ridge in the trace fossil; a rowing foot with interdigital web is used on the illus- tration); the direction of swim is interpreted from changes in relief— lower ends of ridges are thought to be entry spots of limb stirring the sediment; solid arrow shows plausible swimmer’s move direction; dotted arrows show backstroke direction 1 3 1 3 172 G. Sadlok, K. Discussion Swimming vertebrates use a set of movements to propel themselves through the water (Fig. 4). Their functional morphology constrains their swim techniques. Vertebrates swim in undulatory or oscillatory fashion. Fish, tadpoles, salamanders, crocodiles use body/tail lateral flexion— undulatory movements—to swim. Fins of some fish, e.g., tuna tail, as well as limbs of frogs, reptiles, birds and most mammals are propellers being used in an oscillatory fash- ion (Webb 1988). Footmark I is composed of ends of three ridges. These are separated by narrow groves (groves are narrower than the ridges). Footmark II has also three ridges; its length is 7 cm (Fig. 2g, i) and width is 4 cm (Fig. 2g, i). Footmark II has a crescent-like depression (approximately 1–2 mm deep) at one of its narrow ends (rear end; Fig. 2g, i). Millimeter-scale stria- tion occurs on this footmark (Fig. 2g, h). Undulatory body movements of axial propulsion may utilize various parts of a vertebrate body length. Based on the utilized part of the fish body, their swim techniques can be classified under four different categories: anguilliform, sub-carangiform, carangiform and thunniform (see McDo- wall 2003). These different techniques can be reflected in Undichna and Parundichna—sine-shaped trace fossils resulting from undulatory swim behaviors (e.g. Simon et al. 2003; Wisshak et al. 2004; Minter and Braddy 2006). Amphibious swimming tetrapods (e.g., salamander, alli- gator) may use similar techniques and flex the body/tail to swim (Bartholomew et al. 1976; Fish 1984; Hoff et al. 1989). Such amphibious tetrapods typically keep their limbs folded along their bodies when swimming by undu- lation of the body/tail (Fish 1984; Ijspeert et al. 2005). In shallow water, they switch from swimming to walking as their limbs get in contact with the substrate (see Ashley- Ross and Bechtel 2004). Material Pawełczyk Table 1   The length, width and relief measurements of the tetrapod footmarks a These specimens are not considered in detail in the present paper (indistinct morphologies) b Measured as straight line end-to-end Specimen Track/footprint Length (cm) Width (cm) Relief (cm) Figures WNoZ/S/7/62 I 21 14 ≤ 2 2a, c–e WNoZ/S/7/62 II 21 12 ≤ 2 2a, b WNoZ/S/7/62 III 6 12 ≤ 2 2aa WNoZ/S/7/62 IV 9 15 ≤ 2 2aa WNoZ/S/7/258 I Incomplete Incomplete ≤ 1 2g WNoZ/S/7/258 II 7 4 ≤ 1 2g WNoZ/S/7/258 III 3.5 3.5 ≤ 1 2ga Field specimen 1 Multiple ~ 2–20b ~ 1–3 ≤ 2 3a, b, e Field specimen 2 Multiple ~ 4–20b ~ 1–3 ≤ 2 3c–d a These specimens are not considered in detail in the present paper (indistinct morphologies) defined trackways (Fig. 3a, f) and locally display lateral repeti- tion (Fig. 3d, g). I and II is 15 cm (measured between tops of central ridges; Fig. 2g, i). Both sets of ridges are elongated. Their detail description is provided below. Track III is a more isometric imprint (width and length are approximately 3.5 cm) and its digital imprints are curved-backward (Fig. 2g). It has a cres- cent-like depression at its rear end (Fig. 2g, i). Invertebrate trace fossils occur on the surface of sediment around the imprints (Fig. 2g) and some are cut by the imprints (Fig. 2h). Sine‑shaped ridges a Fish swimming with undulatory movements may produce sine-shaped continu- ous traces; b  Tetrapod using undulatory swim technique may pro- duces discontinuous sine-shaped imprints; c  Tetrapod paddles with its erected limbs may leave indistinct scratches; d Sets of scratches may show two morphologies: spread and compact; it is shown how striations on digit marks, variable footmark lengths and vari- able/incomplete trackway patterns (see McAllister and Kirby 1998; Thomson and Lovelace 2014). Thomson and Droser (2015) showed that morphologies of underwater footmarks depend also on substrate properties—firmground conditions allow preservation of fine morphological details, like longi- tudinal striations and displacement rims. Some of the pro- posed criteria, like kick-off scours, are susceptible to subse- quent shallow erosion of mud. Some of the criteria excludes each other as their formation and preservation require differ- ent properties—kick-off scours require water-saturated and easily erodible sediment (see McAllister and Kirby 1998); whereas, the longitudinal striations require firmground set- tings (see Thomson and Droser 2015). Tetrapods may swim also using oscillatory technique— they paddle or row with their limbs (Fish 2000; Fish and Baudinette 2008; Provini et al. 2012). The paddling is when limbs move in parasagittal plane—rowing, perpen- dicular to that plane (Fish 2000) and it may be performed in various ways: quadrupedal, pectoral and pelvic paddling (see Fish 1996, 2000). The paddling, especially its quad- rupedal variant, is less efficient than other techniques and it reflects poor aquatic adaptation of terrestrial tetrapods that use it (Williams 1983; Fish and Baudinette 2008). Paddling is utilized by tetrapods with erect limbs, includ- ing the large ones, like elephants (Fish 1996). Dinosaurs likely used a paddling swim technique as well (e.g., Whyte and Romano 2001; Ezquerra et al. 2007).f Despite the fact that buoyancy affects the limb walk cycle (Ashley-Ross and Bechtel 2004), trackway pattern and its completeness (e.g., Lockley 1991), the detailed analysis may help to decipher tracemaker movements, their swim tech- niques and in some cases, anatomical features. Sine‑shaped ridges These footmarks were observed on lower surfaces of deci- meter-thick sandstone beds (convex hypichnia). One of the beds (Fig. 3a) shows pebble-grade material and millimeter- scale U-shaped burrows associated with the footmarks. The U-burrows and pebble-grade material postdate the footmarks (Fig. 3a, b). The footmarks are sine-shaped ridges (Fig. 3a–h). The measurements of length, width and relief are provided in Table 1. The ridges are relatively deep and blunt on one end and pass gradually into the surrounding sediment on the other or show two blunt ends with one end having lower relief. Their surfaces are smooth. The sides of ridges are steep to weakly inclined and asymmetric. Some overlapping of the ridges may be seen locally (Fig. 3d, g). The footmarks form poorly 1 3 173 Triassic tetrapod trace fossils from central Poland a b c d Fig. 4   Main swimming techniques of vertebrates. a Fish swimming with undulatory movements may produce sine-shaped continu- ous traces; b  Tetrapod using undulatory swim technique may pro- duces discontinuous sine-shaped imprints; c  Tetrapod paddles with its erected limbs may leave indistinct scratches; d Sets of scratches may show two morphologies: spread and compact; it is shown how the foot, during the backstroke, scratches the sediment (dotted line: sediment/water boundary); it is proposed herein that the difference in scratch set morphology may reflect various foot behaviors during the backstroke phase, and variation in foot design (webbed vs. un-webbed feet) d the foot, during the backstroke, scratches the sediment (dotted line: sediment/water boundary); it is proposed herein that the difference in scratch set morphology may reflect various foot behaviors during the backstroke phase, and variation in foot design (webbed vs. un-webbed feet) Fig. 4   Main swimming techniques of vertebrates. a Fish swimming with undulatory movements may produce sine-shaped continu- ous traces; b  Tetrapod using undulatory swim technique may pro- duces discontinuous sine-shaped imprints; c  Tetrapod paddles with its erected limbs may leave indistinct scratches; d Sets of scratches may show two morphologies: spread and compact; it is shown how the foot, during the backstroke, scratches the sediment (dotted line: sediment/water boundary); it is proposed herein that the difference in scratch set morphology may reflect various foot behaviors during the backstroke phase, and variation in foot design (webbed vs. un-webbed feet) Fig. 4   Main swimming techniques of vertebrates. Oscillatory swim technique Imprints left under water may represent walking or swim- ming. There are a few ichnogenera produced by dinosaurs, turtles and crocodiles while moving under partial buoyancy (e.g., Lockley 1991; Foster and Lockley 1997; McCrea et al. 2004; Lockley and Foster 2006; Ezquerra et al. 2007; Avan- zini et al. 2010; Vila et al. 2014). Most of those ichnites (Chelonichnium, Chelonipus, Saltosauripus, Hatcherich- nus), convey enough details of the autopodium anatomy to link them with their potential trackmakers. In those cases, the trackmakers’ limbs were used to support, at least par- tially, their bodies, or to “feel” their way when they sensed they were close to the substrate. However, with increasing Before a sound analysis may be attempted, vertebrate footmarks created under water must be differentiated not only from inorganic structures (cf. Peabody 1947) but also from tracks and undertracks produced on a dry land (cf. McAllister and Kirby 1998). The criteria for the recogni- tion of subaqueous formation involve features resulting from locomotion under buoyancy, movements of digits within the sediment, and action of currents generated by moving limbs. The criteria include: kick-off scours, z-traces, variable preservation of footmarks, posterior overhangs, longitudinal 1 3 174 G. Sadlok, K. Pawełczyk similarity between the two cannot be fully assessed. Hence, a distinction between consecutive footmarks of opposite vs. the same foot cannot be made. Footmarks I and II on speci- men WNoZ/S/7/258 appear similar in size and orientation and, therefore, could be produced by the same tracemaker. Footmarks on the specimen WNoZ/S/7/258 (Fig. 2g–i) differ from those on the specimen WNoZ/S/7/62 (Fig. 2a, g) in the presence of crescent depression associated with the footmark II (Fig. 2g–i). This feature represents likely a cast of sedi- ment displacement rim. The crescent depression and fine, millimeter-scale secondary striation of footmark II (Fig. 2g, h) may suggest a firmground properties of the substrate (cf. Thomson and Droser 2015). buoyancy only digits’ tips can stir the bottom sediment and leave simple, indistinct scratches assignable to ichnogenera like Characichnos or Albertasuchipes—such swim traces cannot be associated with a specific tracemaker (Whyte and Romano 2001; McCrea et al. 2004; Vila et al. 2014; Ezquerra et al. 2007). The sets of parallel ridges shown in Fig. 2 are vertebrate footmarks composed of digital scratches with no foot outline as would be expected from imprints resulting due to weight- bearing phase of walking cycle executed over dry land (cf. Oscillatory swim technique Thulborn and Wade 1989). Irregular depressions (Fig. 2b, c) occur within footprints and indicate that these are the casts of elevations within the imprints. Those elevations could result from sediment of footmark edges collapsing or sliding into the footmark. Therefore, their presence and the presence of fine longitudinal striations seen on footmarks (inset in Fig. 3b) are taken as indications that the footmarks were originally open superficial features in relatively firm mud substrate rather than undertracks where such collapse structures and striations would not have occurred.i The sets of parallel scratches (see footmarks I and II in Fig. 2a) resemble Characichnos with its general morphol- ogy and number of scratches in a footmark (see Whyte and Romano 2001; Bujok 2007). However, the studied mate- rial differs from Characichnos morphology described by Whyte and Romano (2001) with a compact appearance of sets which are composed of scratches contacting each other (Fig. 2). This difference may have behavioral and anatomi- cal significance.f The shallow and superficial invertebrate trace fossils co- occur with the footmarks (see Fig. 2a, g). Some are pre- served within the depressions of the footmarks and post- date them (Fig. 2b, c, e). The footmarks cut the invertebrate trace fossils (Fig. 2d, g, h) and, in places, desiccation cracks (Fig. 2d). In those cases, invertebrate trace fossils and desic- cation cracks predate the footmarks. The presence of shallow invertebrate penetrations within the footmarks supports the view that these imprints have been left under water, close to or at the water–sediment interface. The superficial and/ or shallow invertebrate trace fossils preserved within the footmarks render undertrack origin unlikely. i The propulsive effectiveness of the paddling propeller increases with its area—an interdigital web increases foot’s paddling area and drag during propulsive backstroke in sem- iaquatic tetrapods (Fish 2000, and references therein). To achieve that, digits of webbed foot are spread during the pro- pulsive backstroke (Fig. 4d). Therefore, they would create separated scratches—a typical spread morphology as in type material of Characichnos (cf. Whyte and Romano 2001). Crocodiles have interdigital webs on their feet and Charac- ichnos assigned to them has not only spread scratches but also may in fact show traces of the web itself (cf. McCrea et al. 2004; Vila et al. 2014). Oscillatory swim technique The morphologies of ichnites, their sedimentological con- text and ichnological association suggest the footmarks are not undertracks and that they originated under water when the weight-bearing phase of the walking cycle was inhibited by the buoyancy of water. Contrary, close spacing of the scratches—a compact mor- phology, may indicate that swimmer lacking the webbed foot held its digits close together during the backstroke to increase the foot paddling area and effectiveness. A human palm can serve as analog—narrow gaps between digits increase slightly and optimize its paddling area and drag. However, they cannot increase over a certain threshold (e.g., 10°–12° of interdigital angle) without compromising the effectiveness of the propulsive backstroke (see Sidelnik and Young 2006; Minetti et al. 2009). Therefore, the compact morphology, as in the present footmarks, may result from action of an un-webbed foot with digits held close during the backstroke phase of the limb movement cycle (Fig. 4d)—this could be an example of behavior making up for the poor design of the propelling limb. Footmarks I and II on the specimen WNoZ/S/7/62 are very similar in terms of their morphology and symmetry— both have the shorter digit trace on the same side of their symmetry planes (Fig. 2f) and therefore, they are likely consecutive footmarks left by a single foot of a tracemaker (Fig. 2b, c). A mirror symmetry in morphology would be expected for footmarks and tracks left by opposite feet of the same animal. The footmarks’ long axes are parallel but they do not sit on a single line (dashed lines in Fig. 2f are footmarks axes)—footmarks are separated by 19 cm, meas- ured perpendicular to their axes (see Fig. 2f). This pattern may suggest that the foot scratched the substrate at an angle to the swimming direction (cf. Thomson and Droser, 2015, fig. 3a therein). No such pattern can be assessed for foot- marks I and II from the specimen WNoZ/S/7/258. This is because footmark I is incomplete and morphological Both morphologies—spread and compact—of Charac- ichnos are present in the strata from the Baranów Formation (see, e.g., Kuleta et al. 2005; Bujok et al. 2008). This may 1 3 3 175 Triassic tetrapod trace fossils from central Poland indicate two distinct types of tracemaker produced Charac- ichnos from the unit. well-defined tracks and footmarks with no sine-shaped con- figuration (cf. Lockley and Foster 2006; Xing et al. 2014). Oscillatory swim technique The material studied herein suggests tracemakers with feet having no interdigital web—likely terrestrial tetrapods who crossed the body of water (Fig. 2). The tetrapod swim traces occur in strata comprising also ichnites of Chirotherii- dae ichnofamily (Kuleta et al. 2005; Bujok 2007; Bujok et al. 2008). Archosaurs who produced such tracks (see Sarjeant 1975; Niedźwiedzki and Ptaszyński 2007) had no interdigi- tal web as evidenced by their track morphology (cf. King et al. 2005). The ichnites of this ichnofamily are common in Triassic deposits (Sarjeant 1975; Leonardi and de Oliveira 1990; Ptaszyński 2000; Nicosia and Loi 2003; Fichter and Kunz 2004; King et al. 2005) and their archosaurian track- makers are likely producers of the paddling traces described herein. i The sine-shaped component suggests undulation was likely superimposed on the progressive movement of the tracemaker. Regarding this feature, the sine-shaped swim traces resemble Lunichnium, which is thought to be a swim trace fossil of tetrapods, e.g., amphibians (Minter and Braddy 2006). The trackway pattern is not clear. One can attempt to discern two footmark rows in the specimen illustrated in Fig. 3a. However, trackway pattern is much more elusive in specimen from Fig. 3c. Nevertheless, in both specimens, the ridges are closely spaced and locally overlap each other (Fig. 3d, g). Short distances between repetitive foot–sub- strate contacts may suggest the tracemaker climbed the inclined lake/river bed (cf. Ashley-Ross and Bechtel 2004) or it fought the current and swam upstream. In such a case, the progressive speed would be a resultant vector of trace- maker and the water current speeds. Undulatory swim technique The sine-shaped ridges (Fig. 3) are likely footmarks pro- duced by limbs stirring the bottom mud. Field specimen illustrated in Fig. 3b shows sections of U-shaped burrows associated with the ridges. The burrows represent Diplocra- terion preservation style known from subaqueous deposits of Lower Triassic (see Gradziński and Uchman 1994). The burrows occur around the ridges and on the ridges indicat- ing the invertebrate colonization took place after the ridges had been formed. The pebble-rich horizon occurs a few cen- timeters above the centimeter-thick sandstone layer casting the imprints (Fig. 3a, b). This pebbly horizon suggests the onset of high energy regime with deposition of lag within the channel. Therefore, the sandstone bed illustrates the high energy settings (pebble-grade lag deposition) postdated the period of low energy subaqueous settings when swim traces and U-burrows could form. The sine-shaped morphology suggests that undulatory swim technique was used and an amphibian or amphibious tracemaker produced the studied swim traces. The swim- mers had likely a laterally compressed (deep) tails acting as propellers (cf. Fish 1984; Ashley-Ross and Bechtel 2004). Kuleta et al. (2005) described from the Baranów Forma- tion poorly preserved walking tracks of ?Capitosauroides and assigned the ichnites to amphibians. The studied swim traces support the evidence for the presence of water-adapted animals in the fluvial ecosystem represented by the Baranów Formation. Conclusion In the case of parallel scratches, the tracemakers were likely the same archosaurs that produced tracks and track- ways of Chirotheriidae ichnofamily—the inferred un-webbed foot matches the ichnofamily general track morphology. Fish, F.E. 1984. Kinematics of undulatory swimming in the American alligator. Copeia 1984(4): 839–843. Fish, F.E. 1996. Transitions from drag-based to lift-based propulsion in mammalian swimming. Integrative and Comparative Biology 36(6): 628–641. Sine-shaped, discontinuous imprints were likely pro- duced by swimmers utilizing the undulatory swim technique. Amphibians or amphibious reptiles with laterally flattened tails are likely candidates for the tracemakers. These swim traces provide evidence for the presence of amphibious tet- rapods in the fluvial ecosystem represented by the Lower Triassic Baranów Formation. Fish, F.E. 2000. Biomechanics and energetics in aquatic and semia- quatic mammals: Platypus to whale. Physiological and Biochemi- cal Zoology 73(6): 683–698. Fish, F.E., and R.V. Baudinette. 2008. Energetics of swimming by the ferret: Consequences of forelimb paddling. Comparative Biochem- istry and Physiology Part A: Molecular & Integrative Physiology 150(2): 136–143. Foster, J.R., and M.G. Lockley. 1997. Probable crocodilian tracks and traces from the Morrison Formation (Upper Jurassic) of eastern Utah. Ichnos 5(2): 121–129. Acknowledgements  We are grateful to Martin Lockley (University of Colorado), Sebastian Voigt (Urweltmuseum GEOSKOP/Burg Lichten- berg) and Mike Reich (Munich, Editor-in-Chief of PalZ) and to an anonymous reviewer of PalZ. Their critics, valuable comments and suggestions greatly improved our manuscript. Gradziński, R., and A. Uchman. 1994. Trace fossils from interdune deposits—an example from the Lower Triassic aeolian Tumlin Sandstone, central Poland. Palaeogeography, Palaeoclimatol- ogy, Palaeoecology 108(1–2): 121–138.f Hoff, K.V., N. Huq, V.A. King, and R.J. Wassersug. 1989. The kin- ematics of larval salamander swimming (Ambystomatidae: Caudata). Canadian Journal of Zoology 67(11): 2756–2761. Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. Conclusion The tetrapods swim traces were analyzed from the Lower Triassic fluvial Baranów Formation from the Holy Cross Mountains (central Poland). Two main types of fossilized swim traces can be distinguished in these strata (Figs. 2 and 3): The ridges have smooth surfaces. This may suggest traces of individual digits have not been preserved either due to taphonomic filtration, due to blunt morphology of the foot (e.g., foot with extensive interdigital web and/or no claws) and smothering action of turbulent water flow emerg- ing behind the limb during its backstroke movement (cf. Maglischo 2003; Matsuuchi et al. 2009). The flow deposit- ing the casting sandstone could also smooth the imprint’s morphology. The most characteristic feature of individual ridges is their sine-shaped morphology (Fig. 3a–e). Some shallower ridges (Fig. 3a–d) resemble flute casts (cf. Pollard 1985). However, clustering of ridges (Fig. 3a), sine-shaped morphology of larger ridges (Fig. 3b, d), cross-cutting rela- tionships (Fig. 3d, g) and double well-defined ends seen in some ridges (Fig. 3d) indicate these are trace fossils. Closely spaced relatively short ridges may resemble—at a first glance—tracks/footmarks assignable to turtles. How- ever, the ridges representing digital imprints of turtles form – Parallel scratch sets; – Sine-shaped discontinuous imprints. These two types of ichnites are thought to represent dif- ferent swim techniques and swimmers. The trackways com- posed of scratch sets were likely produced by swimmers paddling with their limbs. These trackmakers, as all animals leaving their traces in the bottom muds, had to move through water which provided buoyancy to them. The studied foot- marks show compact morphology—scratches in a set are in contact. This may indicate the tracemaker’s foot had no interdigital web, contrary to animals responsible for swim 1 176 G. Sadlok, K. Pawełczyk traces composed of widely spaced scratches. Therefore, the tracemakers could be fully terrestrial tetrapods and likely crossed the water body due to their occasional needs (fac- ultative swimmers). Ezquerra, R., S. Doublet, L. Costeur, P. Galton, and F. Pérez-Lorente. 2007. Were nonavian theropod dinosaurs able to swim? Support- ive evidence from an Early Cretaceous trackway, Cameros Basin (La Rioja, Spain). Geology 35(6): 507–510. Fichter, J., and R. Kunz. 2004. New genus and species of chirotheroid tracks in the Detfurth-Formation (Middle Bunter, Lower Triassic) of Central Germany. Ichnos 11(3–4): 183–193. Conclusion If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. Ijspeert, A.J., A. Crespi, and J.M. Cabelguen. 2005. Simulation and robotics studies of salamander locomotion. Neuroinformatics 3(3): 171–195. Jordan, P.A., R.O. Peterson, and K.A. LeDoux. 2010. Swimming wolves, Canis lupus, attack a swimming Moose, Alces alces. The Canadian Field-Naturalist 124(1): 54–56. King, M.J., W.A.S. Sarjeant, D.B. Thompson, and G. Tresise. 2005. A revised systematic ichnotaxonomy and review of the verte- brate footprint ichnofamily Chirotheriidae from the British Tri- assic. Ichnos 12(4): 241–299. Kuleta, M., and J. Nawrocki. 2000. Litostratygrafia i magneto- stratygrafia pstrego piaskowca w północnym obrzezeniu Gór˙ Świętokrzyskich. Kielce-Warszawa: CAG Państwowy Instytut Geologiczny. References Kuleta, M., G. Niedźwiedzki, and T. Ptaszyński. 2005. Nowe stanow- isko z tropami kręgowców z górnego pstrego piaskowca Gór Świętokrzyskich. Przegląd Geologiczny 53(2): 2–6. Ashley-Ross, M.A., and B.F. Bechtel. 2004. Kinematics of the transi- tion between aquatic and terrestrial locomotion in the newt Tari- cha torosa. Journal of Experimental Biology 207(3): 461–474. Leonardi, G., and F.H. de Oliveira. 1990. A revision of the Tri- assic and Jurassic tetrapod footprints of Argentina and a new approach on the age meaning of the Botucatu Formation footprints (Brasil). Revista Brasileira de Geociencias 2(4): 216–229. Avanzini, M., L. Piñuela, J.I. Ruiz-Omeñaca, and J. Garcia-Ramos. 2010. The crocodile track Hatcherichnus, from the Upper Jurassic of Asturias (Spain). New Mexico Museum of Natural History and Science, Bulletin 51: 89–92. Barbraud, C., and H. Weimerskirch. 2001. Emperor penguins and cli- mate change. Nature 411(6834): 183–186. Lockley, M.G. 1991. Tracking dinosaurs: A new look at an ancient world. Cambridge: Cambridge University Press. Bartholomew, G.A., A.F. Bennett, and W.R. Dawson. 1976. Swimming, diving and lactate production of the marine iguana, Amblyrhyn- chus cristatus. Copeia 1976(4): 709–720. Lockley, M.G., and J.R. Foster. 2006. Dinosaur and turtle tracks from the Morrison Formation (Upper Jurassic) of Colorado National Monument, with observations on the taxonomy of vertebrate swim traces. In Paleontology and Geology of the Upper Jurassic Mor- rison Formation, eds. J.R. Foster and S.G. Lucas. New Mexico Museum of Natural History and Science, Bulletin 36: 193–198. Bujok, A. 2007. Zespół skamieniałości śladowych z osadów aluwialno- litoralnych formacji z Baranowa, górny pstry piaskowiec, Góry Świętokrzyskie. Unpublished Master thesis. Sosnowiec: University of Silesia. Maglischo, E.W. 2003. Swimming fastest. Champaign: Human Kinetics. Bujok, A., G. Sadlok, G. Pieńkowski, and G. Niedźwiedzki. 2008. Stop 4—Baranów, Lower Triassic, Upper Spathian. In Ichnological sites of Poland: The Holy Cross Mountains and the Carpathian Flysch. The pre-congress and post-congress field trip guidebook, eds. G. Pieńkowski and A. Uchman, 54–59. Warszawa: Polish Geological Institute. Matsuuchi, K., T. Miwa, T. Nomura, J. Sakakibara, H. Shintani, and B.E. Ungerechts. 2009. Unsteady flow field around a human hand and propulsive force in swimming. Journal of Biomechan- ics 42(1): 42–47. McAllister, J. 1989. Subaqueous vertebrate footmarks from the upper Dakota Formation (Cretaceous) of Kansas, USA. Kansas: 1 3 Triassic tetrapod trace fossils from central Poland 177 Museum of Natural History Occasional Papers, University of Kansas. Somers, M.J., and M.G. Purves. 1996. References Trophic overlap between three syntopic semiaquatic carnivores: cape clawless otter, spotted- necked otter and water mongoose. African Journal of Ecology 34(2): 158–166. McAllister, J., and J. Kirby. 1998. An occurrence of reptile subaque- ous traces in the Moenkopi Formation (Triassic) of Capitol Reef National Park, south central Utah, USA National Park Service, Technical Report NPS/NRGRD/GRDTR-98/1 98, 45–49. Sweeney, J.R., R.L. Marchinton, and J.M. Sweeney. 1971. Responses of radio monitored white-tailed deer chased by hunting dogs. The Journal of Wildlife Management 35(4): 707–716. McCrea, R.T., S.G. Pemberton, and P.J. Currie. 2004. New ichnotaxa of mammal and reptile tracks from the Upper Paleocene of Alberta. Ichnos 11(3–4): 323–339.i Thomson, T.J., and M.L. Droser. 2015. Swimming reptiles make their mark in the Early Triassic: Delayed ecologic recovery increased the preservation potential of vertebrate swim tracks. Geology 43(3): 215–218. McDowall, R.M. 2003. Variation in vertebral number in galaxiid fishes, how fishes swim and a possible reason for pleomerism. Reviews in Fish Biology and Fisheries 13(3): 247–263. Thomson, T.J., and D.M. Lovelace. 2014. Swim track morphotypes and new track localities from the Moenkopi and Red Peak forma- tions (Lower-Middle Triassic) with preliminary interpretations of aquatic behaviors. New Mexico Museum of Natural History and Science Bulletin 62: 103–128. Minetti, A.E., G. Machtsiras, and J.C. Masters. 2009. The optimum finger spacing in human swimming. Journal of Biomechanics 42(13): 2188–2190.i Minter, N.J., and S.J. Braddy. 2006. The fish and amphibian swimming traces Undichna and Lunichnium, with examples from the Lower Permian of New Mexico, USA. Palaeontology 49(5): 1123–1142. Thulborn, R.A., and M. Wade. 1989. A footprint as a history of move- ment. In Dinosaur tracks and traces, eds. D.G. Gillette and M.L. Lockley, 51–56. Cambridge: Cambridge University Press. Nelson, M.E., and L.D. Mech. 1984. Observation of a swimming wolf killing a swimming deer. Journal of Mammalogy 65(1): 143–144. Vallon, L.H., A.K. Rindsberg, and R.G. Bromley. 2016. An updated classification of animal behaviour preserved in substrates. Geo- dinamica Acta 28(1–2): 5–20. Nicosia, U., and M. Loi. 2003. Triassic footprints from Lerici (La Spe- zia, Northern Italy). Ichnos 10(2–4): 127–140. Vila, B., D. Castanera, J. Marmi, J.I. Canudo, and A. Galobart. 2014. Crocodile swim tracks from the latest Cretaceous of Europe. Lethaia 48(2): 256–266.l Niedźwiedzki, G., and T. Ptaszyński. 2007. Large Chirotheriidae tracks in the Early Triassic of Wióry, Holy Cross Mountains, Poland. Acta Geologica Polonica 57(3): 325–342. Walker, R.G., and J.C. Douglas. 1984. Sandy fluvial systems. References In Facies models, 2nd ed, ed. R.G. Walker, 71–89. Toronto: Geoscience Canada. Peabody, F.E. 1947. Current crescents in the Triassic Moenkopi Forma- tion (Arizona). Journal of Sedimentary Research 17(2): 73–76. Pollard, J.E. 1985. Isopodichnus, related arthropod trace fossils and notostracans from Triassic fluvial sediments. Transactions of the Royal Society of Edinburgh, Earth Sciences 76: 272–285. Webb, P. 1988. Simple physical principles and vertebrate aquatic loco- motion. Integrative and Comparative Biology 28(2): 709–725. Provini, P., P. Goupil, V. Hugel, and A. Abouraqchid. 2012. Walking, Paddling, Waddling: 3D Kinematics Anatidae Locomotion (Cal- lonetta leucophrys). Journal of Experimental Zoology Part A: Ecological Genetics and Physiology 317(5): 275–282. Whyte, M.A., and M. Romano. 2001. A dinosaur ichnocoenosis from the Middle Jurassic of Yorkshire, UK. Ichnos 8(3–4): 223–234. Williams, T.M. 1983. Locomotion in the North American mink, a semi-aquatic mammal. I. Swimming energetics and body drag. Journal of Experimental Biology 103(1): 155–168. Ptaszyński, T. 2000. Lower Triassic vertebrate footprints from Wióry, Holy Cross Mountains, Poland. Acta Palaeontologica Polonica 45(2): 151–194. Willson, J.D., C.T. Winne, M.E. Dorcas, and J.W. Gibbons. 2006. Post-drought responses of semi-aquatic snakes inhabiting an iso- lated wetland: insights on different strategies for persistence in a dynamic habitat. Wetlands 26(4): 1071–1078. Sarjeant, W.A.S. 1975. Fossil tracks and impressions of vertebrates. In The study of trace fossils: A synthesis of principles, problems, and procedures in ichnology, ed. R. Frey, 283–324. Berlin: Springer. Wisshak, M., E. Volohonsky, and D. Blomeier. 2004. Acanthodian fish trace fossils from the Early Devonian of Spitsbergen. Acta Palaeontologica Polonica 49(4): 629–634. Seebacher, F., P.G. Elsworth, and C.E. Franklin. 2003. Ontogenetic changes of swimming kinematics in a semi-aquatic reptile (Croco- dylus porosus). Australian Journal of Zoology 51(1): 15–24. Xing, L., M. Avanzini, M.G. Lockley, T. Miyashita, H. Klein, J. Zhang, Q. He, L. Qi, J.D. Divay, and C. Jia. 2014. Early Cretaceous tur- tle tracks and skeletons from the Junggar basin, Xinjiang, China. Palaios 29(4): 137–144. Shukla, U., G. Bachmann, G. Beutler, J. Barnasch, and M. Franz. 2006. Extremely distal fluvial sandstone within the playa system of Arn- stadt Formation (Norian, Late Triassic), central Germany. Facies 52(4): 541–554. Yeager, C.P. 1991. Possible antipredator behavior associated with river crossings by proboscis monkeys (Nasalis larvatus). American Journal of Primatology 24(1): 61–66. Sidelnik, N.O., and B.W. Young. 2006. Optimizing the freestyle swim- ming stroke: the effect of finger spread. Sports Engineering 9(3): 129–135. Simon, T., H. Hagdorn, M.K. Hagdorn, and A. Seilacher. 2003. References Swim- ming trace of a coelacanth fish from the Lower Keuper of South- West Germany. Palaeontology 46(5): 911–926. 1 3 1 3 1 3 3
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https://discovery.ucl.ac.uk/id/eprint/1575239/1/Purton_19_10_2018_Green%20biol.pdf
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Green biologics: The algal chloroplast as a platform for making biopharmaceuticals
Bioengineered
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5,046
Bioengineered ISSN: 2165-5979 (Print) 2165-5987 (Online) Journal homepage: http://www.tandfonline.com/loi/kbie20 Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kbie20 ABSTRACT Most commercial production of recombinant pharmaceutical proteins involves the use of mammalian cell lines, E. coli or yeast as the expression host. However, recent work has demonstrated the potential of eukaryotic microalgae as platforms for light-driven synthesis of such proteins. Expression in the algal chloroplast is particularly attractive since this organelle contains a minimal genome suitable for rapid engineering using synthetic biology approaches; with transgenes precisely targeted to specific genomic loci and amenable to high-level, regulated and stable expression. Furthermore, proteins can be tightly contained and bio-encapsulated in the chloroplast allowing accumulation of proteins otherwise toxic to the host, and opening up possibilities for low-cost, oral delivery of biologics. In this commentary we illustrate the technology with recent examples of hormones, protein antibiotics and immunotoxins successfully produced in the algal chloroplast, and highlight possible future applications. KEYWORDS biopharmaceuticals; chlamydomonas; chloroplast; microalgae; synthetic biology Henry N. Taunt, Laura Stoffels & Saul Purton Henry N. Taunt, Laura Stoffels & Saul Purton To cite this article: Henry N. Taunt, Laura Stoffels & Saul Purton (2018) Green biologics: The algal chloroplast as a platform for making biopharmaceuticals, Bioengineered, 9:1, 48-54, DOI: 10.1080/21655979.2017.1377867 To link to this article: https://doi.org/10.1080/21655979.2017.1377867 © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Accepted author version posted online: 11 Sep 2017. Published online: 29 Sep 2017. Submit your article to this journal Article views: 1324 View Crossmark data Citing articles: 2 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kbie20 BIOENGINEERED 2018, VOL. 9, NO. 1, 48–54 https://doi.org/10.1080/21655979.2017.1377867 Institute of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, Green biologics: The algal chloroplast as a platform for making biopharmaceuticals Henry N. Taunta, Laura Stoffelsb, and Saul Purtonb aAlgenuity, Eden Laboratory, Stewartby, United Kingdom; bAlgal Research Group, Institute of Structural and Molecular Biology, University College London, Gower Street, London, United Kingdom ARTICLE HISTORY Received 7 August 2017 Revised 2 September 2017 Accepted 5 September 2017 © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 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. CONTACT Saul Purton s.purton@ucl.ac.uk Institute of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, U.K. Introduction We have successfully adapted the sys- tem for endolysin and vaccine production in C. reinhardtii (L. Stoffels, B. Parker and S. Purton, submitted). The 40 litre bags are optimally illuminated and sterile 5% CO2/95% air supplied at the base of each bag for phototrophic growth and for mixing. Both batch and continuous operation is possible. ©Supreme Health, New Zealand. Reproduced by permission of Supreme Health, New Zealand. Permission to reuse must be obtained from the rightsholder. Figure 1. A low-cost, single-use photobioreactor system for commercial production of algal biomass. This ‘hanging bag’ sys- tem was developed by the Cawthron Institute, New Zealand for production of microalgae as aquaculture feed and for cultivation of Haematococcus pluvialis – a natural source of the high-value nutraceutical astaxanthin. We have successfully adapted the sys- tem for endolysin and vaccine production in C. reinhardtii (L. Stoffels, B. Parker and S. Purton, submitted). The 40 litre bags are optimally illuminated and sterile 5% CO2/95% air supplied at the base of each bag for phototrophic growth and for mixing. Both batch and continuous operation is possible. ©Supreme Health, New Zealand. Reproduced by permission of Supreme Health, New Zealand. Permission to reuse must be obtained from the rightsholder. Introduction The 40 litre bags are optimally illuminated and sterile 5% CO2/95% air supplied at the base of each bag for phototrophic growth and for mixing. Both batch and continuous operation is possible. ©Supreme Health, New Zealand. Reproduced by permission of Supreme Health, New Zealand. Permission to reuse must be obtained from the rightsholder. reinhardtii. Insertion of transgenes into the small chloroplast genome rather than the nuclear genome offers several clear advantages: not least the ability to do precise and predictable ‘DNA surgery’ in which transgenes are integrated into specific, neu- tral loci within the genome via homologous recom- bination, and stable, high-level, stable expression is readily achieved.11 Furthermore, protein folding and disulphide bond formation occurs readily in the chloroplast allowing the correct assembly of complex therapeutic proteins with multiple domains or multiple subunits, as discussed below. Finally, the chloroplast compartment can serve as a safe sub-cellular site for hyper-accumulation of recombinant protein without affecting the biology of the rest of the cell.12 The growing interest in exploitation of the algal chloroplast is now driving the development of synthetic biology tools by our- selves and other groups that allow a rapid and effi- cient pipeline for design and production of engineered strains. Below we highlight this technol- ogy and give three examples of applications in the field of biopharmaceuticals. Figure 1. A low-cost, single-use photobioreactor system for commercial production of algal biomass. This ‘hanging bag’ sys- tem was developed by the Cawthron Institute, New Zealand for production of microalgae as aquaculture feed and for cultivation of Haematococcus pluvialis – a natural source of the high-value nutraceutical astaxanthin. We have successfully adapted the sys- tem for endolysin and vaccine production in C. reinhardtii (L. Stoffels, B. Parker and S. Purton, submitted). The 40 litre bags are optimally illuminated and sterile 5% CO2/95% air supplied at the base of each bag for phototrophic growth and for mixing. Both batch and continuous operation is possible. ©Supreme Health, New Zealand. Reproduced by permission of Supreme Health, New Zealand. Permission to reuse must be obtained from the rightsholder. Figure 1. A low-cost, single-use photobioreactor system for commercial production of algal biomass. This ‘hanging bag’ sys- tem was developed by the Cawthron Institute, New Zealand for production of microalgae as aquaculture feed and for cultivation of Haematococcus pluvialis – a natural source of the high-value nutraceutical astaxanthin. Introduction in Fig. 1, efficient production of algal biomass can be achieved in a cheap, sterile and disposable polythene tubing system that is easily scaled and managed. Each »40 litre ‘hanging bag’ is bubbled with CO2-enriched air and illuminated directly with sunlight, or indirectly using artificial lighting provided by LEDs powered by sunlight captured using photovoltaic devices. Whilst the latter adds to the capital costs, superior daily bio- mass productivities are obtained through 24 hour illumination using light of optimal intensity and wave- length, and tight control of the culture temperature. Cultivation of the algae uses a simple medium of basic nutrients, thereby keeping media costs as low as $0.002 per liter.8 Importantly, algal species grown commercially for the food ingredients and healthfood markets (e.g. species of Chlorella, Dunaliella and Hae- matococcus) already have GRAS (generally recognized as safe) status. The safety of these species offers the possibility of topical application of a biopharmaceuti- cal such as an anti-microbial protein as a crude cell lysate (e.g. formulated into a cream or spray), and Biopharmaceuticals (protein biologics) is an industry estimated to be worth in excess of $140 billion1 and encompasses products such as monoclonal antibodies, immunotoxins, antigens, hormones, enzymes, clotting factors and bioactive peptides.2 These recombinant proteins are produced mainly using heterotrophic fer- mentation technologies with the biological platforms being either mammalian cell lines such as Chinese Hamster Ovary cells, or microorganisms such as bac- teria or yeasts.3 Whilst these are highly advanced and successful technologies, there is a need for additional platforms that offer new opportunities for the produc- tion of therapeutic proteins. Emerging technologies include virus-mediate transient expression in insect cell lines4 or in tobacco plants,5 and stable expression in the chloroplasts of plants and algae.6,7 The use of unicellular algae as cell factories is par- ticularly attractive as a low-cost, low-tech and sustain- able approach, especially for countries lacking advanced fermentation infrastructures. As illustrated BIOENGINEERED 49 Figure 1. A low-cost, single-use photobioreactor system for commercial production of algal biomass. This ‘hanging bag’ sys- tem was developed by the Cawthron Institute, New Zealand for production of microalgae as aquaculture feed and for cultivation of Haematococcus pluvialis – a natural source of the high-value nutraceutical astaxanthin. We have successfully adapted the sys- tem for endolysin and vaccine production in C. reinhardtii (L. Stoffels, B. Parker and S. Purton, submitted). The C. reinhardtii chloroplast as an emerging synbio platform Chloroplast genomes (or ‘plastomes’) are polyploid circular molecules possessing 100–200 genes, with most encoding core components of the photosyn- thetic apparatus or the organelle’s transcription- translation machinery (Fig. 2). Gene structure and expression is essentially prokaryotic in nature, reflecting the evolution of the chloroplast from a cyanobacterial ancestor. Hence, genes are often arranged as operons, transcribed by a eubacterial- type RNA polymerase and the mRNA translated on 70S ribosomes.13 Chloroplast transformation was first achieved using C. reinhardtii whereby a photo- synthetic mutant carrying a chloroplast gene dele- tion was restored to phototrophy by microparticle bombardment with a plasmid carrying the wild-type gene. Molecular analysis showed that the mutant locus had been repaired through efficient homolo- gous recombination (HR) between sequences on the plastome and the introduced DNA. Since then, C. reinhardtii has been used extensively as a laboratory model for reverse-genetic studies of chloroplast gene expression and photosynthetic function, with therefore avoiding costly investment in purification. Alternatively, it might be possible to use the whole algae for oral delivery (to animals, if not to humans) of vaccines, anti-microbials or hormones – with the dried cells exploited as a natural method of encapsula- tion and storage that overcomes the need for a costly cold chain, and the components of the algal cell possi- bly acting as an effective adjuvant.9 Recent surveys of the literature show that over 50 different biopharmaceuticals have been success- fully produced in microalgae.9,10 Although produc- tion using nuclear genetic engineering is reported for several freshwater and marine species of eukaryotic microalgae, the majority of the research has focused instead on chloroplast engineering using the freshwater green alga Chlamydomonas 50 H. N. TAUNT ET AL. 50 H. N. TAUNT ET AL. Figure 2. The chloroplast genome of Chlamydomonas reinhardtii. Generated from Genbank entry BK000554 using OGDRAW (ogdraw. mpimp-golm.mpg.de). Genes are coloured according to function (e.g. photosystem II genes in dark green), with genes transcribed anti- clockwise on the outer side of the circle; those transcribed clockwise on the inner side. Examples of verified neutral sites for transgene insertion are indicated by arrowheads, with those within the inverted repeat (IR) regions that therefore give rise to two transgene copies per genome shown in light or dark blue. 50 H. N. TAUNT ET AL. Figure 2. The chloroplast genome of Chlamydomonas reinhardtii. Generated from Genbank entry BK000554 using OGDRAW (ogdraw. mpimp-golm.mpg.de). Genes are coloured according to function (e.g. The C. reinhardtii chloroplast as an emerging synbio platform photosystem II genes in dark green), with genes transcribed anti- clockwise on the outer side of the circle; those transcribed clockwise on the inner side. Examples of verified neutral sites for transgene insertion are indicated by arrowheads, with those within the inverted repeat (IR) regions that therefore give rise to two transgene copies per genome shown in light or dark blue. specific gene knockouts or site-directed changes introduced into the plastome through HR-mediated engineering.14 plastome and for regulating the expression of the transgenes.19,20,21 Finally, the development of strate- gies for ‘marker-free’ generation of transgenic lines that avoid the use of antibiotic resistance markers,22 and a technique for bio-containment of the transgene through codon reassignment23 will help to address regulatory issues and public concerns regarding com- mercial cultivation of transgenic microalgae. Further details of these tools are given in Fig. 3. More recently, the focus has shifted to biotechno- logical applications and the development of the C. reinhardtii chloroplast as a protein factory through the addition of novel genes into the plastome to make valuable recombinant products.11 Improvements in the transformation technology have helped to advance this field and we now are beginning to see the application of synthetic biology (synbio) principles. These include gene design in silico using dedicated codon optimization software and validated cis ele- ments such as promoters and untranslated regions.15,16,17 Building the designed constructs in vitro is then aided by rapid assembly of standardized DNA parts using methods such as Golden Gate18 that ensure the ‘one-step’ assembly of multiple parts in the correct order and orientation (Fig. 3). Accompanying this are methods for large-scale refactoring of the Three case studies: Human growth hormone, endolysins and an immunotoxin One element carries a wild-type copy of an essential pho- tosynthetic (p/s) gene allowing phototrophic selection in the recipient chassis that lacks this gene. The synthetic gene-of-interest is codon-optimised and fused to promoter and untranslated region (UTR) parts. Biocontainment can be incorporated into the transgene by replacing one or more tryptophan codons with the UGA stop codon (), thereby preventing function transfer of the gene to other microorganisms. Correct translation in the chloroplast is achieved by inclusion of a part carrying trnWUCA. This gene encodes an orthogo- nal variant of the chloroplast’s tryptophan tRNA that recognises UGA. intrapeptide disulphide bonds, then recombinant pro- duction is feasible using a prokaryotic host. E. coli is currently the preferred platform, although correct folding and bond formation requires export into the periplasm.25 However, the demand for recombinant hGH is huge and growing, with a predicted global market of $4.5 billion by 2022, thereby justifying the exploration of alternative production platforms including chloroplasts. Recent work by our group has demonstrated that functional hGH can be produced in the C. reinhardtii chloroplast by expression of a codon-optimized synthetic gene fused to the promoter and 50 untranslated region of the endogenous psaA gene.22 Yields of hGH in the transformant were approximately 500 mg per liter of culture, so there is a need to increase this significantly before we can com- pete with bacterial platforms. Nevertheless, biological activity could be demonstrated even in crude cell lysates using a standard assay where addition of the lysate specifically stimulated growth of a rat lym- phoma cell line. This work highlights the potential of the algal chloroplast as a future platform for making simple biopharmaceuticals such as hGH, insulin and bioactive peptides. In a second study from our group, the production of endolysins in the C. reinhardtii chloroplast was investigated.26 Endolysins are antibacterial proteins produced during bacteriophage infection that digest the bacterial cell wall for phage progeny release at the end of the lytic cycle. These enzymes typically show a high degree of specificity for the target bacterium of the phage. Furthermore, the emergence of resistance to endolysins appears to be extremely rare. Three case studies: Human growth hormone, endolysins and an immunotoxin Human growth hormone (hGH) is a 22 kDa protein that is produced naturally in the pituitary gland. Defi- ciency of the hormone results in growth defects, but can be successfully treated by administration of recombinant hGH.24 As the only post-translational steps required for biological activity are removal of the N-terminal methionine and formation of two BIOENGINEERED 51 BIOENGINEERED 51 B B C C D D 5’ 3’ E * * E F F G G A H promoter flank L gene-of-interest trnWUCA 5’ UTR 3’ UTR flank R (with p/s marker) deleted p/s gene flank L flank R chassis plastome (p/s deficient) DNA parts 1 ‘one-pot’ assembly of device 2 Introducon into chloroplast 3 Integraon via homologous recombinaon engineered plastome (p/s restored) 5’ 3’ * * transgene device Figure 3. A synbio strategy for creating marker-free transgenic lines that also incorporate a biocontainment feature. Standardised DNA parts are assembled in order using Golden Gate to create the transgene device, with left (L) and right (R) flanking plastome elements (shown as bold lines) added for homologous recombination in the chloroplast. One element carries a wild-type copy of an essential pho- tosynthetic (p/s) gene allowing phototrophic selection in the recipient chassis that lacks this gene. The synthetic gene-of-interest is codon-optimised and fused to promoter and untranslated region (UTR) parts. Biocontainment can be incorporated into the transgene by replacing one or more tryptophan codons with the UGA stop codon (), thereby preventing function transfer of the gene to other microorganisms. Correct translation in the chloroplast is achieved by inclusion of a part carrying trnWUCA. This gene encodes an orthogo- nal variant of the chloroplast’s tryptophan tRNA that recognises UGA. B B C C D D 5’ 3’ E * * E F F G G A H promoter flank L gene-of-interest trnWUCA 5’ UTR 3’ UTR flank R (with p/s marker) deleted p/s gene flank L flank R chassis plastome (p/s deficient) DNA parts 1 ‘one-pot’ assembly of device 2 Introducon into chloroplast 3 Integraon via homologous recombinaon engineered plastome (p/s restored) 5’ 3’ * * transgene device transgene device Figure 3. A synbio strategy for creating marker-free transgenic lines that also incorporate a biocontainment feature. Standardised DNA parts are assembled in order using Golden Gate to create the transgene device, with left (L) and right (R) flanking plastome elements (shown as bold lines) added for homologous recombination in the chloroplast. Resources and future applications Advances in the genetic engineering of the C. rein- hardtii plastome, in particular the application of synbio strategies, have simplified and accelerated the process of creating designer strains expressing therapeutic proteins. In our lab, we have sought to develop a simple, low-cost pipeline that can readily be adopted by other groups, including those in developing countries. Chassis strains and DNA parts are available through the Chlamydomonas Resource Center (www.chlamycollection.org) and our software for codon optimization is free to down- load (github.com/khai-/CUO). Our chloroplast trans- formation protocol simply involves agitation of a cell/ DNA suspension in the presence of glass beads, rather than the use of expensive microparticle bombardment equipment, and we have developed a simple PCR- based method for confirming transgene insertion and homoplasmy of the plastome.22 Our on-going work on scale-up using the hanging bag system shows that this is a cost-effective and easily manageable cultiva- tion method; and biomass productivity could be fur- ther improved through optimization of key parameters such as light, CO2 delivery, mixing and media composition.28 CH2 CH3 CH3 II II III III Ib Ib S S S CH2 variable domains of human antibody against B-cell antigen CD22 hinge and constant domains 2&3 of a human IgG1 antibody domains of Pseudomonas exotoxin A endoplasmic reticulum localization peptide S Figure 4. Illustration of a designer immunotoxin produced in the C. reinhardtii chloroplast showing the multi-domain structure. The human CD22-scFv domain was fused to the hinge and con- stant domains of a human IgG1 and to exotoxin A from Pseudo- monas aeruginosa lacking domain 1a. This created an immunotoxin that formed a homodimer through disulphide bonds between the hinge regions. Redrawn from12. CH2 CH3 CH3 II II III III Ib Ib S S S CH2 variable domains of human antibody against B-cell antigen CD22 hinge and constant domains 2&3 of a human IgG1 antibody domains of Pseudomonas exotoxin A endoplasmic reticulum localization peptide S hinge and constant domains 2&3 of a human IgG1 antibody Currently, recombinant protein yields are low (typi- cally 0.5-5% of total soluble protein) compared to established microbial platforms, but better understand- ing of chloroplast gene regulation and the use of orthogonal mechanisms to induce and drive transgene expression,29 should lead to marked improvements. Indeed, recombinant protein levels achieved in Figure 4. Illustration of a designer immunotoxin produced in the C. reinhardtii chloroplast showing the multi-domain structure. Three case studies: Human growth hormone, endolysins and an immunotoxin Conse- quently, endolysins have potential as protein antibiot- ics, with recombinant forms shown to be highly effective when added to bacterial cultures or bio- films.27 The chloroplast is a particularly attractive plat- form for recombinant production since it mimics the prokaryotic environment where endolysins are pro- duced naturally, but unlike bacterial hosts, it lacks any peptidoglycan cell wall that might be compromised during over-expression of an endolysin gene. Using H. N. TAUNT ET AL. 52 the same strategy as for hGH production, two differ- ent endolysins – Pal (36 kDa) and Cpl1 (40 kDa) – that target the major human pathogen Streptococcus pneumoniae were successfully produced in C. rein- hardtii. Each enzyme showed a high lytic activity against cultures of S. pneumoniae even when pre- sented as crude cell lysates, suggesting that the algal platform could be used for simple, low-cost formula- tions of anti-bacterial creams or sprays targeting topi- cal bacterial infections, or infections of the nasal pharynx. platforms often fail to fold proteins with multiple domains efficiently and are unable to form disulphide bonds. Conversely, production of such cytotoxic pro- teins in eukaryotic hosts such as CHO cells or yeast is not feasible because of the lethal effect of the toxin on the cytosolic translation apparatus. The work of Tran et al.12 demonstrates that the algal chloroplast not only possesses the machinery necessary to fold and assemble complex eukaryotic proteins, but that the 70S ribosomes are unaffected by the toxic protein and the organelle is able to contain the protein preventing any inhibitory effect on the host’s cytosolic ribosomes. The chloroplast therefore offers an attractive platform for efficient production of these highly complex therapeutics. The third case study illustrates a possible niche for the algal chloroplast platform that addresses issues encountered with existing prokaryotic and eukaryotic hosts. Tran et al.12 investigated the synthesis of immu- notoxins in the C. reinhardtii chloroplast. These chi- meric proteins are targeted therapeutics that have applications in cancer treatment, and consist of an antibody domain for binding to the target cell and a cytotoxic enzyme that inhibits proliferation of the cell. As shown in Fig. 4, the immunotoxin is a complex multi-domain protein that requires correct folding and disulphide bond formation to generate the active homodimer. Production of such proteins within bacte- rial hosts is challenging because these expression References [1] Walsh G. Biopharmaceutical benchmarks 2014. Nature Biotechnol. 2014;32:992-1000. doi:10.1038/nbt.3040. [2] Dimitrov DS. Therapeutic proteins. Methods Mol Biol. 2012;899:1-26. doi:10.1007/978-1-61779-921-1_1. PMID:22735943 Possible applications of the algal chloroplast plat- form extend beyond human therapeutics, and are par- ticularly attractive where the cost of production and storage are key issues. For example, microalgae are a natural part of the diet for insect larvae, juvenile shell- fish, fish fry, etc. Consequently, engineered C. rein- hardtii strains have been proposed for oral delivery of toxins to insect pests such as mosquitoes,33,34 or deliv- ery of vaccines and growth hormones to farmed fish and shellfish.35 Similarly, the GRAS status and nutri- tional value of various green algal species opens up the possibility of “functionalized feed” for poultry and livestock whereby dried algae formulated into the feed also contains beneficial vaccines, anti-microbials or dietary enzymes.36 [3] Sanchez-Garcia L, Martın L, Mangues R, Ferrer-Miralles N, Vazquez E, Villaverde A. Recombinant pharmaceuti- cals from microbial cells: a 2015 update. Microb Cell Fact. 2016;15:33. doi:10.1186/s12934-016-0437-3. PMID:26861699 [4] Kost TA, Kemp CW. Fundamentals of baculovirus expression and applications. Adv Exp Med Biol. 2016;896:187-97. doi:10.1007/978-3-319-27216-0_12. PMID:27165326 [5] Chen Q, Davis KR. The potential of plants as a system for the development and production of human biologics. F1000Research. 2016;5(F1000 Faculty Rev):912. doi:10.12688/f1000research.8010.1. [6] Zhang B, Shanmugaraj B, Daniell H. Expression and functional evaluation of biopharmaceuticals made in plant chloroplasts. Curr Opin Chem Biol. 2017;38:17-23. doi:10.1016/j.cbpa.2017.02.007. PMID:28229907 To date, no biopharmaceuticals produced in micro- algae has been approved for commercial production and only a handful have been tested in animal experi- ments. Significant further research and development of microalgal platforms is therefore required. How- ever, conventional production of protein biologics is expensive and technically demanding – requiring cap- ital-intensive fermentation facilities, and costly down- stream processing, cold storage and transportation, and sterile delivery methods. To meet the future needs of the global population, alternative low-tech, low-cost and sustainable production systems such as microal- gae must be considered. [7] Almaraz-Delgado AL, Flores-Uribe J, Perez-Espa~na VH, Salgado-Manjarrez E, Badillo-Corona JA. Production of therapeutic proteins in the chloroplast of Chlamydomo- nas reinhardtii. AMB Express. 2014;4:57. doi:10.1186/ s13568-014-0057-4. PMID:25136510 [8] Rosales-Mendoza S. Algae-based biopharmaceuticals. Plant Biotechnol J. 2017;15:1130-40. [9] Yan N, Fan C, Chen Y, Hu Z. The potential for microalgae as bioreactors to produce pharmaceuticals. Int J Mol Sci. 2016;17:E962. doi:10.3390/ijms17060962. PMID:27322258 [9] Yan N, Fan C, Chen Y, Hu Z. No potential conflicts of interest were disclosed. No potential conflicts of interest were disclosed. [12] Tran M, Van C, Barrera DJ, Pettersson PL, Peinado CD, Bui J, Mayfield SP. Production of unique immunotoxin cancer therapeutics in algal chloroplasts. Proc Natl Acad Sci USA. 2013;110:E15-22. doi:10.1073/pnas.1214638110. PMID:23236148 Funding Chloroplast research in the Purton group is supported by the UK’s Biotechnology and Biological Sciences Research Council (grants BB/L002957/1, BB/F016948/1 and BB/L013789/1). Resources and future applications The human CD22-scFv domain was fused to the hinge and con- stant domains of a human IgG1 and to exotoxin A from Pseudo- monas aeruginosa lacking domain 1a. This created an immunotoxin that formed a homodimer through disulphide bonds between the hinge regions. Redrawn from12. BIOENGINEERED 53 BIOENGINEERED 53 chloroplasts of tobacco have been reported as high as 70% of total soluble protein.30 In addition, protein pro- ductivity could be improved through genetic enhance- ment (“domestication”) of the chassis strains to improve their performance in photobioreactors.31 Alternatively, the chloroplast engineering technology could eventually be transferred to faster-growing and more robust native species of green algae such as Chlo- rella that are better suited to intensive commercial cultivation.32 References The potential for microalgae as bioreactors to produce pharmaceuticals. Int J Mol Sci. 2016;17:E962. doi:10.3390/ijms17060962. PMID:27322258 [10] Hempel F, Maier UG. Microalgae as solar-powered pro- tein factories. Adv Exp Med Biol. 2016;896:241-62. doi: 10.1007/978-3-319-27216-0_16. PMID 27165330 [10] Hempel F, Maier UG. Microalgae as solar-powered pro- tein factories. Adv Exp Med Biol. 2016;896:241-62. doi: 10.1007/978-3-319-27216-0_16. PMID 27165330 [11] Rasala BA, Mayfield SP. Photosynthetic biomanufactur- ing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses. Photosynth Res. 2015;123:227-39. doi:10.1007/s11120-014-9994-7. PMID:24659086 Acknowledgments We thank Supreme Health, New Zealand for their support and advice regarding the cultivation system, and for permission to use the photograph in Fig. 1. [13] Green BR. Chloroplast genomes of photosynthetic eukar- yotes. Plant J. 2011;66:34-44. doi:10.1111/j.1365- 313X.2011.04541.x. PMID:21443621 [13] Green BR. Chloroplast genomes of photosynthetic eukar- yotes. Plant J. 2011;66:34-44. doi:10.1111/j.1365- 313X.2011.04541.x. PMID:21443621 54 H. N. TAUNT ET AL. 4 H. N. TAUNT ET AL. H. N. TAUNT ET AL. 54 growth hormone. Protein Expr Purif. 2013;87:129-35. doi:10.1016/j.pep.2012.11.002. PMID:23168094 [14] Purton S. Tools and techniques for chloroplast transfor- mation of Chlamydomonas. Adv Exp Med Biol. 2007;616:34-45. doi:10.1007/978-0-387-75532-8_4. PMID:18161489 [26] Stoffels L, Taunt HN, Charalambous B, Purton S. Syn- thesis of antimicrobial bacteriophage proteins against Streptococcus pneumoniae in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J 2017; [Epub ahead of print]. doi:10.1111/pbi.12703. PMID:28160380 [15] Purton S, Szaub JB, Wannathong T, Young R, Economou CK. Genetic engineering of algal chloroplasts: progress and prospects. Rus J Plant Physiol. 2013;60:521-8. doi:10.1134/S1021443713040146. [27] Roach DR, Donovan DM. Antimicrobial bacterio- phage-derived proteins and therapeutic applications. Bacteriophage. 2015;5:e1062590. doi:10.1080/ 21597081.2015.1062590. PMID:26442196 [16] Rasala BA, Muto M, Sullivan J, Mayfield SP. Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 50 untranslated region optimization. Plant Biotechnol J. 2011;9:674-83. doi:10.1111/j.1467-7652.2011.00620.x. PMID:21535358 [28] Fu W, Gudmundsson O, Feist AM, Herjolfsson G, Brynjolfsson S, Palsson BØ. Maximizing biomass pro- ductivity and cell density of Chlorella vulgaris by using light-emitting diode-based photobioreactor. J Biotech- nol. 2012;161:242-9. doi:10.1016/j.jbiotec.2012.07.004. PMID:22796827 [17] Specht EA, Mayfield SP. Synthetic oligonucleotide librar- ies reveal novel regulatory elements in Chlamydomonas chloroplast mRNAs. ACS Synth Biol. 2013;2:34-46. doi:10.1021/sb300069k. PMID:23656324 [18] Casini A, Storch M, Baldwin GS, Ellis T. Bricks and blue- prints: methods and standards for DNA assembly. Nat Rev Mol Cell Biol. 2015;16:568-76. doi:10.1038/nrm4014. PMID:26081612 [29] Bock R. Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology. Annu Rev Plant Biol. 2015;66:211-41. doi:10.1146/ annurev-arplant-050213-040212. PMID:25494465 [30] Oey M, Lohse M, Kreikemeyer B, Bock R. Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J. 2009;57:436-45.doi:10.1111/j.1365-313X.2008.03702.x. PMID:18939966 [19] O’Neill BM, Mikkelson KL, Gutierrez NM, Cunning- ham JL, Wolff KL, Szyjka SJ, Yohn CB, Redding KE, Mendez MJ. An exogenous chloroplast genome for complex sequence manipulation in algae. Nucleic Acids Res. 2012;40:2782-92. doi:10.1093/nar/gkr1008. PMID:22116061 [31] Cazzaniga S, Dall’Ostoa L, Szaub J, Scibilia L, Ballottari M, Purton S, Bassi R. Acknowledgments Domestication of the green alga Chlorella sorokiniana: reduction of antenna size improves light-use efficiency in a photobioreactor. Biotechnol Biofuels. 2014;7:157. doi:10.1186/s13068-014- 0157-z. PMID:25352913 [20] Gimpel JA, Nour-Eldin HH, Scranton MA, Li D, May- field SP. Refactoring the six-gene photosystem II core in the chloroplast of the green algae Chlamydomonas rein- hardtii. ACS Synth Biol. 2016;5:589-96. doi:10.1021/ acssynbio.5b00076. PMID:26214707 [32] Dyo YM, Vonlanthen SE, Purton S, Zayadan BK. Evalu- ating new isolates of microalgae from Kazakhstan for bio- diesel production. Rus J Plant Physiol. 2013;60:582-7. doi:10.1134/S1021443713040031. [21] Ramundo S, Rochaix JD. Controlling expression of genes in the unicellular alga Chlamydomonas reinhard- tii with a vitamin-repressible riboswitch. Methods Enzymol. 2015;550:267-81. doi:10.1016/bs. mie.2014.10.035. PMID:25605390 [33] Kumar A, Wang S, Ou R, Samrakandi M, Beerntsen BT, Sayre RT. Development of an RNAi based micro- algal larvicide to control mosquitoes. Malaria World J. 2013;4:6 [22] Wannathong T, Waterhouse JC, Young REB, Economou CK, Purton S. New tools for chloroplast genetic engineer- ing allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Appl Microbiol Biotechnol. 2016;100:5467-77. doi:10.1007/s00253-016- 7354-6. PMID:26887319 [34] Kang S, Odom OW, Thangamani S, Herrin DL. Toward mosquito control with a green alga: Expres- sion of Cry toxins of Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast of Chlamydomonas. J Appl Phycol. 2017;29:1377-89.doi:10.1007/s10811- 016-1008-z. PMID:28713202 [23] Young REB, Purton S. Codon reassignment to facilitate genetic engineering and biocontainment in the chloro- plast of Chlamydomonas reinhardtii. Plant Biotechnol J. 2016;14:1251-60. doi:10.1111/pbi.12490. PMID:26471875 [35] Siripornadulsil S, Dabrowski K, Sayre R. Microalgal vac- cines. Adv Exp Med Biol. 2007;616:122-8. doi:10.1007/ 978-0-387-75532-8_11. PMID:18161496 [24] Richmond E, Rogol AD. Treatment of growth hormone deficiency in children, adolescents and at the transitional age. Best Pract Res Clin Endocrinol Metab. 2016;30:749- 55. doi:10.1016/j.beem.2016.11.005. PMID:27974188 [36] Erpel F, Restovic F, Arce-Johnson P. Development of phy- tase-expressing Chlamydomonas reinhardtii for monogas- tric animal nutrition. BMC Biotechnol. 2016;16:29. doi:10.1186/s12896-016-0258-9. PMID:26969115 j [25] Sockolosky JT, Szoka FC. Periplasmic production via the pET expression system of soluble, bioactive human
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‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools in Canada
Journal of religious education
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Journal of Religious Education (2022) 70:327–340 https://doi.org/10.1007/s40839-022-00182-w Journal of Religious Education (2022) 70:327–340 https://doi.org/10.1007/s40839-022-00182-w Abstract Pope Francis met representatives of the Indigenous peoples of Canada in Rome in April 2022 and in Canada in July 2022. At these meetings he offered sincere apologies for the ways in which the Catholic Church had colluded with the strategy of cultural assimila­ tion of the Indigenous people in Canada. This was especially abhorrent in the residential schools, operated by the Catholic Church (and other Christian churches), that aimed to ‘evangelise’ and ‘civilise’ the Indigenous children. He emphasised that this was a ‘disas­ trous error’ and the ‘overall effects of the policies linked to the residential schools were catastrophic’. He was very clear at both meetings that this abuse was ‘incompatible with the Gospel of Jesus Christ’. These Catholic residential schools and the impact of these schools on the Indigenous people are discussed in this article. Keywords  Catholic residential schools · Canada · Pope Francis Keywords  Catholic residential schools · Canada · Pope Francis ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools in Canada Stephen J. McKinney1 Accepted: 15 September 2022 / Published online: 29 October 2022 © The Author(s) 2022 Stephen J. McKinney Stephen.mckinney@glasgow.ac.uk 1 School of Education, University of Glasgow, Glasgow, Scotland Stephen J. McKinney Stephen.mckinney@glasgow.ac.uk 1 School of Education, University of Glasgow, Glasgow, Scotland 2  Forgotten histories and missing voices There are a number of ways to frame or contextualise the history of the Catholic residen­ tial schools in Canada and other parts of the world. They can be framed as a (misguided) opportunity for evangelisation, for spreading the word of God, or as destructive products of a rapacious colonial system and as instruments of power and social control (Aquash, 2013; Johnson, 2018). These are, of course, highly relevant frames and will be referred to in this article. There are other ways to frame this history using the lenses of forgotten histories and missing voices. Forgotten histories and missing voices are very closely related, but there is a distinction to be drawn between them. All remembering is selective, and this can induce selective forgetting (Stone et al., 2017). Remembering is often used to support the sense of the ‘self-concept’ for an individual per­ son. The self-concept includes the following dimensions but is not synonymous with any of them: self-esteem; self-worth; self-image; ideal self; identities or roles and personal traits and qualities (Ackerman, 2018). A person acts and interacts with others based on their own self-concept and, if others accept this self-concept in interactions, they help to affirm the self-concept for that person. The remembering associated with the self-concept can be a combination of positive and negative memories, but an emphasis on more positive memo­ ries can preclude more negative memories. By extension and adaptation, this can be applied to the concept of an institution that can be held by those in leadership positions or positions of power – and this enables a deeper understanding of the internal conception and external projection of the institution. The insti­ tution can act and interact based on the held concept and this can be affirmed in interactions. The held concept and projection of an institution, for example, can be premised on the view that the institution is socially just, and the remembering and recall will be about the positive times that demonstrate this social justice. This then can lead to a hesitancy or a failure to recall times when social justice was not demonstrated – which would obviously contradict the held concept of the institution. This leads to a forgetting or supressing of histories or parts of histories. Forgotten histories refers to unacknowledged, overlooked, obscured or hidden histories that have not been recalled or remembered. 1  Introduction The visit of Pope Francis to Canada in July 2022 has assumed significant historical impor­ tance. During the visit, the Pope apologised to the Indigenous people for the role of the Catholic Church and religious communities in the cultural and religious abuses they have experienced. These abuses were experienced by many children who had been placed in the Indigenous residential schools. This article discusses the context for these apologies and the apologies themselves in three parts. First, the article will use forgotten histories and missing voices as a framework. This framework will enable a deeper understanding of the institu­ tional significance of the revelations about the treatment of the Indigenous children in the residential schools. Second, the rationale for the residential schools and the treatment of the Indigenous children will be scrutinised, with a focus on religious abuse. Third, the apologies 1 3 3 3 328 S. J. McKinney of Pope Francis will be examined, and some concluding remarks are offered about why it is important to remember this history. of Pope Francis will be examined, and some concluding remarks are offered about why it is important to remember this history. 1 3 2  Forgotten histories and missing voices They may have been forgotten because they were about persons or groups of persons who were not deemed to be important or relevant in the ways in which the narratives of the history of the institution have been configured and been projected. For example, the stories of lower placed members of the institution or women may have been forgotten because other narratives were more prominent, such as hierarchical and male dominated narratives. Or they were forgotten because the history was supressed as it exposed uncomfortable or shameful attitudes, actions or events that disrupted the held concept and projection of the institution. There is a danger that the held conception becomes a form of self-deception. These forgotten histories will include the missing voices from the past which were not considered to be important or were deliberately excluded. It would be a mistake to consign missing voices solely to past periods of time, to periods of forgotten history. Missing voices can refer to the present and voices that are missing 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 329 in the contemporary world. Within the Catholic Church, missing voices still refers to the voices of women, including Catholic women religious who are scrutinised, at times, and excluded by male hierarchies (Brigham, 2015). There are signs that Pope Francis is attempt­ ing to address this, and good recent examples have included the appointment of four women (three religious and a lay woman) as consultors to the secretariat of the Synod of Bishops and, at the time of writing, his intention to appoint two women to the Dicastery that helps him select bishops (Lamb, 2019, Vatican news, 2022a). Missing voices still refers to the voices of children and groups of children who are not heard. Very recently it has referred to the voices of children who identify as LGBTQ in Catholic schools (Huchting & Fisher, 2019). A focus on forgotten histories and missing voices can have a positive outcome in the sense that some extraordinary histories can be uncovered, and missing voices heard and highlighted. The life of Saint Katherine Drexel, foundress of the sisters of the Blessed Sac­ rament in America is a good example of a Catholic educator who began to listen to the missing voices. She divided opinion during her lifetime and her legacy continues to divide opinion (Lipperini, 2013; Morton, 2014, 2022). 2  Forgotten histories and missing voices For some she brought the message of Christ to the unchurched black masses, to others she ‘imposed the U.S. Catholic Church’s Jim Crow policies on Black education in the South’. Morton (2022, 19–20) argues for a move away from binary understandings and a more nuanced vision of the process of her life work: In the Afro-Catholic Diaspora, Mother Katherine is neither hero nor villain; she is a beloved witness to this self-determined Black Catholic education…Mother Katherine experienced a shift from being a missionary to unchurched black souls to becoming an accomplice to the holistic survival of Black people – mind, body, and spirit. Within this analysis of the work of Saint Katherine one of the greatest parts of the devel­ opment of her vocation was that she began to listen to the missing voices of the African Americans. Within this analysis of the work of Saint Katherine one of the greatest parts of the devel­ opment of her vocation was that she began to listen to the missing voices of the African Americans. While it is helpful to examine the more positive outcomes of a nuanced approach to for­ gotten histories, it is clear that forgotten histories and missing voices often refer to darker histories that have been deliberately forgotten or suppressed. Forgotten histories and abuses of power are being uncovered in the sex abuse scandals that have emerged in many Chris­ tian churches. The sex abuse has taken many forms and the impact of the scandals, certainly in the Catholic Church, has been exacerbated by the attempts to cover up or to deny, and even to bury the scandals (Dale & Alpert, 2007; Formicola, 2016) In other words trying to ensure they remain forgotten histories and that the missing voices of the victims of abuse are suppressed. This is beginning to be researched from different sociological, legal and theological perspectives (Moran, 2019; Formicola, 2020). 3  Indigenous children in Catholic schools – Australia and the USA Forgotten histories and missing voices are revealed in the contemporary histories of the treatment of the children of Indigenous peoples by the Catholic Church in various locations including Canada, USA and Australia. In all three countries, the aim was to assimilate, not integrate, the Indigenous children into state languages, cultures and even accepted forms of 1 3 330 S. J. McKinney religion in the 19th and 20th centuries. This was partly a legacy of the Doctrine of Discovery and a series of papal bulls that had been used from the 15th century to legally justify the colonisation by the European powers (Miller, 2019). religion in the 19th and 20th centuries. This was partly a legacy of the Doctrine of Discovery and a series of papal bulls that had been used from the 15th century to legally justify the colonisation by the European powers (Miller, 2019). This proposed assimilation was of a limited nature, in that it did not provide equal oppor­ tunities for the Indigenous children. It usually meant assimilation into the lowest socio- economic status and conditions: the boys were prepared for manual labour or farming, and the girls were prepared for some form of domestic service (Smith, 2009). It is useful to note that the assimilation in the early days did not extend to citizenship as this was only granted to all Indians in the United States in 1924 and in Canada in 1960 (Dawson, 2012). In Aus­ tralia the 1967 Referendum granted rights and equality to the Aboriginal people and Torres Strait Islanders – citizenship was granted in different states at different times prior to this (Gordon, 2018). Zucker (2008) notes that the assimilation policy in Australia did not recognise the Aboriginal culture. The Aborigines were considered to have no culture, no history and to be without spirituality. The Indigenous people would be improved by adopting the superior white way of life and the Christian religion. Indigenous children were separated from their parents to be educated into Christianity and in many places by Catholic missionaries. While different stories have emerged, and some suggest that the children were often treated better in the homes run by the Catholic Church than government institutions, the children were still separated from their families and for a specific purpose – evangelisation and civilisation. 3  Indigenous children in Catholic schools – Australia and the USA A similar story unfolds in the United States of America where a recent investigation has confirmed that the United States was responsible for supporting 408 boarding schools across 37 states between 1819 and 1969 (United States Department of the Interior, 2022). The Catholic Church was funded to run some of the Federal Indian boarding schools, as were other Christian denominations. Smith (2009, 5–6) argues that this was conceived as part of a deliberate choice of cultural genocide rather than physical genocide because, in the context of the United States of America, cultural genocide was perceived to be more economically efficient. Cultural genocide or ‘ethnocide’ does not involve the mass death of physical geno­ cide but the attempt to ‘destroy culture, language and religion’ and steal land and outlaw customs (MacDonald & Hudson, 2012, 442). The rejection of the spirituality and religious practices of the children and the imposition of Catholic Christianity were components of the cultural genocide that had tragic consequences. The recent investigation has prompted the United States bishops to seek a greater understanding of this history, promising to share their findings and create a greater culture of inclusion (Ruff, 2022). 4  Indigenous children in Catholic schools – Canada In Canada the strategies to ‘educate’ the Indigenous children (First Nations, Métis, and Inuit children) began before the opening of the residential schools in the late nineteenth century. There was an unsuccessful attempt at a Catholic boarding school for Aboriginal boys in the early 17th century (The Truth and Reconciliation Commission of Canada, 2015a). Sir Pere­ grine Maitland, the lieutenant Governor of Upper Canada proposed a plan in the early 1820s to ‘civilise’ the Indians that would focus on the young as it was expected that this would be more successful than focussing on adults (Hutchings, 2016). Maitland’s chief advisor, John Strachan, who would become Toronto’s first Anglican bishop, believed that Aboriginal 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 331 children should be separated from their families and raised by pious white people. A number of small boarding schools, operated by different denominations, were set up in the early to mid 19th century. children should be separated from their families and raised by pious white people. A number of small boarding schools, operated by different denominations, were set up in the early to mid 19th century. In the latter part of the 19th century, Nicholas Davin was commissioned by Sir John A. Macdonald Canada’s first prime minister, to write a report on Industrial Schools for Indi­ ans and Half-Breeds (published in 1879) (Indian Residential School History and Dialogue Centre Collections, 2022). Davin drew on the example of the Industrial schools in America which were deemed to be a ‘principal feature of the policy of aggressive civilisation’ (Davin, 1979). The evidence from America was that the day school did not work ‘because the influ­ ence of the wigwam was stronger than the influence of the school’. Christian churches were, in the opinion of Davin, best placed to supplant Aboriginal spirituality with a better one (Johnson, 2018). Macdonald justified the residential schools in 1883 as follows: When the school is on the reserve the child lives with its parents, who are savages; he is surrounded by savages, and though he may learn to read and write his habits, and training and mode of thought are Indian. He is simply a savage who can read and write. 4  Indigenous children in Catholic schools – Canada It has been strongly pressed on myself, as the head of the Department, that Indian children should be withdrawn as much as possible from the parental influence, and the only way to do that would be to put them in central training industrial schools where they will acquire the habits and modes of thought of white men. The first industrial residential school was opened by the Canadian government in Battlefield in 1883 and the strategy to ‘Christianise’ and ‘civilise’ would be put into operation in many more Government funded residential schools, the majority of which were operated by the Christian churches until 1969 (White & Peters, 2009; Morcom, 2017). Over 150,000 chil­ dren attended these schools between the late nineteenth century and the late 1990s when the remaining federally supported schools were finally closed. These schools were founded on the principles of the inherent superiority of whites over the uncivilised and savage Indians and attempted to implement the policy of the ‘aggressive civilisation’ of the children, as described by Davin in his 1879 report (Bombay et al., 2014; Hanson et al., 2020). Similar to America, this was an attempt at cultural genocide. Part of the impetus for the cultural genocide was to ensure the acquisition of the lands occupied by the Indigenous peoples and the natural resources on these lands (Rose, 2018). The Indian Act of 1920 allowed the government to ‘compel any First Nations child to attend residential school’, but it was never compulsory for the children to attend residential schools and some children attended day schools (The Truth and Reconciliation Commission of Canada, 2015a, 62). These government funded residential schools were run on behalf of the government by the Catholic Church, the Anglican Church, the Presbyterian Church, the Methodist Church and later the United Church and other denominations. The Catholic Church was responsible for approximately 60% of the residential schools, the Anglican Church 30% and the remaining 10% was between the other denominations (Feir, 2016). The schools provided religious instruction and basic academic and industrial skills. On arrival the children were given a new name (Euro-Canadian) to replace their Aborigi­ nal name and were given a number (The Truth and Reconciliation Commission of Canada, 2015a). It is reported that in many schools the numbers were used daily rather than names. Their hair was cut, and they swapped their traditional clothing for uniform. 4  Indigenous children in Catholic schools – Canada In many of the 1 3 3 332 S. J. McKinney schools, children were punished if they practiced their own customs/culture or used their own language. Most of the children spoke an Aboriginal language and had scant or no understanding of French or English, the languages used in the schools. This meant that the arrival at the school was a bewildering and frightening experience for the children as they could not communicate and could not understand what was being said to them. Over time, some children lost fluency in their own language or abandoned their own tongue. There were exceptions and in one of the Alberta schools, Fr Mullen translated the Bible and hymns into Cree for the children. In some schools the children were free to converse in their own languages in the playground. Brothers and sisters were separated in the schools. There was a high death rate due to maltreatment, poor sanitation, under nourishment and inadequate health care (Mosby & Galloway, 2017; Wilk et al., 2017). Clearly the basic needs were not met for many of the children in terms of care, nourishment and health. Many of the survivors of the residential schools would suffer from long term physical and mental health problems. 5  Truth and Reconciliation Commission of Canada The Truth and Reconciliation Commission of Canada was set up in 2008 to reveal the ‘complex history and on-going legacy of the church-run residential schools to the Canadian people’ and ‘guide and inspire’ a process that would lead to reconciliation. The Commission travelled throughout Canada for six years to hear the voices of the Aboriginal people who had been educated in the residential schools and produced a lengthy report in 2015 with ninety-four Calls to Action (The Truth and Reconciliation Commission of Canada, 2015a). A previous Commission had produced the Report of the Royal Commission on Aboriginal Peoples in 1996 that urged for a reconciliation process in Canada. This 400-page report and the 440 recommendations initially received some media attention but was largely ignored by the government (Hurley & Wherrett, 1999). Nevertheless, this report did begin the process of raising wider awareness of the treatment of the Aboriginal people (McGregor, 2018). The Truth and Reconciliation Commission of Canada provided a second opportunity to examine and understand how the Aboriginal people had been treated and seek reconciliation. The Truth and Reconciliation Commission of Canada (2015a, 1) used the term cultural genocide and distinguished cultural genocide from physical and biological genocide: Physical genocide is the mass killing of the members of a targeted group, and biologi­ cal genocide is the destruction of the group’s reproductive capacity. Cultural genocide is the destruction of those structures and practices that allow the group to continue as a group. States that engage in cultural genocide set out to destroy the political and social institutions of the targeted group. Land is seized, and populations are forcibly transferred, and their movement is restricted. Languages are banned. Spiritual lead­ ers are persecuted, spiritual practices are forbidden, and objects of spiritual value are confiscated and destroyed. And, most significantly to the issue at hand, families are disrupted to prevent the transmission of cultural values and identity from one genera­ tion to the next. 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 333 The Commission concluded that this separation of children from their families meant that ‘the Canadian Government essentially declared the Aboriginal people to be unfit parents’ (The Truth and Reconciliation Commission of Canada, 2015a 4). The Commission heard over 6,000 testimonies from former students of the residential schools, family members, community members and interested participants. 5  Truth and Reconciliation Commission of Canada Some of the testimonies were from former staff of the residential schools. The missing voices began to be heard in the testimonies of the adults who had been forced to attend the residential schools as children (Sedehi, 2019). The testimonies speak about physical, sexual, mental, cultural and religious abuse. 6  Religious abuse This section draws on the testimonies of survivors of Catholic residential schools as recounted to The Truth and Reconciliation Commission of Canada (The Truth and Recon­ ciliation Commission of Canada, 2015b). Some former students appreciated the Christian upbringing they received through religious education and the daily rituals of prayer. How­ ever, there is also evidence of religious abuse that took a number of forms. First, the children were not allowed to practice their own religion and spirituality, and some were threatened with punishment if they participated in their own rituals. In some cases, this even applied to participation in rituals that were practiced outside school term time (Johnson, 2018). Sarah McLeod recalls returning to Kamloops school with a small totem pole, a birthday present. This was taken from her by one of the nuns and discarded; the nun described it as being ‘all devil’. Second, the imposition of a strict regime of daily Christian prayer was common: morn­ ing and evening prayers, before and after meals and before classes. Antonette White who had attended the Catholic school at Kuper Island near Vancouver Island, British Columbia commented: I think the worst thing, is the praying. It’s, it’s like you pray, pray, pray, and yet there’s still no peace in that prayer of what they made you do. Noel Starblanket attended the Lebret (Qu’Appelle, St. Paul’s, Whitecalf) Industrial School. This was operated by the Missionary Oblates of Mary Immaculate and the Grey Nuns from 1884 to 1973 (University of Regina, 2022). He recalls a very similar strict regime, as does Victoria Boucher-Grant who attended Fort William (St. Joseph’s) school, Ontario: I learned how to pray. I learned how to, this became a way of life, kneeling on my knees, and praying to, to some, some God that made me feel guilty because I was, I was not a very clean person. Third, fear of the judgment of God and the images of the devil and hell were used to coerce children in a number of ways. Joseph Martin Larocque at the Qu’Appelle school remembers being scared of images of the devil with a pitchfork in religious education classes. Father Lacombe’s instructional ladder, a pictorial catechism, was used in a number of the Catholic schools. This represented stairs leading to heaven and Jesus and the angels. At the bottom of 1 3 334 S. J. 6  Religious abuse McKinney the stairs was fire, and according to Fred Bass, Indian people. Bass stated that he was told at the Roman Catholic school, Kamsack, Sastatchewan that if he and the other children did not change their ways, they would end up in the fire. Strict prohibition of the children’s spiri­ tuality and the denial of their right to practice their rituals and the imposition of Catholic religious instruction and practice, then, were used as weapons to evangelise and catechise. 7  The Christian churches and Canada apologise The apologies by the Christian churches were for the collusion with the assimilationist poli­ cies of colonialism and for the work of the churches in the residential schools. These apolo­ gies began with the United Church of Canada in 1986 apologising for being an agent of colonialism (Bush, 2015). At this point, there was no explicit apology for the collusion with the Residential school system. Later apologies included contrition for involvement in the residential schools. Two Anglican bishops apologised in 1991 and 1993 and the Presbyte­ rian Church in 1994. In the Catholic Church the apologies were issued, most notably, by the Missionary Oblates of Mary Immaculate and different dioceses. On 24 July, 1991 Fr Doug Crosby, then President of the Oblates apologised on behalf of the members of the order for the damage caused by the schools they operated (Robinson, 2019). He apologised for three things. In the first instance, he apologised for the existence of the schools that separated chil­ dren from their families. In the second instance, he apologised for the physical and sexual abuse. In the third instance, he apologised for dismissing the Indigenous religious tradition and the attempts at assimilation. On June 11, 2008 Stephen Harper, the Prime Minister of Canada, apologised on behalf of Canada for the role of the residential schools in the policy of assimilation. 1 3 9  Pope Francis and the apologies to the Indigenous people in Canada The Truth and Reconciliation Commission of Canada (2015a, 221) noted that that the Roman Catholic Church did not have a single spokesperson and the apologies had been issued by different diocese and the religious organisations. On April 29, 2009, Pope Benedict XVI met with Mr Phil Fontaine, the Grand Chief of the Assembly of First Nations of Canada and The Most Reverend James Weisbgerber, President of the Canadian Conference of Bishops (Vatican, 2009). In the short communique that was released, the Pope expressed his ‘sorrow at the anguish caused’ by the church involvement in the residential schools. These words of regret were not an official apology and he did not use the word apology. The Commission contrasted this with the pastoral letter of Benedict issued in Ireland in 2010 as a response to the child abuse in the church in Ireland. In this letter, the Pope stated: ‘You have suffered grievously and I am truly sorry’ (Pope Benedict, 2010, Sect. 6). This led the Commission to develop Call to Action number 58 which called upon the Pope to issue an apology to survivors and their families and communities for the ‘spiritual, cultural, emotional, physi­ cal and sexual abuse’ of the children in the Catholic residential schools. They called for an apology similar to the pastoral letter of 2010 and that the apology should be delivered by the Pope one year after he received the report of the Commission (The Truth and Reconciliation Commission of Canada, 2015a, 223, 330). On July 24 to 30, 2022, Pope Francis visited Canada on an apostolic journey. On July 25, he met with the Indigenous people (First Nation, Métis and Inuit) of Canada and described his visit as a penitential pilgrimage (Pope Francis, 2022a). The Pope had previously met some representatives of the Indigenous people in Rome in March/April 2022. At that meet­ ing in Rome, the Pope apologised for the ‘suffering, hardship, discrimination’ and abuse experienced by the people and he drew attention to the role of the residential schools in the attempt to rob the Indigenous people of their cultural identity. Pope Francis stated that all of this had made him feel indignation and shame very strongly (Pope Francis, 2022b). He was emphatic that when confronted with historical memory we must have a commitment to learn from past mistakes. 8  The unmarked graves The report of the discovery of unmarked graves of 215 Indigenous children in 2021 at the site of Kamloops Indian Residential School in South Central British Columbia, and later at the sites of other former residential schools, once again highlighted the tragic consequences of the injustices of the residential schools (Thorne & Moss, 2022). Kamloops was run by the Catholic Church. The exact number of children who died may never be known because of the practice of poor record keeping (Supernant, 2022). There are parallels in other parts of the world. In Scotland, the unmarked grave of possi­ bly as many as 400 children is in the cemetery of St. Mary’s Parish Church Lanark (Gamble, 2018). The children had been residents at the Smyllum Park School run by the Daughters of Charity as an orphanage and for poor children (1864–1981) and they were buried in the ‘Smyllum plot’ in the graveyard. While the children appeared to have died from natural causes and economic cost may explain the lack of headstones, this is within the context of the grim evidence of emotional, physical and sexual abuse towards children in the school that has been uncovered in the Scottish Child Abuse Inquiry Case Study no. 1 (Scottish Child Abuse Inquiry, 2018). The unmarked graves in Canada and Scotland are chilling indicators that the children were not to be remembered and would be part of forgotten history. This recovered history 1 3 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 335 of the unmarked graves and the abuse experienced by many of the Indigenous children in the residential homes in Canada demonstrates how far removed the care for the children was from the conception of social justice that is espoused by Catholic Social Teaching. The inherent God-given human dignity of all individuals as revealed in sacred scripture which underpins contemporary Catholic Social Teaching was not recognised (Genesis 1:26–27; Pontifical Council for Justice and Peace, 2004). Christian ‘dignity’ was imposed with a European cultural form of Catholic Christianity with terrible consequences. 9  Pope Francis and the apologies to the Indigenous people in Canada The Pope was very clear that the abuses the Indigenous people had experienced were ‘contrary to the Gospel of Jesus Christ’. In his address at Maskwacis in Canada on July 25, 2022, the Pope apologised again for the devastation of the Indigenous families and their culture caused by the assimilationist policies and the residential schools. He drew attention to the role of Catholic Church and the religious communities in the cultural destruction and the physical, verbal, psychological and spiritual abuse of the children in their care. Pope Francis emphasised that the ‘overall effects of the policies linked to the residential schools were catastrophic’ and that this was a ‘disastrous error’. Once again, he was very clear that this abuse was ‘incompatible with the 3 3 336 S. J. McKinney Gospel of Jesus Christ’. Pope Francis recognised the importance of memory and recovering the forgotten histories (Pope Francis, 2022a). The Pope acknowledged that the public apol­ ogy is only a first step in a process that must seek to ensure there is no repetition of the dam­ aging culture and practices that have caused such harm for a vulnerable group of people. In the second address on July 25, 2022, at a meeting with Indigenous peoples and mem­ bers of the parish community of Sacred Heart Edmonton, the Pope again recalled the role of the residential schools in ‘robbing communities and individuals of their cultural and spiri­ tual identity’ (Pope Francis, 2022c). This was part of the ‘violation of dignity, the experience of evil, the betrayal of trust’ and shameful for believers. He does not offer a simple solution but urges that the way forward is to be reconciled in the crucified Christ, and he used this powerful image to stress the betrayal that took place in the residential schools: This is the way forward: to look together to Christ, to love betrayed and crucified for our sake; to look to Christ, crucified in the many students of the residential schools. If we truly want to be reconciled with one another and with ourselves, to be reconciled with the past, with wrongs endured and memories wounded, with traumatic experi­ ences that no human consolation can ever heal, our eyes must be lifted to the crucified Jesus; peace must be attained at the altar of his cross. 9  Pope Francis and the apologies to the Indigenous people in Canada Pope Francis, despite limited physical mobility, undertook the penitential pilgrimage to deliver frequent apologies to the Indigenous Peoples for the harm inflicted on them and damage caused by the cultural suppression in the residential schools. The repeated apologies emphasised the severity of the harm and damage. His final public statements were delivered in a Homily at Vespers with Bishops, Priests, Deacons, Consecrated Persons, Seminarians, and Pastoral Workers in the Cathedral of Notre Dame in Quebec on July 28, 2022 (Pope Francis, 2022d). He discussed the cultural coercion of the Indigenous peoples and also asked forgiveness of the victims of sexual abuse. 10  Concluding remarks This article has examined the history of the residential schools in Canada, the meetings of Pope Francis with the Indigenous people of Canada and his apologies for the cultural and religious injustices imposed on the Indigenous people and the grievous harm caused by the residential schools. This has been discussed within the frameworks of forgotten histories and the missing voices of the survivors of the Catholic residential schools. The Pope has listened to the voices and urges that the history is investigated, and a healing process can begin. The Pope did not refer to the term cultural genocide during the encounters with Indig­ enous people in Rome and Canada, despite this being the term used by the Truth and Recon­ ciliation Commission of Canada. However, he did use this term in his response to a reporter on the flight back to Rome from Canada. Pope Francis said the following (Vatican News, 2022b): …I didn’t use the word because it didn’t come to my mind, but I described the geno­ cide and asked for forgiveness, pardon for this activity that is genocidal. For example, 1 3 337 …and yet there’s still no peace’ Catholic Indigenous Residential Schools… I condemned this too: taking away children, changing culture, changing mentality, changing traditions, changing a race, let’s put it that way, an entire culture. Yes, geno­ cide is a technical word. I didn’t use it because it didn’t come to my mind, but I described it… It’s true, yes, yes, it’s genocide. It is vitally important that the forgotten history of the Indigenous Residential schools is being recovered and the missing voices are heard. Education is needed to ensure that people, especially non-Indigenous people in Canada and beyond, are aware of this history (Neufeld et al., 2022). This history impacts not only on the Canadian people but also on the associ­ ated Christian churches and, for the purposes of this study, especially the Catholic Church. Education in this history is crucial for current and future generations – that they avoid the deeply harmful mistakes of the past (Kuhl, 2017).i Significant numbers of the children did not receive an adequate education in the harsh environment of many of the residential schools. 10  Concluding remarks The education in the residential schools, for the most part, did not cohere with the vision of education described by Pope Francis in his address to the parish community of Sacred Heart Edmonton (Pope Francis, 2022c): Education must always start from respect and the promotion of talents already present in individuals. It is not, nor can it ever be, something pre-packaged and imposed. For education is an adventure, in which we explore and discover together the mystery of life. Further, the Catholic culture and religious education in the residential schools did not cohere with the vision of Catholic education based on the love of Christ that is outlined in the series of contemporary church documents on Catholic education. This series begins with Gravissi­ mum Educationis (1965) through The Catholic School (1977) to the Identity of the Catholic School for a Culture of Dialogue (2022) (Pope Paul VI, 1965; The Sacred Congregation for Catholic Education, 1977; Congregation for Catholic Education 2022).f These documents offer a vision of Catholic education that is free of coercion; an educa­ tion that is not imposed to support a strategy of cultural genocide. This education respects the inherent God-given dignity of every person and aims to ensure that all people have opportunities to develop their talents and gifts, enabling them to contribute to the com­ mon good of society. Often, the children in the residential schools were not provided with sufficient opportunities to develop their talents and gifts - opportunities which would have enabled them to fulfil their potential and make a meaningful contribution to the common good of society (Brady, 2010). Funding  The author did not receive support from any organisation for the submitted work. Declarations Conflict of interest  The author declares that he has no conflict of interest. The author has no relevant financial or non-financial interests to disclose. 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Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. irshdc.ubc.ca/index.php/Detail/objects/9427 (2017). Residential schools and the effects on Indigenous health and well-being in Canada—a scoping review. Public Health Reviews, 38(8), 1–23. https://doi.org/10.1186/ s40985-017-0055-6 Zucker, M. (2008). Open hearts: the Catholic church and the stolen generation in the Kimberley. Journal of the Australian Catholic Historical Society, 29, 23–37. https://link.gale.com/apps/doc/A190243415/ AONE?u=glasuni&sid=googleScholar&xid=eca28645 Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3 1
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Physiological potential of bean seeds as a function of population densities on winter crop season, in northern Minas Gerais
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415 415 Original Article Hugo Tiago Ribeiro AMARO1; Andréia Márcia Santos de Souza DAVID2; Abner José de CARVALHO3; Ignacio ASPIAZÚ3; Izabel Costa SILVA NETA4; Marcos Gleidson Pereira dos SANTOS5 1. Engenheiro Agrônomo, Doutorando em Fitotecnia, Universidade Federal de Viçosa – UFV, Viçosa, MG, Brasil, htiagoamaro@yahoo.com.br; 2. Engenheira Agrônoma, Professora Doutora do Departamento de Ciências Agrárias, Universidade Estadual de Montes Claros – UNIMONTES, Janaúba, MG, Brasil; 3. Engenheiro Agrônomo, Professor Doutor do Departamento de Ciências Agrárias – UNIMONTES, Janaúba, MG, Brasil; 4. Engenheira Agrônoma, Doutoranda em Agronomia/Fitotecnia, Universidade Federal de Lavras – UFLA, Lavras, MG, Brasil; 5. Engenheiro Agrônomo, Doutorando em Fitotecnia – UFV, Viçosa, MG, Brasil. ABSTRACT: For adapting well to various soil and climatic conditions in Brazil and the tradition of high consumption, bean plays a key role in feeding the population. Despite the different environmental conditions, there are few specific cultivars for each planting season, particularly when aimed at the production of quality seeds. The objective of this study was to evaluate the physiological potential of bean seeds from five population densities, under the edafoclimatic conditions of Janaúba, northern Minas Gerais. The experiment was conducted on winter crop season, with planting in August 2011. The experimental design was a randomized block in 3 x 5 factorial, with four replicates. The treatments consisted of three cultivars of common bean with different growth types (Ouro Vermelho, Ouro Negro and BRSMG Madrepérola), and five densities (100, 200, 300, 400 and 500 thousand plants ha-1). Seeds were evaluated for water content, germination and vigor (tests of first count, seedling emergence, emergence speed and accelerated aging). From the results, it can be concluded that the bean seeds of the cultivar Ouro Negro present superior physiological potential in relation to the cultivars Ouro Vermelho and BRSMG Madrepérola. Increasing the sowing density up to 300 thousand plants ha-1 is suitable for the production of bean seeds on winter crop season, representing a good alternative for cultivation in the region. KEYWORDS: Phaseolus vulgaris L. Plant populations. Germination. Force. DESEMPENHO FISIOLÓGICO DE SEMENTES DE FEIJÃO-COMUM CULTIVADO EM DIFERENTES DENSIDADES POPULACIONAIS NA SAFRA DE INVERNO, NO NORTE DE MINAS GERAIS Hugo Tiago Ribeiro AMARO1; Andréia Márcia Santos de Souza DAVID2; Abner José de CARVALHO3; Ignacio ASPIAZÚ3; Izabel Costa SILVA NETA4; Marcos Gleidson Pereira dos SANTOS5 MATERIAL AND METHODS Base Mg)/T] x 100, in which T = K + Ca + Mg + (H+Al); H+Al extracted by a solution of calcium acetate 0,5 mol L- planting. Based on the results of chemical analyzes of samples collected in the experimental area, it was observed that there was no need for lime application. The beans fertilization was based on official recommendation for the State of Minas Gerais for Level 3 technology (CHAGAS et al., 1999) and consisted on the application of 375 kg ha- 1 of the 04-30-10 formulation at planting, plus 40 kg ha-1 of nitrogen using urea as a source, applied to the soil in the form of a continuous bead during the V3- V4 stage of the crop. In addition, it was carried out a foliar application of 40 g ha-1 of molybdenum, using sodium molybdate as a source. Before sowing, the seeds were treated with mixtures of the fungicides carboxin and thiram at a dosage of 300 mL 100 kg-1 of seeds. The experimental design was a randomized block in a 3 x 5 factorial scheme, with four replications. The treatments consisted of three cultivars of common bean with different types of growth (Ouro Vermelho, Ouro Negro and BRSMG Madrepérola), and five population densities (100, 200, 300, 400 and 500 thousand plants ha-1). The cultivar Ouro Vermelho has grains of the type red commercial group, 80-90 days cycle, semiprostrated size and growth habit type II/III (indeterminate). On the other hand, the Ouro Negro presents grains of the black type commercial group, 80-100 days cycle, and growth habit type III (indeterminate) and prostrated size (PAULA JUNIOR et al., 2010). The BRSMG Madrepérola presents grains of the carioca commercial group, 80 days cycle, prostratedsize and, growth habit type III (indeterminate). Sowing was done manually, using hand seeders, adopting the optimum sowing density with the aid of graduated wooden rulers marked for each sowing density predetermined by the treatments. At each point on the graduated ruler, two seeds were sown. After seedling emergence, thinning was done, leaving one plant per position. Each plot consisted of six rows with 5 m long, spaced 0.5 m apart, making a total area of 15 m2, being the useful area considered as the plants located in the fourth and fifth rows of each plot. INTRODUCTION crop to climatic variations, use of seeds with unknown origin, crop implantation in soils with high acidity and inappropriate use of population density in the production areas , particularly for semierect to erect bean cultivars (SHIMADA et al., 2000). The common bean (Phaseolus vulgaris L.), by adapting well to various soil and climatic conditions in Brazil and by the high consumption tradition, plays a key role in the diet of the Brazilian population and in the demand for manpower (SENA et al., 2008). It is one of the major agricultural activities in the country, with its chain of production, processing and marketing generating employment and income, especially to lower income classes. Brazil stands out as the largest producer and consumer of beans. Considering the sown area and expected yield, the production of beans in the 2011/12 season should be 3.137 million tons (CONAB, 2012), with average yield estimated at 873 kg ha-1. However, this value can be considered low, since in fields where a high technological level is used, values three times bigger have been obtained. Among the reasons pointed out for low yield in Brazil are extreme sensitivity of the One of the first practices to be studied to adapt a species to a cultivation condition is the arrangement of plants, by the changes it provides in the microclimate, in the availability of light, nutrients, water, temperature and the interaction of plants with other biotic factors (JAUER et al., 2006). However, the best arrangement of plants depends on the intrinsic characteristics of the cultivar, such as size, growth habit and plant architecture, as well as the crop management system (BEZERRA et al., 2009), factors which will determine the ability for using available resources. In addition, weather conditions represent another factor limiting production of bean cultivars with different growth habits, with effects also on the physiological quality of seeds. Therefore, planning Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 Received: 03/04/13 Accepted: 05/09/14 416 Physiological potential... AMARO, H. T. T. et al Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 MATERIAL AND METHODS the planting season must coincide with favorable climatic conditions in different growing seasons , since variations in temperature, relative humidity and precipitation can damage the seeds, harming its vigor. The experiment was conducted in winter- spring season (winter crop season), planted in August 2011, in the municipality of Janaúba, northern Minas Gerais, with the following coordinates: 15° 47' 50'' S latitude and 43° 18' 31'' W longitude, and altitude of 516 m. The climate of the region according to Koppen's classification is "Aw" (tropical with dry winter), with average annual temperature and precipitation of 25 ºC and 900 mm, respectively. Climatic data collected during the experiment are shown in Figure 1. g Despite the different environmental conditions, there are few specific cultivars for each growing season, especially when aimed at the production of quality seeds. Thus, to enable the effective employment of these cultivars by farmers, it is necessary to test them in different edaphoclimatic regions, as well as adapt them to current production systems, typically developed for carioca bean cultivars, which predominantly exhibit growing habits II/III or III (ALVES et al., 2009). Hence, this study was conducted to evaluate the physiologic performance of common bean seeds grown in different population densities in winter crop season, in northern Minas Gerais. The soil of the experimental area was classified as an eutrophic Red Latosol (Eutrustox) (EMBRAPA, 2006), its main chemical characteristics are presented in Table 1. Table 1. Chemical characterization of the soil samples of the experimental areas removed in the 0 to 20 cm depth. Janaúba, MG. 2012. Soil property(1) M.O pH P K Al3+ Ca2+ Mg2+ Zn Fe Mn Cu B H+Al V m Prem dag kg-1 - mg dm-3 ........ cmolc dm-3…... ......……. mg dm-3..................... ...... cmolc dm-3 .... ... % .... - 1,7 5,3 8,7 96 0,1 4,1 1,0 3,3 44,7 9,9 3,6 11,5 1,6 77 1,0 34,5 (1) P, K, Fe, Zn, Mn and Cu extracted by Mehlich-1 solution. Al, Mg, and Ca extracted by HCl 1 mol L-1. B extracted by hot-water. Base saturation = [(K + Ca + Mg)/T] x 100, in which T = K + Ca + Mg + (H+Al); H+Al extracted by a solution of calcium acetate 0,5 mol L- 1, pH 7,0. d Cu extracted by Mehlich-1 solution. Al, Mg, and Ca extracted by HCl 1 mol L-1. B extracted by hot-water. MATERIAL AND METHODS Soil tillage was conventional, consisting of one plowing and two harrowings, held before Weed control was accomplished with the application of a tank mixture of the herbicides Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 417 AMARO, H. T. T. et al Physiological potential... AMARO, H. T. T. et al fomesafen and fluazifop-p-butyl at a dosage of 125 g ha-1, 25 days after emergence. Complementary irrigation by conventional aspersion were used and constantly monitored, all phytosanitary measures being taken according to the necessity of the crop. screen fixed and suspended inside a plastic box containing 40 mL of distilled water, kept at 41 ºC and 100% relative humidity for 72 hours inside a germinator (MARCOS FILHO, 1999). After this period, the seeds were removed from the chamber and germinated under the same conditions described for the germination test, being then determined the percentage of normal seedlings on the fifth day after installation of the test and the results expressed in percentage. Plants belonging to the useful area of each plot were manually harvested, and then submitted to threshing for obtaining seeds. Due to differences in maturation periods that occurred between cultivars, the plots were harvested as soon as maturity was reached, which led to not harvesting all plots at a single time. The seeds were cleaned and dried, and then taken to the Seed Analysis Laboratory to evaluate the physiological quality, by means of the following tests and/or determinations: p g The data were not transformed because they met the assumptions of the normality and homogeneity tests, being subjected to analysis of variance. The effects of population densities were studied by regression analysis, choosing the appropriate models to represent them according to their biological behavior, to the significance of the model coefficients and to the coefficient of determination (R²), and the effects of cultivars were studied by Tukey test at 5% (p < 0.05) significance level. Water content was determined as prescribed in the Rules for Seed Analysis - RAS (BRAZIL, 2009), using the oven method at 105 ± 3 ° C for 24 hours, with four replications of 50 seeds, being the results expressed in percentage. For the germination test, four replicates of 50 seeds were distributed evenly over germination paper in roll form, moistened with distilled water at a ratio of 2.5 times its initial weight. MATERIAL AND METHODS The rolls were placed in a germination chamber preset at an alternated temperature of 25 ºC and constant light. Evaluations were made on the fifth and ninth days after installation of the test and the results expressed as percentage of normal seedlings, according to the RAS (BRAZIL, 2009). RESULTS AND DISCUSSION Table 2 shows the analysis of variance of the data referring to the physiological quality of the seeds. It can be noted that, with the exception of the variable first germination count, there was significant effect of the variation source cultivar (CV) on the other evaluated characteristics. Sowing density (D) influenced significantly water content, thousand seeds mass and accelerated aging, while the double interaction CV x D was significant only for seed water content. The results of the first count test were obtained by the number of normal seedlings, determined during the first count of the germination test, ie, on the seventh day after installation (BRAZIL, 2009). The development of the interaction CV x D, studying the effects of cultivars inside each sowing density, revealed that the seed water content showed different behavior from the studied cultivars, according to the data shown in Table 3. The seeds of the cultivars Ouro Negro and Madrepérola, which show prostrate size and growth habit type III, showed higher water contents in relation to the seeds from the cultivar Ouro Vermelho in most of the population densities. These results are certainly related to the size and growth habit of the cultivars. The seedling emergence test was conducted under laboratory conditions, and sowing carried out at a depth of 3 cm in plastic trays containing washed and sterilized sand as substrate, moistened with water equivalent to 60% of the retention capacity, and the moisture was maintained by daily irrigations (BRAZIL, 2009). Four replications of 50 seeds were used and the results were obtained by the number of normal seedlings emerged, determined during the ninth day after the installation of the test, being the results expressed in percentage. In these cultivars, it is common to observe a higher contact of the pods with the soil in the period before harvest. This behavior, associated to the fact that harvest was performed in a period with frequent rainfall in the region (Figure 1), certainly contributed to the high water content values found in this work. The emergence speed index was conducted together with the seedling emergence test, noting daily, at the same time, the number of seedlings that showed visible hypocotyl. At the end of the test, the emergence speed index was calculated using the formula proposed by Maguire (1962). For the accelerated aging test, 200 seeds per treatment were distributed on the surface of a metal Biosci. Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 RESULTS AND DISCUSSION J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 rst the Apr. 2015 mination f es during 26 ** 0 * 5 ns 4 5 0 Apr. 2015 mination f es during E 26 ** 0 * 5 ns 4 5 0 ândia, v. 31, n. 2, p. 415-424, Mar./Apr. s (MTS), germination (GE), germina s cultivars grown in five densities du ESI AE 68,84 ** 1095,26 * 2,44 ns 348,90 * 2,08 ns 238,85 ns 3,67 132,44 2,66 117,15 12,68 22,30 erais. Janaúba, MG. 2012. 1) 500 15,4 B 20,2 A 14,3 B 16,6 RO, H. T. T. et al Biosci. J. nt (WC), mass of one thousan lerated aging (AE) of seeds o Squares FC SE 304,06 ns 375,26 284,26 ns 58,93 199,81 ns 47,68 274,31 80,60 212,69 71,21 25,60 9,51 winter season, in northern Mi ulation densities (thousand plan 200 300 4 2,6 B 15,8 B 15 9,0 A 21,3 A 21 8,0 A 19,0 A 19 6,5 18,7 18 y by Tukey test at 5% probability. iosci. J hern M and pla 4 15 21 19 18 ability. ousan eeds o SE 375,2 58,9 47,6 80,6 71,2 9,51 ern M nd pla 4 15 21 19 18 ility. sci. J 15 21 19 18 lity. 419 AMARO, H. T. T. et al Physiological potential... Figure 1. Daily variation of precipitation and temperature averages (maximum, average and minimum) during the year 2011. Data obtained from the Climatologic Station of the Minas Gerais Agricultural Research Agency (EPAMIG), northern regional unit of Minas Gerais, Nova Porteirinha, MG. Figure 1. Daily variation of precipitation and temperature averages (maximum, average and minimum) during the year 2011. Data obtained from the Climatologic Station of the Minas Gerais Agricultural Research Agency (EPAMIG), northern regional unit of Minas Gerais, Nova Porteirinha, MG. Sowing season must be adjusted in such a way that maturation and harvest of the seeds occur under mild temperature conditions, associated to lower precipitation indexes, according to França Neto et al (2007), factors which will determine harvesting seeds with ideal water content. On the contrary, harvest and subsequent storage of the beans seeds with water content superior to 13% can result in damages caused by changes in cell metabolism, with an increase in enzymatic and respiratory activities, allowing the development of fungi, with negative effects on seed quality. RESULTS AND DISCUSSION Evaluating the effect of population densities in each cultivar, it can be observed significative difference only for the cultivar Madrepérola (Figure 2). Results of water content fitted to a quadratic model, in which increasing sowing densities caused increase in water content until the density of 300 thousand plants ha-1, reaching 19,3% of moisture. Figure 2. Water content (%) of beans seeds of the cultivar Madrepérola, grown in five sowing densities in the winter season, in northern Minas Gerais. Janaúba, MG. 2012. Figure 2. Water content (%) of beans seeds of the cultivar Madrepérola, grown in five sowing densities in the winter season, in northern Minas Gerais. Janaúba, MG. 2012. Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 420 Physiological potential... AMARO, H. T. T. et al Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 Physiological potential... Physiological potential... AMARO, H. T. T. et al the mass of one hundred seeds. Probably, this result is explained by the more limitant climatic factors during the filling stage of the seeds (Figure 1). Carvalho et al (1998), verifying the effect of spacing and sowing season on the production of beans seeds, observed different behavior of the cultivars regarding the mass of one thousand seeds. The same authors found lower values when the cultivars were sown in the winter season, agreeing with the results of this work. The studied cultivars differed as for the mass of one thousand seeds (Table 4). The higher results were found in the seeds originated from the cultivars Ouro Vermelho and Madrepérola, showing results of 213,48 and 220,42, respectively. In a general way, in can be highlighted that this characteristic is a feature of each cultivar, as inform Paula Junior et al (2010). However, the values reached in this work for the cultivars were inferior to the described by these authors, when compared to Table 4. Mass (g) of one thousand seeds of beans cultivars grown during winter season, in northern Minas Gerais. Janaúba, MG. 2012. Variable Cultivars Ouro Vermelho Ouro Negro Madrepérola MTS 213,48 a 190,66 b 220,42 a Means followed by different letters in the line differ significantly by Tukey test at 5% probability. Physiological potential... Physiological potential... only the seeds of the cultivar Ouro Negro showed germination superior to the standard considered as ideal for the commercialization of basic beans seeds. In this sense, strategies to obtain higher responses of the crops to environmental conditions involve optimization of light interception by soil cover (SANTOS; FAGERIA, 2008), which can be obtained by managing plant population, a factor that should be very well elucidated when evaluating seed performance, as already mentioned. In general, the low values observed for seed germination percentage of the other studied cultivars is certainly associated to the climatic conditions verified during the period before seed harvest. The greater contact of plants, flowers and pods with the soil led to pod deterioration due to the occurrence of high temperatures and excessive rainfall, concentrated after physiological maturity of the plants (Figure 1). Seeds germination showed differentiated behavior among the studied cultivars (Table 5). The seeds of the cultivar Ouro Negro presented higher performance in germination, while the seeds of the cultivars Ouro Vermelho and Madrepérola did not differ from each other. It can also be observed that Table 5. Average results, in percentage, of germination (GE) and first count (FC) of beans seeds grown during winter season, in northern Minas Gerais. Janaúba, MG. 2012. Variable Cultivars Ouro Vermelho Ouro Negro Madrepérola GE 63 b 76 a 60 b FC 56 a 61 a 54 a Means followed by different letters in the line differ significantly by Tukey test at 5% probability. Table 5. Average results, in percentage, of germination (GE) and first count (FC) of beans seeds grown during winter season, in northern Minas Gerais. Janaúba, MG. 2012. Variable Cultivars Ouro Vermelho Ouro Negro Madrepérola GE 63 b 76 a 60 b FC 56 a 61 a 54 a Means followed by different letters in the line differ significantly by Tukey test at 5% probability. Table 5. Average results, in percentage, of germination (GE) and first count (FC) of beans winter season, in northern Minas Gerais. Janaúba, MG. 2012. In soybeans seeds, França Neto et al (2007) report that exposing the seed to alternated cycles of elevated and low humidity before harvest, due to the occurrence of frequent rains or to daily fluctuations of high and low relative humidity of the air, will result in its deterioration by humidity, with direct consequences in seed quality. evaluated cultivars did not suffer any effect of sowing densities. Physiological potential... These results suggest that these characteristics can be little affected by changes in plant population, being more influenced by genetic characteristics and climatic conditions. Regarding vigor (seedling emergence, emergence speed índex and accelerated aging), it can be noted a differentiated behavior among the analyzed cultivars (Table 6). In accordance with the germination results, the cultivar Ouro Negro showed higher physiological potential of the seeds, with 93% of seedling emergence, while the cultivars Ouro Vermelho and Madrepérola showed 88 and 85% of seedling emergence, respectively. It is important to highlight that to sow beans in the winter season can be justified by harvesting the grains in a period that there is a shortage of the product in the region. This way, new alternatives of sowing seasons are being researched, in function of the necessity of rotation with other crops and the possibility of a third “season” in only one agricultural year, also aiming seed production. As it was expected, the seeds of the cultivar Ouro Negro showed superior performance for the results of emergence speed index, while for the other cultivars there was no statistically significant difference (Table 6). The seeds of the studied cultivars did not differ on the results of germination first count (Table 5). It can also be observed that the germination and germination first count of the Table 6. Average test results of seedling emergence (SE), emergence speed index (ESI) and accelerated aging (EA) of beans seeds grown during winter season, in northern Minas Gerais. Janaúba, MG. 2012. Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 Table 6. Average test results of seedling emergence (SE), emergence speed index (ESI) and accelerated aging (EA) of beans seeds grown during winter season, in northern Minas Gerais. Janaúba, MG. 2012. Variable Cultivars Ouro Vermelho Ouro Negro Madrepérola SE (%) 88 b 93 a 85 b ESI 12,2 b 14,9 a 11,4 b AE (%) 48 ab 56 a 41 b Means followed by different letters in the line differ significantly by Tukey test at 5% probability. (EA) of beans seeds grown during winter season, in northern Minas Gerais. Janaúba, MG. 2012. Variable Cultivars Ouro Vermelho Ouro Negro Madrepérola SE (%) 88 b 93 a 85 b ESI 12,2 b 14,9 a 11,4 b AE (%) 48 ab 56 a 41 b Means followed by different letters in the line differ significantly by Tukey test at 5% probability. Variable Variable Additionally, Jauer et al (2002) concluded that seed size did not affect the germination and vigor of bean cultivars TPS Bionobre, TPS Nobre e Xamego. The mass of one thousand seeds was influenced significantly by the sowing density, showing that this characteristic is affected by changes in plant population (Figure 3). Increasing sowing density caused increase in the mass of one thousand seeds until the density of 300 thousand plants ha-1, in which it was found the best result (214,25 g), with a reduction in the values from this point. Possibly this reduction is related to a higher competition for water, light and nutrients, caused by the increasing sowing densities. g Although there is a great number of papers evaluating the effect of plant population on beans production, there is still no agreement as for the correct management of this parameter when evaluating the physiological potential of seeds. Several studies have been indicating contradictory results of mass of one thousand seeds in relation to plant population. Crusciol et al (2002) and Vazquez et al (2008) did not find variations in the mass of seeds in function of the evaluated plant populations. On the other hand, Tourino et al (2002), evaluating the effect of sowing density and uniformity on productivity and agronomical characteristics of soybeans, found decreases in the mass of seeds. The mass of one thousand seeds is a measure of quality used for different purposes, among them the comparison of the quality of different lots of seeds, as well as determining crops yields. In this sense, Carvalho e Nagakawa (2000) highlight that the bigger seeds have higher amount of reserves, being, therefore, the more vigorous. Figure 3. Mass (g) of one thousand seeds of the beans cultivars (means of the three cultivars), grown in five sowing densities during the winter season, in northern Minas Gerais. Janaúba, MG. 2012. Figure 3. Mass (g) of one thousand seeds of the beans cultivars (means of the three cultivars), grown in five sowing densities during the winter season, in northern Minas Gerais. Janaúba, MG. 2012. Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 421 AMARO, H. T. T. et al AMARO, H. T. T. et al Physiological potential... Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 Physiological potential... Physiological potential... plants ha-1, reaching the maximum germination percentage (53%) after seed aging. From this density, there were reductions in the observed values, verifying 42% of germination after accelerated aging in the density of 500 thousand plants ha-1. The higher plant populations certainly led to a higher intraspecific competition and higher lodging caused by the high density of the plants in the field, and that probably hindered seed quality. Accelerated aging is recognized as one of the most important tests to evaluate seed vigor of several species, being able to provide information with a high consistency level. In this test, it is considered that seed lots with high vigor maintain their viability when submitted, over time periods, to severe conditions of temperature and relative air humidity (MARCOS FILHO, 2005). In a similar way to the germination test, the low results observed in seed germination after accelerated aging is certainly related to the climatic conditions observed during the experiment (Figure 1). In this sense, Marcos Filho (2005) reports that at temperatures superior to 30 ºC during the period of transferring dry matter from the plant to the seeds, which are related to the reduction in photosynthetic rate and photoassimilates translocation, usually siginificative after flowering in several species, such as the leguminous. The climatic conditions of each region, as humidity and temperature, vary with the seasons of the year. Thus, determining the sowing season, in a given region, means to expose the crop to the most diverse environment conditions during its development. As reported, the studies evaluating the effects of plant populations on the physiological potential of seeds are rare and, in a certain way, contradictory. In their work, Vazquez et al (2008) concluded that variations in plant populations did not cause changes in the germination percentage and accelerated aging in soybeans seeds. On the other hand, Lazarini et al (2001) reported that the sowing density affected seed vigor when evaluated by the accelerated aging and the germination speed index. It is important to point out that, in order to obtain higher responses to the technologies that may result in higher grain yields for the beans crop, using the adequate plant population is a crucial factor. Physiological potential... Due to its great importance in the crop implantation phase, the best plant arrangement in the different production environments is a matter that still gets lots of interest in research, because, if executed in an inadequate way, it can compromise the subsequent phases of plant physiological development, with effects also on seed performance. Figure 4 shows the regression equation and its coefficient of determination (R2 = 0,85) for the accelerated aging in function of sowing densities. The results assumed a quadratic behavior, in which increasing sowing density caused an increase in germination until the density of 262.5 thousand Figure 4. Accelerated aging (%) of beans seeds cultivars (means of the three cultivars), grown during the winter season, in northern Minas Gerais. Janaúba, MG. 2012. Figure 4. Accelerated aging (%) of beans seeds cultivars (means of the three cultivars), grown during the winter season, in northern Minas Gerais. Janaúba, MG. 2012. Physiological potential... Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 422 AMARO, H. T. T. et al Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 CONCLUSIONS to the cultivars Ouro Vermelho and BRSMG Madrepérola. The beans seeds of the cultivar Ouro Negro present superior physiological potential in relation Increasing sowing density up to 300 thousand plants ha-1 is adequate for producing beans Increasing sowing density up to 300 thousand plants ha-1 is adequate for producing beans Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 423 AMARO, H. T. T. et al Physiological potential... AMARO, H. T. T. et al scholarship to the first author of the paper; to the Minas Gerais Research Support Foundation (FAPEMIG) and the National Council for Scientific and Technological Development (CNPq) for grants and financial support for the accomplishment of this work. seeds in the winter season, representing a good alternative for cultivation in the region. ACKNOWLEDGEMENTS To the Coordination for the Improvement of Higher Level Personnel (CAPES), by granting the RESUMO: Por se adaptar bem às mais variadas condições edafoclimáticas do Brasil e pela alta tradição de consumo, o feijoeiro desempenha papel fundamental na alimentação da população brasileira. Apesar das condições ambientais distintas, existem poucas cultivares específicas para cada época de plantio, principalmente quando visa à produção de sementes de qualidade. Objetivou-se com este trabalho avaliar o desempenho fisiológico de sementes de feijão-comum cultivado em cinco densidades populacionais, nas condições edafoclimáticas de Janaúba, Norte de Minas Gerais. O experimento foi conduzido na safra de inverno, com plantio em agosto de 2011. O delineamento experimental utilizado foi em blocos casualizados, em esquema fatorial 3 x 5, com quatro repetições. Os tratamentos consistiram em três cultivares de feijão-comum de diferentes tipos de crescimento (Ouro Vermelho, Ouro Negro e BRSMG Madrepérola), e cinco densidades populacionais (100, 200, 300, 400 e 500 mil plantas ha-1). As sementes foram avaliadas quanto ao teor de água, à germinação e ao vigor (testes de primeira contagem, emergência de plântulas, índice de velocidade de emergência e envelhecimento acelerado). Diante dos resultados, conclui-se que as sementes de feijão da cultivar Ouro Negro apresentam desempenho fisiológico superior em comparação com as cultivares Ouro Vermelho e BRSMG Madrepérola. O aumento da densidade de semeadura até 300 mil plantas ha-1 é adequado para a produção de sementes de feijão na safra de inverno, representando boa alternativa para o cultivo na região. PALAVRAS-CHAVE: Phaseolus vulgaris L. População de plantas. Germinação. Vigor. PALAVRAS-CHAVE: Phaseolus vulgaris L. População de plantas. Germinação. Vigor. REFERENCES ALVES, A. F.; ANDRADE, M. J. B.; RODRIGUES, J. R. M.; VIEIRA, N. M. B. Densidades populacionais para cultivares alternativas de feijoeiro no Norte de Minas Gerais. Ciência e Agrotecnologia, Lavras, v. 33, n. 6, p. 1495-1502, nov/dez. 2009. BEZERRA, A. A. C.; TÁVORA, F. J. A. F.; FREIRE FILHO, F. R.; RIBEIRO, V. Q. Características de dossel e de rendimento em feijão-caupi ereto em diferentes densidades populacionais. Pesquisa Agropecuária Brasileira, Brasília, v. 44, n. 10, p. 1239-1245, out. 2009. http://dx.doi.org/10.1590/S0100- 204X2009001000005 BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para Análise de Sementes. Brasília: DNDV/CLAV, 2009. 365 p. CARVALHO, N. M.; NAKAGAWA, J. (Ed.). Sementes: ciência, tecnologia e produção. 4 ed. Jaboticabal: FUNEP, 2000. 588 p. CARVALHO, M. A. C.; ARF, O.; SÁ, M. E. Efeito do espaçamento e épocas de semeadura sobre o desempenho do feijão. II. Qualidade fisiológica das sementes. Revista Brasileira de Sementes, v. 20, n.1, p. 202-208, 1998. CHAGAS, J. M.; BRAGA, J. M.; VIEIRA, C.; SALGADO, L. T.; JUNQUEIRA NETO, A.; ARAÚJO, G. A. A.; ANDRADE, M. J. B.; LANA; R. M. Q.; RIBEIRO, A. C. Feijão. In: RIBEIRO, A. C.; GUIMARÃES, P. T. G.; ALVAREZ, V. V. H. (Ed.). Recomendações para o uso de corretivos e fertilizantes em Minas Gerais: 5a aproximação. Viçosa, MG: Comissão de Fertilidade do Solo do Estado de Minas Gerais, 1999, p. 306-307. CONAB - Companhia Nacional de Abastecimento. Acompanhamento de safra brasileira: grãos, oitavo levantamento, maio 2012/Companhia Nacional de Abastecimento – Brasília: Conab, 2012. 36 p. Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 424 Physiological potential... AMARO, H. T. T. et al CRUSCIOL, C . A. C.; LAZARINI, E.; BUZO, C. L.; SÁ, M. E. Produção e qualidade fisiológica de sementes de soja avaliadas na semeadura de inverno. Scientia Agricola, Piracicaba, v. 59, n. 1, p. 75-96, jan./mar. 2002. EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA – EMBRAPA. Sistema Brasileiro de Classificação de Solos. 2. ed. Rio de Janeiro: Embrapa Solos, 2006. 306 p. j g p j EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA – EMBRAPA. Sistema Brasileiro de Classificação de Solos. 2. ed. Rio de Janeiro: Embrapa Solos, 2006. 306 p. FRANÇA NETO, J. B.; KRZYZANOWSKI, F. C.; PÁDUA, G. P.; COSTA, N. P.; HENNING, A. A. Tecnologia da produção de semente de soja de alta qualidade - Série Sementes. Circular técnica, Londrina, 2007. JAUER, A.; DUTRA, L. M. C.; ZABOT, L.; LUCCA FILHO, O. Biosci. J., Uberlândia, v. 31, n. 2, p. 415-424, Mar./Apr. 2015 REFERENCES A.; UHRY, D.; LUDWIG, M. P.; FARIAS, J. R. Comportamento de cultivar Pérola de feijoeiro comum (Phaseolus vulgaris L.) em quatro densidades de semeadura na safrinha em Santa Maria – RS. Revista da FZVA, Uruguaiana, v. 13, n. 1, p. 12-23, 2006. JAUER, A.; MENEZES, N. L.; GARCIA, D. C. Tamanho das sementes na qualidade fisiológica de cultivares de feijoeiro comum. Revista da FZVA, Uruguaiana, v. 9, n. 1, p. 65-72, 2002. LAZARINI, E.; CRUSCIOL, C. A. C.; BUZO, C. L.; SÁ, M. E. Qualidade fisiológica de sementes de cultivares de soja semeadas em diferentes densidades no período de primavera e de outono após a colheita e o armazenamento. Revista Brasileira de Sementes, v. 23, n. 1, p. 68-75, 2001. MAGUIRE, J. D. Speed of germination-aid in selection and evaluation for seedling emergence and vigor. Crop Science, Madison, v. 2, n.1 p. 176-177, 1962. http://dx.doi.org/10.2135/cropsci1962.0011183X000200020033x MARCOS FILHO, J. Fisiologia de Sementes de Plantas Cultivadas. 1. ed. Piracicaba: FEAL MARCOS FILHO, J. Fisiologia de Sementes de Plantas Cultivadas. 1. ed. Piracicaba: FEALQ, 2005. 495 p. MARCOS FILHO, J. Teste de envelhecimento acelerado. In: KRZYZANOWSKI, F. C.; VIEIRA, R. D.; FRANÇA NETO, J. B. (Ed.). Vigor de sementes: conceitos e testes. Londrina ABRATES, 1999. cap. 3, p.1- 24. PAULA JÚNIOR, T. J.; CARNEIRO, J. E. S.; VIEIRA, R. F.; ABREU, A. F. B.; RAMALHO, M. A. P.; PELOSO, M. J.; TEIXEIRA, H. Cultivares de feijão-comum para Minas Gerais. Belo Horizonte: EPAMIG, 2010, 39 p. SANTOS, A. B.; FAGERIA, N. K. Características fisiológicas do feijoeiro em várzeas tropicais afetadas por doses e manejo de nitrogênio. Ciência e Agrotecnologia, Lavras, v. 32, n. 1, p. 23-31, jan/fev. 2008. SENA, M. R.; ABREU, A. F. B.; RAMALHO, M. A. P.; BRUZI, A. T. Envolvimento de agricultores no processo seletivo de novas linhagens de feijoeiro. Ciência e Agrotecnologia, Lavras, v. 32, n. 2, p. 407- 412, mar./abr. 2008. SHIMADA, M. M.; ARF, O.; SÁ, M. E. Componentes do rendimento e desenvolvimento do feijoeiro de porte ereto sob diferentes densidades populacionais. Bragantia, Campinas, v. 59, n. 2, p. 181-187, 2000. http://dx.doi.org/10.1590/S0006-87052000000200009 TOURINO, M. C. C.; REZENDE, P. M.; SALVADOR, N. Espaçamento, densidade e uniformidade de semeadura na produtividade e características agronômicas da soja. Pesquisa Agropecuária Brasileira, Brasília, v. 37, n. 8, p. 1071-1077, ago. 2002. VAZQUEZ, G. H.; CARVALHO, N. M.; BORBA, M. M. Z. Redução na população de plantas sobre a produtividade e a qualidade fisiológica da semente se soja. Revista Brasileira de Sementes, Londrina, v. 30, n. 2, p. 01-011, 2008.
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Carcass traits, fatty acid composition, gene expression, oxidative stability and quality attributes of different muscles in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves and their combination
Asian-Australasian journal of animal sciences
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Carcass traits, fatty acid composition, gene expression, oxidative stability and quality attributes of different muscles in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves and their combination Kifah Jumaah Odhaib 1,2, Kazeem Dauda Adeyemi 1,3, and Awis Qurni Sazili 1,4,5,* Kifah Jumaah Odhaib 1,2, Kazeem Dauda Adeyemi 1,3, and Awis Qurni Sazili 1,4,5,* * Corresponding Author: Awis Qurni Sazili Tel: +60-3-89474870, Fax: +60-3-89381024, E-mail: awis@upm.edu.my Objective: This study examined the influence of dietary supplementation of Nigella sativa seeds, Rosmarinus officinalis leaves and their combination on carcass attributes, fatty acid (FA) composition, gene expression, lipid oxidation and physicochemical properties of longissimus dorsi (LD), semitendinosus (ST), and supraspinatus (SS) muscles in Dorper lambs. Objective: This study examined the influence of dietary supplementation of Nigella sativa seeds, Rosmarinus officinalis leaves and their combination on carcass attributes, fatty acid (FA) composition, gene expression, lipid oxidation and physicochemical properties of longissimus dorsi (LD), semitendinosus (ST), and supraspinatus (SS) muscles in Dorper lambs. Methods: Twenty-four Dorper lambs (18.68±0.6 kg, 4 to 5 months old) were randomly assigned to a concentrate mixture containing either, no supplement (control, T1), 1% Rosmarinus officinalis leaves (T2), 1% Nigella sativa seeds (T3), or 1% Rosmarinus officinalis leaves+1% Nigella sativa seeds (T4) on a dry matter basis. The lambs were fed the treatments with urea-treated rice straw for 90 days, slaughtered and the muscles were subjected to a 7 d postmortem chill storage. h 1 Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2 Department of Physiology, College of Veterinary Medicine, University of Basrah, 61004 Basrah, Iraq 3 Department of Animal Production, University of Ilorin, PMB 1515 Ilorin, Nigeria 4 Laboratory of Sustainable Animal Production and Biodiversity, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 5 Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia p p p Methods: Twenty-four Dorper lambs (18.68±0.6 kg, 4 to 5 months old) were randomly assigned to a concentrate mixture containing either, no supplement (control, T1), 1% Rosmarinus officinalis leaves (T2), 1% Nigella sativa seeds (T3), or 1% Rosmarinus officinalis leaves+1% Nigella sativa seeds (T4) on a dry matter basis. The lambs were fed the treatments with urea-treated rice straw for 90 days, slaughtered and the muscles were subjected to a 7 d postmortem chill storage. h p g Results: The T2 lambs had greater (p<0.05) slaughter and cold carcass weights than the con­trol lambs. Dietary supplements did not affect (p>0.05) chill loss, dressing percentage, carcass composition, intramuscular fat and muscle pH in Dorper lambs. ORCID ORCID Kazeem Dauda Adeyemi https://orcid.org/0000-0002-6719-2081 Awis Qurni Sazili https://orcid.org/0000-0002-7362-0855 Submitted Jun 17, 2017; Revised Jul 30, 2017; Accepted Oct 22, 2017 Submitted Jun 17, 2017; Revised Jul 30, 2017; Accepted Oct 22, 2017 g p p Conclusion: Dietary supplementation of Nigella sativa seeds and Rosmarinus officinalis leaves had beneficial effects on meat quality in Dorper lambs. Copyright © 2018 by Asian-Australasian Journal of Animal Sciences This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and repro- duction in any medium, provided the original work is properly cited. Open Access Asian-Australas J Anim Sci Vol. 31, No. 8:1345-1357 August 2018 https://doi.org/10.5713/ajas.17.0468 pISSN 1011-2367 eISSN 1976-5517 Open Access Carcass traits, fatty acid composition, gene expression, oxidative stability and quality attributes of different muscles in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves and their combination Meat from supple­ mented lambs had lower (p<0.05) cooking and drip losses, shear force, lightness, and lipid oxidation and greater (p<0.05) redness compared with the control meat. The impact of dietary supplements on muscle FA varied with muscle type. Diet had no effect (p>0.05) on the expression of stearoyl-CoA desaturase and lipoprotein lipase genes in LD and ST muscles in Dorper lambs. The T2 and T3 diets up regulated the expression of AMP-activated protein kinase alpha 2 gene in LD and ST muscles and up regulated the expression of sterol regulatory element-binding protein 1 in ST muscle in Dorper lambs. fi Corresponding Author: Awis Qurni Sazili Tel: +60-3-89474870, Fax: +60-3-89381024, E-mail: awis@upm.edu.my 1 Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2 Department of Physiology, College of Veterinary Medicine, University of Basrah, 61004 Basrah, Iraq 3 Department of Animal Production, University of Ilorin, PMB 1515 Ilorin, Nigeria 4 Laboratory of Sustainable Animal Production and Biodiversity, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 5 Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Keywords: Dorper Lambs; Gene Expression; Meat Quality; Nigella sativa; Rosmarinus officinalis Copyright © 2018 by Asian-Australasian Journal of Animal Sciences Experimental diet and management of animals Experimental diet and management of animals g Twenty-four, entire male Dorper lambs with average initial body weight of 18.68±0.6 kg and 4 to 5 months old were used for the trial. Each lamb was housed in individual pens (1.3 m ×0.9 m) provided with drinking and feeding facilities. The experimental diets were formulated to meet the nutritional requirements of lambs in line with NRC [15] recommendation. The lambs were randomly allotted to one of the four experi­ mental diets namely, a concentrate mixture (55% yellow corn, 20% soybean meal, 20% rice bran, 3% palm oil, 1% CaCO3, 0.5% NaCl, 0.5% minerals-vitamins mix) without an additive (control, T1), concentrate mixture+1% (dry matter [DM] of concentrate) Rosmarinus officinalis leaves (T2), concentrate mixture+1% (DM of concentrate) Nigella sativa seeds (T3), concentrate mixture+1% (DM of concentrate) Rosmarinus officinalis leaves+1% (DM of concentrate) Nigella sativa seeds (T4). Each lamb received concentrate at 1% of body weight with ad libitum urea-treated rice straw daily for 90 d follow­ ing two weeks of acclimatization. The concentrate was offered to the lambs in equal proportion in two splits at 0800 and 1600 hours. All lambs had ad libitum access to water and mineral block. Nigella sativa (NS) and Rosmarinus officinalis (RO) contain myriad phytochemicals whose antioxidant, therapeutic, an­ timicrobial, antitumor and anti-inflammatory properties have been documented [8,9]. Dietary supplementation of RO and NS improved body weight gain and lean to fat ratio in lambs [10,11]. Nonetheless, there is limited investigation on the ef­ fects of dietary supplementation of NS seeds and RO leaves on the physicochemical properties and oxidative stability of meat in ruminants. The use of medicinal plants as antioxidant in foods is favoured due to the hazardous effects of synthetic antioxidants on human health [6,7]. h Determination of chemical composition and phytochemical contents of dietary treatmentsh The feed samples were dried at 60°C for 48 h to determine the DM content, ground to pass a through a 1 mm screen and analysed for protein, ether extract, crude protein and ash ac­ cording to the method of AOAC [16]. The acid detergent fibre and neutral detergent fibre were analysed by the protocol of Van Soest et al [17]. The total phenol and tannin contents were determined following the procedure of Makkar et al [18]. The chemical composition and phytochemical contents of the di­ etary treatments, additives and urea treated rice straw are shown in Table 1. Determination of fatty acid composition of dietary treatmentsh The total lipids in dietary treatments were extracted in chloro­ form:methanol (2:1, v/v) mixture following the protocol described by Adeyemi et al [1]. The extracted lipid was trans­ methylated to fatty acid methyl esters using 2 mL 14% BF3 and 2 mL 0.66 N KOH in methanol following the protocol of AOAC [16]. The chromatography settings, the column and the stan­ dard used were as described by Adeyemi et al [1]. The FA composition of the dietary treatments is presented in Table 2. Experimental diet and management of animals There has been a renewed interest in the manipulation of the fatty acid (FA) composition of ruminant meat to meet the prevailing consumers’ demands [1]. Plant polyphenols, such as those found in NS and RO, when supplemented in ruminant diets could manipulate rumen biohydrogenation of unsaturated FAs thereby modifying the FA composition of ruminant meat [12]. The changes in muscle FAs due to feeding strategies are implicated in the expression of lipogenic genes [13,14]. An improved understanding of the genes and the underlying mechanisms involved in fat metabolism would allow a better control of the content and composition of FA in ruminant meat [13,14]. Therefore, the objective of this study was to deter­ mine the effects of NS seeds, RO leaves and their combination on carcass traits, FA composition, expression of lipogenic genes, physicochemical properties and lipid oxidation in longissimus dorsi (LD), semitendinosus (ST), and supraspinatus (SS) mus­ cles in Dorper lambs. Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 and Ethics. The care of the Dorper lambs was in accordance to Malaysian standards. Dietary supplementation of medicinal plants to livestock has been advocated as an effective strategy for improving production performance [4] of livestock and the quality and storage stability of animal products [5]. It has been established that nutritional strategy is more effective in enhancing the oxi­ dative stability of meat when compared to exogenous addition of antioxidants because dietary antioxidants are preferentially deposited where they are most needed [5,6]. In addition, di­ etary intervention remains the most effective strategy to modify the oxidative stability of intact muscle foods, where the use of exogenous antioxidant may be difficult or practically im­ possible [1,6]. Nonetheless, the effects of medicinal plants on livestock product quality are highly variable and inconsistent in the published literatures [5-7]. These scenarios have created the impetus for further research in diverse production systems to allow informed choices and tailored decisions in the use of medicinal plants for the improvement of the healthiness and storage stability animal products. fi INTRODUCTION Ruminant meat is a good source of animal protein, which is valued in many cultural culinary traditions [1]. Nonetheless, in recent times, its consumption has been linked with the inci­ dence of chronic diseases [2] in humans thereby triggering a lack of consumer confidence in ruminant meat. In addition, the meat industry has been adversely affected by food scares relating to the residual effects of antibiotic growth promoters used in animal nutrition [3]. Thus, enhancing the safety, nutritional and sensory quality of ruminant meat in order to meet the rapidly changing requirements of consumers have been the subject of research in recent times. 1345 www.ajas.info Animal welfareh Animal welfareh Slaughtering and carcass analysis Slaughtering and carcass analysis This study was conducted following the guidelines of the Re­ search Policy of Universiti Putra Malaysia on Animal Welfare On the last day of the feeding trial, the lambs were fasted over­ On the last day of the feeding trial, the lambs were fasted over­ 1346    www.ajas.info Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 Table 1. Chemical composition of dietary treatments, urea treated rice straw, Nigella sativa seeds and Rosmarinus officinalis leaves Parameter T1 1) T2 T3 T4 UTRS NS RO Chemical composition (% DM) Dry matter 90.00 90.38 90.05 90.46 96.58 92.62 91.63 Organic matter 94.83 94.85 94.76 94.83 87.06 96.09 93.95 Ash 5.15 5.12 5.24 5.16 12.94 3.91 6.05 Crude protein 16.96 16.86 17.03 16.92 4.98 22.70 5.59 Ether extract 3.74 3.80 3.70 3.70 1.63 9.034 4.23 Crude fibre 3.07 3.08 3.15 3.13 36.25 6.60 13.40 Neutral detergent fibre 38.93 39.25 46.59 47.16 80.75 35.30 36.64 Acid detergent fibre 8.88 7.38 6.98 8.99 48.57 21.24 19.08 Phytochemical compounds Total polyphenol (mg/g) 3.16 12.35 19.08 34.86 - 37.69 43.29 Non-tannin polyphenol (mg/g) 0.98 4.30 3.61 7.88 - 2.16 10.71 Tannin polyphenol (mg/g) 2.18 8.05 15.47 26.98 - 35.53 32.58 NS, Nigella sativa seeds; RO, Rosmarinus officinalis leaves; UTRS, urea treated rice straw; DM, dry matter. 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. ble 1. Chemical composition of dietary treatments, urea treated rice straw, Nigella sativa seeds and Rosmarinus officinalis leaves NS, Nigella sativa seeds; RO, Rosmarinus officinalis leaves; UTRS, urea treated rice straw; DM, dry matter. 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves night with ad libitum access to water and slaughtered according to the Halal procedure as described in MS1500:2009 [19]. After evisceration, each carcass was split along the vertebra column into right and left halves. The right half was used for carcass analyses as described by Adeyemi et al [20]. Table 2. Slaughtering and carcass analysis Fatty acid composition (% of total FA) of dietary treatments Fatty acid Dietary treatment T1 1) T2 T3 T4 C14:0 0.89 1.22 0.76 0.82 C16:0 36.44 31.24 31.89 32.53 C16:1 0.57 0.72 0.62 0.67 C18:0 8.67 7.61 7.89 8.16 C18:1n-9 40.30 46.59 46.62 46.59 C18:2n-6 7.14 7.19 6.18 5.44 C18:3n-3 1.70 1.43 1.47 1.49 C20:4n-6 1.53 1.09 1.33 1.23 C20:5n-3 0.10 0.30 0.92 0.71 C22:5n-3 1.94 1.36 1.50 1.52 C22:6n-3 0.75 0.93 0.81 0.89 Sum and ratio of FA 2) ΣSFA 45.99 40.07 40.54 41.51 ΣUFA 54.00 59.92 59.45 58.49 ΣMUFA 40.87 47.32 47.24 47.22 ΣPUFA 13.13 12.62 12.21 11.27 Σn-3 4.47 4.01 4.70 4.61 Σn-6 8.66 8.60 7.51 6.67 n-6:n-3 1.94 2.13 1.62 1.44 UFA:SFA 1.18 1.50 1.47 1.41 PUFA:SFA 0.29 0.32 0.30 0.27 Total FA (mg/g) 1,679.61 1,230.10 1,372.04 1,655.40 SFA, saturated fatty acids; UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Ros­ marinus officinalis leaves. 2) ΣSFA = C14:0+C16:0+C18:0; ΣMUFA = C16:1+C18:1+C18:1 trans-11; ΣUFA = C16:1+C18:1+Σn-3+Σn-6; ΣPUFA = Σn-3+Σn-6; Σn-3 = C18:3n- 3+C20:5n-3+C22:5n-3+C22:6n-3; Σn-6 = C18:2n-6+C20:4n-6; n-6:n-3 = (C18:2n-6+C20:4n-6)÷(C18:3n-3+C20:5n-3+C22:5n-3+C22:6n-3). Table 2. Fatty acid composition (% of total FA) of dietary treatments Muscle sampling and storage of meattt g g Meat samples were left intact on the left half of each carcass until a particular postmortem storage was reached. The SS muscle was sampled from the right forelimb. The right LD muscle was excised from the 6th to 8th lumbar vertebra. The ST muscle was sampled at the posterior face of the left hind limb. On day 0, 90 g of each muscle sample was removed from each carcass, trimmed free of epimyseal connective tissue and external fat and divided into three parts. The first part (10 g) was pulverized in liquid nitrogen with porcelain mortar and pestle to produce a homogenous powder, stored at –80°C until analysis and assigned for the determination of muscle pH, FA composition and lipid oxidation. The second part (30 g) was vacuum packaged and stored in a chiller at 4°C±1°C and used to determine drip loss. The third part (50 g) was used to de­ termine cooking loss, colour, and shear force on d 0. Upon the completion of each storage period, muscle cuts (60 g) were removed from the carcass, trimmed free of epimyseal con­ nective tissue and external fat and sectioned into two parts. The first part (10 g) was pulverized in liquid nitrogen and as­ signed as described earlier. The second portion (50 g) was used to determine colour coordinates, cooking loss and shear force. Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 shown in Table 3. stances (TBARS) using QuantiChromTM TBARS Assay Kit (DTBA-100, BioAssay Systems, Hayward, CA, USA) in line with the manufacturer’s procedure. The muscle FA composi­ tion was determined as described earlier. stances (TBARS) using QuantiChromTM TBARS Assay Kit (DTBA-100, BioAssay Systems, Hayward, CA, USA) in line with the manufacturer’s procedure. The muscle FA composi­ tion was determined as described earlier. Statistical analysish The experiment followed a completely randomized design. The gene expression data was checked for normality prior to subjecting it to the generalized linear model (GLM) of SAS [22]. Data obtained from carcass traits and muscle FA were subjected to the GLM procedure of SAS [22]. Data for physi­ cochemical properties were analyzed using the PROC MIXED procedure of SAS [22] in which diet and postmortem storage days and their first order interaction were fitted as fixed effects in a repeated measure. Means were separated using the “PDIFF” option of the “LSMEANS” statement of the MIXED proce­ dure. Tukey HSD test was used to adjust the means. The level of significance difference was set at p<0.05. Carcass traits Th fi l b d The final body weight and carcass characteristics of Dorper lambs fed different medicinal plants are shown in Table 4. Dorper lambs fed 1% RO leaves had greater (p<0.05) final body weight compared with those fed other diets. Dietary supplementation of medicinal plants had no effect (p>0.05) on the hot carcass weight, chill loss, dressing percentage, per­ centages of shoulder, legs, breast, loin and neck in Dorper lambs. The proportion of lean, bone and fat in the neck, loin and breast cuts in Dorper lambs were similar (p>0.05) between the diets. Dietary treatments had no effect (p>0.05) on the pro­ portion of bone and fat in the shoulder and leg cuts of Dorper lambs. The T3 lambs had greater (p<0.05) lean in the leg cut compared with lambs fed other dietary treatments. RNA extraction from muscle samples and quantitative real-time polymerase chain reaction Total RNA from LD and ST muscles (pulverized in liquid ni­ trogen and stored at –80°C) was extracted and purified using The RNeasy Fibrous Tissue Kit (cat. no. 74704) following the manufacturer’s protocol. The concentration and purity of the RNA was assessed using a NanoDrop ND-1000 UV-Vis Spec­ trophotometer (NanoDrop Technologies, Wilmington, DE, USA) at 260/280 nm absorbance. The purified RNA was kept at –80°C until further analysis. The reverse transcription of total RNA to complementary DNA was done using Quantitate Reversed Transcription Kit (Qiagen, Hilden, Germany) as per the manufacturer’s protocol. Gene expression was carried out using Quantitative real-time polymerase chain reaction (PCR). The PCR reaction was performed on a total volume of 20 μL using the iQTMSYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). Each 20 μL PCR reaction contained 10 μL 2× SYBR Green Master Mix, 1 μL forward primer, 1 μL reverse primer, 5 μL template cDNA and 3 mL RNase-free water. The PCR conditions for all genes were, ini­ tial denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing for 30 s, and extension at 72°C for 30 s with a single fluorescence detection point at the end of the relevant annealing section. At the end of the PCR run, the temperature was increased from 70°C to 95°C at the rate of 0.5°C/min, and the fluorescence was measured at every 5 s interval to construct the melting curve. The com­ parative CT method (ΔΔCT) expression of the investigated genes was normalized with the endogenous control hypo­ xanthine phosphoribosyltransferase 1. CT values are means of duplicate measurements. Comparative CT quantification was determined by the ΔΔCT method. The primers used are Determination of muscle pH, colour coordinates, drip and cooking losses, shear force, lipid oxidation and fatty acid composition Muscle pH, meat colour coordinates, drip loss, cooking loss and shear force were determined following the protocol de­ scribed by Lokman et al [21]. Lipid oxidation in the muscle samples was quantified as 2-thiobarbituric acid reactive sub­ 1347 www.ajas.info www.ajas.info Muscle fatty acid compositionh The FA composition of LD muscle in Dorper lambs fed diet supplemented with NS seeds, RO leaves and their combination is shown in Table 5. Except for the concentration of C18:1n-9, Table 3. Target genes and sequences of primers Gene No. Targets genes Primers Amplicon (bp) Annealing temperature (°C) Accession No. 1 LPL F-5′aatgaagagatgaacggaacg-3′ 119 60 NM_001009394 R-5′gcactttccaaccaggatgt-3′ 2 SCD F- 5´cccagctgtcagagaaaagg- 3´ 115 60 AJ001048 R- 5´gatgaagcacaacagcagga- 3´ 3 SREBF1 F-5´ctgctatgcaggcagcac- 3´ 99 60 GU206528 R- 5´ggttgatgggcagcttgt- 3´ 4 YWHAZ F-5´tgtaggagcccgtaggtcatct-3´ 102 60 AY970970 R-5´ttctctctgtattctcgagccatct-3 5 PRKAA2 F-5´accctcccatttgatgatga-3´ 97 60 NM_001112816 R-5´tggcaacagaacgattgaga-3′ F: forward, R: reverse; LPL, lipoprotein lipase; SCD, stearoyl-CoA desaturase; SREBF1, sterol regulatory element-binding transcription factor 1; YWHAZ, tyrosine 3-monooxy­ genase/tryptophan 5-monooxygenase activation protein zeta; PRKAA2, AMP-activated protewin kinase alpha 2. F: forward, R: reverse; LPL, lipoprotein lipase; SCD, stearoyl-CoA desaturase; SREBF1, sterol regulatory element-binding transcription factor 1; YWHAZ, tyrosine 3-monooxy­ genase/tryptophan 5-monooxygenase activation protein zeta; PRKAA2, AMP-activated protewin kinase alpha 2. Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 Table 4. Muscle fatty acid compositionh Carcass traits in Dorper lambs fed diets containing Nigella sativa seeds, Rosmarinus officinalis leaves and their combination Parameter T1 1) T2 T3 T4 SEM p value Slaughter weight (kg) 31.97 b 34.80 a 31.85 b 33.00 b 0.388 0.001 Hot carcass weight (kg) 13.15 15.10 13.90 14.65 0.500 0.072 Cold carcass weight (kg) 12.37 b 14.10 a 13.17 ab 13.95 a 0.161 0.009 Chill loss (%) 5.60 6.62 5.28 4.69 0.788 0.792 Dressing (%) 41.08 43.39 43.58 44.38 0.325 0.337 Neck (%) 8.44 7.12 7.23 7.37 0.302 0.423 Legs (%) 29.36 30.00 27.79 30.43 0.917 0.758 Shoulder (%) 22.00 23.13 23.59 23.30 0.632 0.823 Loin (%) 18.20 20.46 22.55 18.56 0.903 0.342 Breast and flank (%) 22.77 20.83 20.64 20.04 0.608 0.444 Composition of prime cuts (%) Leg lean 67.49 b 68.36 b 72.15 a 68.99 b 0.55 0.001 Leg bone 21.89 20.91 19.56 21.77 1.01 0.396 Leg fat 10.61 10.73 8.28 9.22 0.330 0.146 Neck lean 54.10 52.97 57.74 58.49 0.722 0.122 Neck bone 42.34 39.90 39.47 37.67 0.688 0.295 Neck fat 3.55 3.96 2.78 3.83 0.210 0.342 Shoulder lean 56.09 60.81 56.51 58.45 0.803 0.126 Shoulder bone 33.11 26.73 35.70 34.29 1.026 0.202 Shoulder fat 10.79 11.11 9.72 9.25 0.428 0.416 Loin lean 56.64 60.18 58.18 57.88 1.073 0.791 Loin bone 29.38 25.65 24.07 26.06 0.46 0.358 Loin fat 12.85 13.60 13.51 13.30 0.389 0.499 Breast lean 63.27 67.05 65.33 62.95 0.547 0.153 Breast bone 25.56 21.11 21.97 24.19 0.718 0.286 Breast fat 11.16 11.82 12.69 12.85 0.219 0.141 SEM, standard error of means. 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means having different superscripts along the same row are significantly different (p<0.05). . Carcass traits in Dorper lambs fed diets containing Nigella sativa seeds, Rosmarinus officinalis leaves and their combination other treatments. Diet had no effect (p>0.05) on IMF in ST muscle in Dorper lambs. h which differed between the diets, supplementation of medici­ nal plants did not affect the composition of most FA and the intramuscular fat (IMF) in LD muscle in Dorper lambs. The percentage of C18:1n-9 in the LD muscle of lambs fed RO leaves was greater (p<0.05) than that of the control lambs. Muscle fatty acid compositionh The LD muscle of the T3 and T4 lambs had similar percentage of C18:1n-9, which did not differ from those of lambs, fed other dietary treatments. The FA composition of SS muscle in Dorper lambs fed diet supplemented with NS seeds, RO leaves and their combination is presented in Table 7. Dietary supplements had no signifi­ cant effect (p>0.05) on the IMF and FA composition of SS muscle in Dorper lambs. The FA composition of ST muscle in Dorper lambs fed diet supplemented with NS seeds, RO leaves and their com­ bination is presented in Table 6. The concentration of C18:0 was greater (p<0.05) in the ST muscle of the control lambs compared with those fed the NS seeds. The concentration of C18:0 in the meat of T2 and T4 lambs did not differ from those fed other dietary treatments. The ST muscle of T2 and T3 lambs had similar concentration of C18:1n-9 and total monounsatu­ rated FAs, which were significantly different (p<0.05) from those of lambs fed the T1 and T4 diets. The ST muscle of T3 lambs had greater (p<0.05) concentration of C18:3n-3 com­ pared with the control lambs. The concentration of C18:3n-3 in the ST muscle of T4 lambs did not differ from those fed Parameter Treatment SEM p value T1 1) T2 T3 T4 C14:0 2.39 2.65 2.64 3.55 0.450 0.132 C16:0 25.16 25.77 25.42 25.72 1.783 0.906 C16:1 2.84 2.01 2.49 2.66 0.452 0.377 C17:0 1.02 0.95 1.04 1.09 0.009 0.328 C18:0 21.73 19.93 21.54 21.44 5.321 0.679 C18:1n-9 36.78 b 40.85 a 37.85 ab 37.67 ab 5.386 0.026 C18:2n-6 5.33 3.85 4.42 4.37 1.942 0.530 C18:3n-3 0.39 0.32 0.37 0.35 0.012 0.675 C20:4n-6 3.04 2.28 2.65 1.94 1.273 0.570 C20:5n-3 0.40 0.32 0.59 0.32 0.104 0.608 C22:5n-3 0.34 0.48 0.49 0.21 0.058 0.348 C22:6n-3 0.32 0.43 0.34 0.26 0.056 0.795 Sum and ratio of FA 2) ΣSFA 50.56 49.46 50.81 52.23 11.159 0.711 ΣUFA 49.45 50.53 49.19 47.78 11.164 0.715 ΣMUFA 39.62 42.86 40.34 40.33 6.074 0.309 ΣPUFA 9.83 7.67 8.86 7.45 9.59 0.682 Σn-3 1.46 1.53 1.79 1.13 0.584 0.688 Σn-6 8.36 6.13 7.06 6.30 5.99 0.578 n-6:n-3 5.78 4.26 4.58 5.55 1.35 0.239 UFA:SFA 0.98 1.03 0.97 0.92 0.017 0.693 PUFA:SFA 0.19 0.15 0.18 0.14 0.005 0.743 IMF (g/100 g) 5.14 5.24 5.20 5.04 0.012 0.119 SEM, standard error of means; SFA, saturated fatty acids; UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Ros­ marinus officinalis leaves. 2) ΣSFA = C14:0+C16:0+C18:0, ΣMUFA = C16:1+C18:1+C18:1 trans-11; ΣUFA = C16:1+C18:1+Σn-3+Σn-6; ΣPUFA = Σn-3+Σn-6; Σn-3 = C18:3n- 3+C20:5n-3+C22:5n-3+C22:6n-3; Σn-6 = C18:2n-6+C20:4n-6; n-6:n-3 = (C18:2n-6+C20:4n-6)÷(C18:3n-3+C20:5n-3+C22:5n-3+C22:6n-3). a,b,c Means having different superscripts along the same row are significantly different (p<0.05). mented lambs. In ST muscle, the control lambs had similar (p>0.05) drip loss as those fed dietary RO leaves. The ST mus­ cle in the T3 and T4 lambs had lower (p<0.05) drip loss than those fed the T1 and T2 diets. The percentage drip loss de­ creased (p<0.05) over postmortem storage of LD, ST, and SS muscles. There was no significant interaction between diet and postmortem storage days for drip loss in different muscles in Dorper lambs. The shear force in the LD and ST muscles of the control lambs was greater (p<0.05) than that of the supplemented lambs. Dietary treatments had no effect (p>0.05) on the sheer force of SS muscle in Dorper lambs. Regardless of muscle, the shear force decreased (p<0.05) over postmortem storage. i Parameter Treatment SEM p value T1 1) T2 T3 T4 C14:0 2.99 3.31 4.11 4.63 0.761 0.075 C16:0 26.41 26.31 26.89 26.43 1.050 0.854 C16:1 2.35 b 3.20 a 3.03 a 2.59 ab 0.168 0.045 C17:0 1.02 1.06 1.05 1.08 0.018 0.926 C18:0 23.76 a 19.79 b 19.44 b 21.89 b 6.357 0.010 C18:1n-9 37.09 b 38.86 a 39.27 a 34.58 b 4.770 0.039 C18:2n-6 3.63 4.03 3.17 4.65 1.538 0.417 C18:3n-3 0.28 b 0.29 b 0.42 a 0.37 ab 0.0049 0.046 C20:4n-6 1.74 2.12 1.56 2.270 0.394 0.387 C20:5n-3 0.13 0.16 0.15 0.357 0.025 0.20 C22:5n-3 0.24 0.26 0.31 0.270 0.019 0.933 C22:6n-3 0.24 0.34 0.20 0.262 0.007 0.193 Sum and ratio 2) ΣSFA 54.29 50.74 51.90 54.65 8.209 0.214 ΣUFA 45.69 49.25 48.10 45.35 8.194 0.212 ΣMUFA 39.44 b 42.05 a 42.29 a 37.17 b 5.58 0.030 ΣPUFA 6.25 7.19 5.81 8.18 3.55 0.33 Σn-3 0.89 1.05 1.08 1.26 0.050 0.20 Σn-6 5.36 6.15 4.73 6.92 3.056 0.35 n-6:n-3 5.99 5.91 4.34 5.54 2.270 0.417 UFA:SFA 0.85 0.98 0.94 0.83 0.011 0.21 PUFA:SFA 0.12 0.14 0.12 0.15 0.001 0.54 IMF (g/100 g) 5.34 5.09 5.23 5.03 0.117 0.220 SEM, standard error of means; SFA, saturated fatty acids; UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Ros­ marinus officinalis leaves. 2) ΣSFA = C14:0+C16:0+C18:0; ΣMUFA = C16:1+C18:1+C18:1 trans-11; ΣUFA = C16:1+C18:1+Σn-3+Σn-6; ΣPUFA = Σn-3+Σn-6; Σn-3 = C18:3n- 3+C20:5n-3+C22:5n-3+C22:6n-3; Σn-6 = C18:2n-6+C20:4n-6; n-6:n-3 = (C18:2n-6+C20:4n-6)÷(C18:3n-3+C20:5n-3+C22:5n-3+C22:6n-3). a,b,c Means having different superscripts along the same row are significantly different (p<0.05). Physicochemical traits of different muscles in Dorper lambsh The physicochemical properties and oxidative stability of LD, ST, and SS muscles in Dorper lambs fed diet supplemented with NS seeds, RO leaves and their combination are presented in Table 8. Dietary supplements had no significant effect (p> 0.05) on the muscle pH in different muscles in Dorper lambs. Regardless of muscle type, the pH on d 0 was greater (p<0.05) than that observed on d 1 and 7 postmortem. The interaction between diet and postmortem storage on muscle pH was not significant (p>0.05). h i   The percentage drip loss in the LD and SS muscles of the control lambs was greater (p<0.05) than those of the supple­ 1349 www.ajas.info semitendinosus muscle in Doper lambs fed diets containing Nigella sativa Rosemarinus officinalis leaves and their combination Parameter Treatment SEM p v T1 1) T2 T3 T4 C14:0 2.99 3.31 4.11 4.63 0.761 0 C16:0 26.41 26.31 26.89 26.43 1.050 0 C16:1 2.35 b 3.20 a 3.03 a 2.59 ab 0.168 0 C17:0 1.02 1.06 1.05 1.08 0.018 0 C18:0 23.76 a 19.79 b 19.44 b 21.89 b 6.357 0 C18:1n-9 37.09 b 38.86 a 39.27 a 34.58 b 4.770 0 C18:2n-6 3.63 4.03 3.17 4.65 1.538 0 C18:3n-3 0.28 b 0.29 b 0.42 a 0.37 ab 0.0049 0 C20:4n-6 1.74 2.12 1.56 2.270 0.394 0 C20:5n-3 0.13 0.16 0.15 0.357 0.025 0 C22:5n-3 0.24 0.26 0.31 0.270 0.019 0 C22:6n-3 0.24 0.34 0.20 0.262 0.007 0 Sum and ratio 2) ΣSFA 54.29 50.74 51.90 54.65 8.209 0 ΣUFA 45.69 49.25 48.10 45.35 8.194 0 ΣMUFA 39.44 b 42.05 a 42.29 a 37.17 b 5.58 0 ΣPUFA 6.25 7.19 5.81 8.18 3.55 0 Σn-3 0.89 1.05 1.08 1.26 0.050 0 Σn-6 5.36 6.15 4.73 6.92 3.056 0 n-6:n-3 5.99 5.91 4.34 5.54 2.270 0 UFA:SFA 0.85 0.98 0.94 0.83 0.011 0 PUFA:SFA 0.12 0.14 0.12 0.15 0.001 0 IMF (g/100 g) 5.34 5.09 5.23 5.03 0.117 0 SEM, standard error of means; SFA, saturated fatty acids; UFA, unsaturated f acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acid 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% R marinus officinalis leaves. 2) ΣSFA = C14:0+C16:0+C18:0; ΣMUFA = C16:1+C18:1+C18:1 trans-11 ΣUFA = C16:1+C18:1+Σn-3+Σn-6; ΣPUFA = Σn-3+Σn-6; Σn-3 = C18:3 3+C20:5n-3+C22:5n-3+C22:6n-3; Σn-6 = C18:2n-6+C20:4n-6; n-6:n-3 = (C18:2n-6+C20:4n-6)÷(C18:3n-3+C20:5n-3+C22:5n-3+C22:6n-3). a,b,c Means having different superscripts along the same row are significantly different (p<0.05). 1350    www.ajas.info Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 Table 5. Fatty acid composition (% of total FA) and intramuscular fat (IMF) of longissimus dorsi muscle in Doper lambs fed diets containing Nigella sativa seeds, Rosemarinus officinalis leaves and their combination Table 6. Fatty acid composition (% of total FA) and intramuscular fat (IMF) of semitendinosus muscle in Doper lambs fed diets containing Nigella sativa seeds, Rosemarinus officinalis leaves and their combination longissimus dorsi muscle in Doper lambs fed diets containing Nigella sativa seeds, Rosemarinus officinalis leaves and their combination Parameter Treatment SEM p value T1 1) T2 T3 T4 C14:0 2.39 2.65 2.64 3.55 0.450 0.132 C16:0 25.16 25.77 25.42 25.72 1.783 0.906 C16:1 2.84 2.01 2.49 2.66 0.452 0.377 C17:0 1.02 0.95 1.04 1.09 0.009 0.328 C18:0 21.73 19.93 21.54 21.44 5.321 0.679 C18:1n-9 36.78 b 40.85 a 37.85 ab 37.67 ab 5.386 0.026 C18:2n-6 5.33 3.85 4.42 4.37 1.942 0.530 C18:3n-3 0.39 0.32 0.37 0.35 0.012 0.675 C20:4n-6 3.04 2.28 2.65 1.94 1.273 0.570 C20:5n-3 0.40 0.32 0.59 0.32 0.104 0.608 C22:5n-3 0.34 0.48 0.49 0.21 0.058 0.348 C22:6n-3 0.32 0.43 0.34 0.26 0.056 0.795 Sum and ratio of FA 2) ΣSFA 50.56 49.46 50.81 52.23 11.159 0.711 ΣUFA 49.45 50.53 49.19 47.78 11.164 0.715 ΣMUFA 39.62 42.86 40.34 40.33 6.074 0.309 ΣPUFA 9.83 7.67 8.86 7.45 9.59 0.682 Σn-3 1.46 1.53 1.79 1.13 0.584 0.688 Σn-6 8.36 6.13 7.06 6.30 5.99 0.578 n-6:n-3 5.78 4.26 4.58 5.55 1.35 0.239 UFA:SFA 0.98 1.03 0.97 0.92 0.017 0.693 PUFA:SFA 0.19 0.15 0.18 0.14 0.005 0.743 IMF (g/100 g) 5.14 5.24 5.20 5.04 0.012 0.119 SEM, standard error of means; SFA, saturated fatty acids; UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Ros­ marinus officinalis leaves. 2) ΣSFA = C14:0+C16:0+C18:0, ΣMUFA = C16:1+C18:1+C18:1 trans-11; ΣUFA = C16:1+C18:1+Σn-3+Σn-6; ΣPUFA = Σn-3+Σn-6; Σn-3 = C18:3n- 3+C20:5n-3+C22:5n-3+C22:6n-3; Σn-6 = C18:2n-6+C20:4n-6; n-6:n-3 = (C18:2n-6+C20:4n-6)÷(C18:3n-3+C20:5n-3+C22:5n-3+C22:6n-3). a,b,c Means having different superscripts along the same row are significantly different (p<0.05). Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 Table 7. Fatty acid composition (% of total FA) and intramuscular fat (IMF) of supraspinatus muscle in Doper lambs fed diets containing Nigella sativa seeds, Rosemarinus officinalis leaves and their combination Gene expression in muscles Gene expression in muscles The mRNA expression of lipoprotein lipase (LPL) in LD and ST muscles in Dorper lambs fed diet supplemented with NS seeds, RO leaves and their combination are presented in Figure 1 and 2, respectively. Dietary supplementation of NS seeds, RO leaves and their combination did not have significant effect (p>0.05) on the mRNA expression of LPL gene in the LD (Figure 1) and ST (Figure 2) muscles in Dorper lambs. h Parameter Treatment SEM p value T1 1) T2 T3 T4 C14:0 3.71 2.72 3.00 4.05 0.558 0.090 C16:0 26.03 24.29 24.03 25.64 2.010 0.176 C16:1 2.97 2.56 2.07 2.98 0.362 0.162 C17:0 1.10 1.07 1.00 1.08 0.015 0.693 C18:0 21.29 19.61 22.01 20.37 10.364 0.738 C18:1n-9 37.59 40.10 38.49 36.09 11.566 0.437 C18:2n-6 3.97 5.70 5.68 5.78 3.620 0.494 C18:3n-3 0.36 0.37 0.39 0.38 0.007 0.970 C20:4n-6 1.78 2.37 2.18 2.41 0.638 0.672 C20:5n-3 0.12 0.28 0.25 0.06 0.027 0.227 C22:5n-3 0.392 0.35 0.35 0.31 0.033 0.947 C22:6n-3 0.24 0.30 0.24 0.42 0.025 0.362 Sum and ratio 2) ΣSFA 52.55 47.94 50.33 51.54 11.405 0.294 ΣUFA 47.44 52.05 49.67 48.45 11.405 0.294 ΣMUFA 40.57 42.67 40.56 39.07 13.173 0.590 ΣPUFA 6.86 9.38 9.10 9.37 8.663 0.577 Σn-3 1.11 1.31 1.24 1.18 0.114 0.866 Σn-6 5.75 8.07 7.86 8.20 7.224 0.547 n-6:n-3 5.03 6.17 6.44 6.91 2.430 0.407 UFA:SFA 0.91 1.09 0.99 0.94 0.014 0.240 PUFA:SFA 0.13 0.19 0.18 0.18 0.003 0.506 IMF (g/100 g) 5.34 5.32 5.23 5.23 0.310 0.310 SEM, standard error of means. SFA, saturated fatty acids; UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Ros­ marinus officinalis leaves. 2) ΣSFA = C14:0+C16:0+C18:0; ΣMUFA = C16:1+C18:1+C18:1 trans-11; ΣUFA = C16:1+C18:1+Σn-3+Σn-6; ΣPUFA = Σn-3+Σn-6; Σn-3 = C18:3n- 3+C20:5n-3+C22:5n-3+C22:6n-3; Σn-6 = C18:2n-6+C20:4n-6; n-6:n-3 = (C18:2n-6+C20:4n-6)÷(C18:3n-3+C20:5n-3+C22:5n-3+C22:6n-3). The mRNA expression of stearoyl-CoA desaturase (SCD) in LD and ST muscles in Dorper lambs fed diet supplemented with NS seeds, RO leaves and their combination is presented in Figure 3 and 4, respectively. The mRNA expression of SCD in LD (Figure 3) and ST (Figure 4) muscles in Dorper lambs did not differ (p>0.05) among dietary treatments.h f   The relative expression of sterol regulatory element bind­ ing transcription factor 1 (SREBF1) in LD muscle in Dorper lambs was not influenced (p>0.05) by dietary supplementa­ tion of RO leaves, NS seeds and their combination (Figure 5). Contrarily, the relative expression of SREBF1 in ST muscle was influenced by dietary supplements (Figure 6). The mRNA expression of SREBF1 in the SM muscle of Dorper lambs fed T2 and T3 diets was greater (p<0.05) than in the SM muscle of the control lambs. The mRNA expression of SREBF1 in the SM muscle of Dorper lambs fed diet supplemented with blend of RO leaves and NS seeds was not significantly different (p> 0.05) from those fed other dietary treatments. h The expression of the AMP-activated protein kinase alpha 2 (PRKAA2) gene in the LD (Figure 7) and SM (Figure 8) muscles of Dorper lambs differ (p<0.05) among the dietary treatments. The relative expression of PKRAA2 in LD muscle of Dorper lambs fed T2 and T3 diets was greater (p<0.05) than in the LD muscle of the control lambs. The mRNA expression of PRKAA2 in the LD muscle of Dorper lambs fed diet sup­ plemented with blend of RO leaves and NS seeds was not significantly different (p>0.05) from those fed other dietary treatments. The relative expression of PRKAA2 was greater (p<0.05) in the ST muscle of lambs fed diet supplemented with NS seeds compared with those fed the control diet and T4 diet. The expression of PRKAA2 in the ST muscle of lambs fed diet supplemented with RO leaves did not differ (p>0.05) from that in the ST muscle of lambs fed other dietary treatments. was greater than those of lambs fed other dietary treatments. Lightness increased (p<0.05) over postmortem storage. No significant interaction (p>0.05) between diet and postmortem storage on meat lightness was observed. Dietary treatments had no significant effects (p>0.05) on muscle yellowness in Dorper lambs. The muscle yellowness in LD, ST, and SS muscles on d 7 was lower than that observed on d 0 and 1 postmortem. There was no significant interaction (p>0.05) between diet and postmortem storage for meat yellowness in Dorper lambs. h In­ teraction between diet and postmortem storage on shear force of different muscles in Dorper lambs was not significant (p> 0.05). h The LD, ST, and SS muscles of the control lambs had lower (p<0.05) redness than the muscles of the supplemented lambs. Meat redness decreased (p<0.05) as postmortem storage pro­ gressed. There was no significant interaction (p>0.05) between diet and postmortem storage for the redness of meat in Dorper lambs. The lightness of the LD muscle in the control lambs was greater (p<0.05) than that of lambs fed other dietary treat­ ments. The lightness of the ST and SS muscles in the T4 lambs Dietary treatments had no effect (p>0.05) on the cooking loss of SS muscles in Dorper lambs. Cooking loss in LD and ST muscles of the control lambs was greater (p<0.05) than that of the supplemented lambs. Cooking loss in LD, ST, and SS muscle in Dorper lambs increased (p<0.05) over postmortem storage. There was no significant interaction (p>0.05) between diet and postmortem storage for cooking loss in different mus­ cles in Dorper lambs. 1350    www.ajas.info Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 Table 8. Physicochemical properties and lipid oxidation in longissimus dorsi, semitendinosus and supraspinatus muscles in Dorper lambs fed diet supplemented with Rosmarinus officinalis leaves, Nigella sativa seeds and their combination Parameter Muscle Dietary treatments SEM Storage days SEM p value T1 1) T2 T3 T4 0 1 7 Diet (D) Storage (S) D×S pH (unit) LD 5.92 5.99 5.99 5.92 0.03 6.11 a 5.90 b 5.86 b 0.03 0.228 <0.0001 0.956 ST 5.95 6.05 6.01 6.00 0.03 6.10 a 6.00 b 5.91 c 0.03 0.223 0.001 0.838 SS 6.15 6.19 6.18 6.12 0.03 6.20 a 6.10 b 5.95 c 0.03 0.4626 <0.0001 0.982 Drip loss (%) LD 5.19 a 3.28 b 3.27 b 2.95 c 0.08 - 3.80 a 2.40 b 0.23 0.002 0.002 0.240 ST 3.51 a 3.50 a 2.16 b 2.11 b 0.05 - 3.93 a 2.58 b 0.12 0.032 0.006 0.112 SS 3.64 a 2.36 b 2.89 b 2.25 b 0.07 - 3.95 a 2.00 b 0.23 0.003 0.015 0.321 Cooking loss (%) LD 33.26 a 31.18 b 29.95 b 30.44 b 0.81 24.72 c 27.69 b 41.21 a 0.70 0.034 <0.0001 0.783 ST 39.54 a 37.81 b 37.95 b 36.72 b 0.92 27.75 c 37.01 b 47.01 a 0.79 0.019 <0.0001 0.079 SS 32.67 31.33 32.84 34.61 1.20 27.83 b 30.56 b 40.19 a 1.03 0.305 <0.0001 0.148 Shear force (kg) LD 1.10 a 1.03 b 1.03 b 0.93 c 0.05 1.26 a 1.00 b 0.80 c 0.04 0.031 <0.0001 0.439 ST 1.25 a 1.13 b 1.15 b 0.98 b 0.06 1.22 a 1.20 a 0.97 b 0.05 0.027 0.002 0.414 SS 0.76 0.76 0.71 0.76 0.03 0.90 a 0.70 b 0.49 c 0.03 0.551 0.166 0.260 Yellowness (b*) LD 11.07 11.91 11.46 11.32 0.13 11.77 a 11.87 a 9.93 b 0.11 0.271 <0.0001 0.091 ST 10.69 9.92 9.60 10.10 0.19 10.73 a 10.73 a 9.55 b 0.17 0.201 <0.0001 0.210 SS 11.70 11.34 11.04 11.65 0.26 11.11 b 12.13 a 11.05 b 0.23 0.2587 0.0023 0.672 Lightness (L*) LD 38.62 a 35.44 b 34.41 b 32.67 c 0.53 31.43 c 36.29 b 38.14 a 0.46 <0.0001 <0.0001 0.148 ST 41.74 a 40.97 a 40.69 a 39.00 b 0.57 39.97 b 40.18 b 41.65 a 0.48 0.012 0.041 0.551 SS 38.11 a 38.83 a 38.97 a 36.71 b 0.42 34.58 b 39.52 a 40.36 a 0.37 0.0021 <0.0001 0.174 Redness (a*) LD 12.98 b 13.95 a 13.72 a 14.08 a 0.24 14.39 a 12.67 b 11.45 b 0.21 0.012 <0.0001 0.142 ST 13.18 b 14.55 a 14.64 a 14.83 a 0.24 14.63 a 13.68 b 12.34 c 0.21 <0.0001 <0.0001 0.094 SS 13.02 b 14.72 a 14.26 a 14.49 a 0.21 14.94 a 13.95 b 12.73 c 0.18 <0.0001 <0.0001 0.250 TBARS (mg MDA/kg) LD 0.53 a 0.42 b 0.39 b 0.41 b 0.01 0.32 b 0.32 b 0.72 a 0.04 0.0428 <0.0001 0.992 ST 0.56 a 0.40 b 0.37 b 0.41 b 0.01 0.34 a 0.34 a 0.57 b 0.05 0.039 0.014 0.877 SS 0.58 a 0.45 b 0.41 b 0.44 b 0.01 0.41 0.41 0.55 0.02 0.033 0.100 0.993 SEM, standard error of means; LD, longissimus dorsi; ST, semitendinosus; SS, supraspinatus. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800 T1 T2 T3 T4 Relative mRNA levels Dietary treatments LPL expressions of lipoprotein lipase (LPL) target gene in longissimus dorsi of Dorper va seeds, Rosmarinus officinalis leaves and their blend. T1, basal diet; T2, basal officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+ 1 % % Rosmarinus officinalis leaves. Figure 2. The relative expressions of lipoprotein lipase (LPL) target gene in semitendinosus muscle in of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. 0 0.5 1 1.5 2 2.5 3 T1 T2 T3 T4 Relative mRNA levels Dietary treatments LPL Figure 2. The relative expressions of lipoprotein lipase (LPL) target gene in semitendinosus m Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, bas basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, ba Nigella sativa seeds+1% Rosmarinus officinalis leaves. Figure 2. The relative expressions of lipoprotein lipase (LPL) target gene in semitendinosus muscle in of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. 0 0.5 1 1.5 2 2.5 3 T1 T2 T3 T4 Relative mRNA levels Dietary treatments LPL ative expressions of lipoprotein lipase (LPL) target gene in semitendinosus m Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, ba marinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, b 0 0.5 1 1.5 2 2.5 3 T1 T2 T3 T4 Relative mRNA levels Dietary treatments LPL Figure 2. The relative expressions of lipoprotein lipase (LPL) target gene in semitendinosus muscle in of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. tive expressions of lipoprotein lipase (LPL) target gene in semitendinosus m Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. DISCUSSION Dorper lambs fed 1% RO leaves had greater final body weight compared with those fed other diets. This observation could be attributed to the greater feed intake and efficiency in the T2 lambs as observed during the feeding trial. The greater slaughter weight in the T2 lambs could be responsible for their greater cold carcass weight. The current observation concurs with the findings of Allam et al [11] who observed that dietary RO improved final body weight in Awassi lambs. Despite the The TBARS value in the LD, ST, and SS muscles of the con­ trol lambs was greater (p<0.05) than those of supplemented lambs. The concentration of TBARS in LD, ST, and SS muscles of Dorper lambs increased (p<0.05) as postmortem storage progressed. Interaction between diet and postmortem storage was not significant (p>0.05) for muscle lipid oxidation in Dorper lambs. 1351 www.ajas.info 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means having different superscripts along the same row for each factor are significantly different (p<0.05). SEM, standard error of means; LD, longissimus dorsi; ST, semitendinosus; SS, supraspinatus. 1) T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means having different superscripts along the same row for each factor are significantly different (p<0.05). is consistent with that of Hassan et al [10] who observed that dietary supplementation of NS (7.5 g NS/kg DM) had no effect on the carcass traits in Karadi lambs. 27 changes in slaughter and cold carcass weights among the treat­ ments, chill loss, dressing percentage, percentages of shoulder, breast, neck and legs and the proportion of lean, bone and fat in the primal cuts of Dorper lambs did not differ. This obser­ vation suggests that the dietary supplements did not affect tissue partitioning in Dorper lambs. The current observation Herein, dietary supplementation of medicinal plants did not affect IMF and carcass fatness in Dorper lambs. This sug­ gests that the muscle FA composition was not confounded by IMF and carcass fatness. The similar IMF and carcass fat­ Figure 1. The relative expressions of lipoprotein lipase (LPL) target gene in longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves and their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800 T1 T2 T3 T4 Relative mRNA levels Dietary treatments LPL expressions of lipoprotein lipase (LPL) target gene in longissimus dorsi of Dor a seeds, Rosmarinus officinalis leaves and their blend. T1, basal diet; T2, basa fficinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+ 1 % % Rosmarinus officinalis leaves. Figure 2. The Dorper lambs basal diet+1% 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800 T1 T2 T3 T4 Relative mRNA levels Dietary treatments LPL 1352    www.ajas.info Figure 1. The relative expressions of lipoprotein lipase (LPL) target gene in longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves and their blend. The relative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target ge dorsi muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their ble T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, bas sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are sign (p<0.05). Figure 8. The relative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target gene in semitendinosus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are significantly different (p<0.05). 0 0.5 1 1.5 2 2.5 3 3.5 4 T1 T2 T3 T4 Relative mRNA levels Dietary treatments PRKAA2 b b ab a Figure 8. The relative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target g muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blen basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, ba Figure 4. The relative expressions of stearoyl-CoA desaturase (SCD) target gene in semitendinosus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. 0 0.5 1 1.5 2 2.5 3 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SCD expressions of stearoyl-CoA desaturase (SCD) target gene in semitendin a sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T3 b l di t+1% Ni ll ti d T4 b l di t+1% Ni ll Figure 7. The dorsi muscle in T2, basal diet+ sativa seeds+1 0.000 0.500 1.000 1.500 2.000 2.500 3.000 T1 T2 T3 T4 Relative mRNA levels Dietarytreatment PRKAA2 a a ab b 0 0.5 1 1.5 2 2.5 3 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SCD Figure 4. The relative expressions of stearoyl-CoA desaturase (SCD) target gene in semitendinosus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. Figure in semi basal d di t+1% 0 0.5 1 1.5 2 2.5 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SCD 0 0.5 1 1.5 2 2.5 3 3.5 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SREBF1 a a b ab Figure 3. The relative expressions of stearoyl-CoA desaturase (SCD) target gene in longissimus dorsi in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. expressions of stearoyl-CoA desaturase (SCD) target gene in longissim a sativa seeds, Rosmarinus officinalis leaves or their blend. T1, ba us officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, b osmarinus officinalis leaves Figure in semi basal d orsi in et; T2, diet+1% Figure 6. The relative expressions of sterol regulatory element-binding transcription factor 1 (SREBF1) target gene in semitendinosus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are significantly different (p<0.05). e relative expressions of sterol regulatory element-binding transcription factor 1 (SRE osus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves o 2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa gella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with differen different (p<0.05). ness could be due to the similar energy content of the dietary treatments. Irrespective of dietary treatment and muscle type, C18:1n-9 was the most abundant FA followed by C16:0 and C18:0. Similar observation was documented in chevon [23] and lamb meat [6]. g The muscle FA of Doper lambs fed diets supplemented with medicinal plants was inconsistent. The FA content of SS mus­ cle was unaffected by dietary supplements. This observation is 30 s muscle in basal diet+1% a seeds+1% 31 Figure 7. The relative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target gene in longissimus dorsi muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are significantly different (p<0.05). T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% eeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. Figure 8. The relative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target gene in semitendinosus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are significantly different (p<0.05). 0 0.5 1 1.5 2 2.5 3 3.5 4 T1 T2 T3 T4 Relative mRNA levels Dietary treatments PRKAA2 b b ab a Figure 8. The relative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target g muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blen basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, ba sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are s or 1 (SREBF1) officinalis leaves basal diet+1% Figure 8. The relative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target gene in semitendinosus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are significantly different (p<0.05). 0 0.5 1 1.5 2 2.5 3 3.5 4 T1 T2 T3 T4 Relative mRNA levels Dietary treatments PRKAA2 b b ab a elative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) targe per lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their b Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, 0 0.5 1 1.5 2 2.5 3 3.5 4 T1 T2 T3 T4 Relative mRNA levels Dietary treatments PRKAA2 b b ab a Figure 5. The relative expressions of sterol regulatory element-binding transcription factor 1 (SREBF1) target gene in longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. T1, ba marinus officinalis leaves; T3 basal diet+1% Nigella sativa seeds; T4 b Figure 1. The relative expressions of lipoprotein lipase (LPL) target gene in longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves and their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. xpressions of lipoprotein lipase (LPL) target gene in longissimus dorsi of Do seeds, Rosmarinus officinalis leaves and their blend. T1, basal diet; T2, bas icinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+ 1 % Figure 2. Th Dorper lambs basal diet+1% 1352 www.ajas.info 29 dorsi in diet; T2, diet+1% Figure 6. The relative expressions of sterol regulatory element-binding transcription factor 1 (SREBF1) target gene in semitendinosus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are significantly different (p<0.05). 0 0.5 1 1.5 2 2.5 3 3.5 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SREBF1 a a b ab he relative expressions of sterol regulatory element-binding transcription factor 1 (SRE nosus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves o T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different different (p<0.05). Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 Figure 3. The relative expressions of stearoyl-CoA desaturase (SCD) target gene in longissimus dorsi in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. 0 0.5 1 1.5 2 2.5 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SCD expressions of stearoyl-CoA desaturase (SCD) target gene in longissim la sativa seeds, Rosmarinus officinalis leaves or their blend. T1, ba us officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, b Rosmarinus officinalis leaves. 0.000 0.500 1.000 1.500 2.000 2.500 3.000 T1 T2 T3 T4 Relative mRNA levels Dietarytreatment PRKAA2 a a ab b tive expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target orper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their b Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, b Rosmarinus officinalis leaves. a,b,c Means with different superscript are s Figure 4. The relative expressions of stearoyl-CoA desaturase (SCD) target gene in semitendinosus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. 0 0.5 1 1.5 2 2.5 3 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SCD expressions of stearoyl-CoA desaturase (SCD) target gene in semitendinosus muscle in la sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% eaves. Figure 5. The relative expressions of sterol regulatory element-binding transcription factor 1 (SREBF1) target gene in longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. 31 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SREBF1 e relative expressions of sterol regulatory element-binding transcription factor 1 (SREBF1) longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves d. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% eeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. Figure 7. The relative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target gene in longissimus dorsi muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are significantly different (p<0.05). 0.000 0.500 1.000 1.500 2.000 2.500 3.000 T1 T2 T3 T4 Relative mRNA levels Dietarytreatment PRKAA2 a a ab b Figure 7. expressions of stearoyl-CoA desaturase (SCD) target gene in semitend sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet Figure 7. The dorsi muscle T2, basal diet sativa seeds+ s muscle in basal diet+1% a seeds+1% 31 Figure 7. The relative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target gene in longissimus dorsi muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are significantly different (p<0.05). ive expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target rper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their b Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, b Rosmarinus officinalis leaves. a,b,c Means with different superscript are s 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SREBF1 Figure 5. The relative expressions of sterol regulatory element-binding transcription factor 1 (SREBF1) target gene in longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SREBF1 relative expressions of sterol regulatory element-binding transcription longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmarin . T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; eeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves Figure 8. muscle in basal diet+ www ajas info 1353 Figure 5. The relative expressions of sterol regulatory element-binding transcription factor 1 (SREBF1) target gene in longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800 T1 T2 T3 T4 Relative mRNA levels Dietary treatments SREBF1 e relative expressions of sterol regulatory element-binding transcription factor 1 (SREBF1) longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves d. relative expressions of sterol regulatory element-binding transcription longissimus dorsi of Dorper lambs fed Nigella sativa seeds, Rosmari T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; eds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leave Figure 8. muscle in or 1 (SREBF1) fficinalis leaves basal diet+1% Figure 8. The relative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) target gene in semitendinosus muscle in Dorper lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their blend. T1, basal diet; T2, basal diet+1% Rosmarinus officinalis leaves; T3, basal diet+1% Nigella sativa seeds; T4, basal diet+1% Nigella sativa seeds+1% Rosmarinus officinalis leaves. a,b,c Means with different superscript are significantly different (p<0.05). elative expressions of AMP-activated protein kinase alpha 2 (PRKAA2) targe er lambs fed Nigella sativa seeds, Rosmarinus officinalis leaves or their b 1353 cript ar www.ajas.info  with different sup Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 consistent with that of Karami et al [5], who reported that di­ etary supplementation of turmeric and Andrographis paniculata leaves had minimal impact on the muscle FA composition of longissimus dorsi muscle in Kacang goats. In contrast, dietary thyme in pregnant and lactating Segurena ewes increased the concentration of polyunsaturated FAs in the meat from the lambs [6]. h electric point of most proteins thereby affecting their ability to hold water [26]. This observation could also be due to stearic effects, in which there is a reduction in the available space for water resulting from the formation of crosslinks between thin and thick filaments during the development of rigor [25,26]. The increase in drip loss over chill storage is in tandem with the report in goats [26]. However, cooking loss was reduced [26] during postmortem storage of chevon.h The changes in the concentration of C18:1n-9 in the LD muscles of Dorper lambs could be attributed to the changes in the ruminal concentration of the FA. The ruminal con­ centration of C18:1n-9 was greater in the rumen of the T2 lambs as observed during the feeding trial. Dietary supple­ mentation of medicinal plants reduced the concentration of C18:0 and increased the concentration of C18:1 in the ST mus­ cle of Doper lambs. This observation could be attributed to the phenolic compounds in the supplements, which have the capacity to reduce the biohydrogenation of FAs in the rumen. Similar observation was reported in the LD muscle of goats fed different parts of Andographis paniculata [12].fh The LD and ST muscles in supplemented lambs had lower shear force than those from the control lambs. The higher ten­ derness in the meat of supplemented lambs could be due to the lower cooking loss of the meat samples. Adequate water in muscle increases juiciness on mastication, which enhances tenderness [25,26]. Reduced cooking loss would possibly en­ hance tenderness because a given cross-sectional area of a meat sample would have less structural components and more water [26]. In line with the current observation, Yusuf [12] observed that dietary Andrographis paniculata improved ten­ derness in chevon. In addition, dietary supplementation of quercetin improved the tenderness of longissimus muscle of beef cattle [24]. Contrarily, dietary treatments had no effect on the shear force value of SS muscle in Dorper lambs. The shear force of different muscles reduced over chill storage. This observation could be attributed to the weakening of myofi­ brillar structures by endogenous muscle proteinases [25,26]. h Diets had no effect on the muscle pH in Dorper lambs. This could be attributed to the similar energy content of the di­ etary treatments and the similar management and slaughter conditions employed during the trial. The pH values observed in the current study fall within the pH of normal meat as re­ ported in goats [1] and beef [24]. The current observation corroborates the findings of Karami et al [5] who observed that dietary Andrographis paniculata and turmeric powder had no effect on muscle pH in chevon. Contrarily, dietary sup­ plementation of quercetin increased the pH of longissimus muscle in Holstein Friesian cattle [24]. Postmortem storage influenced muscle pH in Dorper lambs. The pre-rigor pH was greater than the post-rigor pH. This observation could be attributed to postmortem glycolysis, which requires the conversion of glycogen to lactic acid [25]. Similar observa­ tion was observed in chevon [1].h The meat from the supplemented lambs had greater red­ ness than the meat from the control lambs. This observation could be due to the antioxidant effect of the polyphenols in the supplements, which prevented oxidative deterioration as depicted in the TBARS data. Similarly, dietary Moringa oleifera [7] turmeric and Andrographis paniculata [5] leaves improved the redness of chevon. Contrarily, dietary quercetin did not affect the colour coordinates of longissimus muscle in Holstein Friesian cattle [24]. 354    www.ajas.info The current observation is consis­ tent with the report of Bhuiyan et al [34] who observed that the expression of SREBP1 had a positive relationship with the C18:1n-9 and polyunsaturated fatty acids contents in different muscles in Hanwoo cattle.h Diets can alter the synthesis and deposition of FA in mus­ cles which can modulate the expression of lipogenic genes [13,14]. It is expected that a better knowledge of the genes and mechanisms involved would allow a better control of the content and composition of FA in ruminant meat [14]. In the current study, we limited our investigation on lipogenic gene expression to the LD and ST muscles because dietary sup­ plements had no significant effect on the FA composition of SS muscles in Dorper lambs. f The PRKAA2 plays an important role in the regulation of FA and cholesterol [35]. Dietary supplementation of medici­ nal plants affected the expression of PRKAA2 gene in LD and ST muscles in Dorper lambs. Lambs fed the T2 and T3 diets had greater expression of PRKAA2 gene in LD muscle than the lambs fed the control and blend of RO leaves and NS seeds. Similar trend was observed in the ST muscle of Dorper lambs. The current observation could be attributed to the greater pro­ portion of C18:1n-9 in the muscle of lambs fed T2 and T3 diets compared to those fed the control and T4 diets. This observa­ tion contradicts the findings of Dervishi et al [13] who observed that differences in feeding system and FA composition of mus­ cles did not alter the expression of PRKAA2 gene in lambs. Dietary supplementation of medicinal plants did not affect the expression of LPL gene in LD and ST muscles in Dorper lambs. This observation could be attributed to the similar muscle IMF among the treatments. LPL is the rate limiting enzyme for the import of triglyceride (TAG) derived FAs by muscle, for utilization, and adipose tissue, for storage, and plays an important role in the differentiation and maturation of adi­ pose cells [28]. Lipoprotein lipase serves as a central factor in hydrolysis of triacylglycerol and uptake of free FAs from the plasma [29]. Moreover, it controls the triglycerides partitioning between adipose tissue and muscles [30]. Chill storage influenced the colour co­ ordinates of mutton. The redness and yellowness of chevon decreased while the lightness increased over chill storage. This finding could be due to the oxidative deterioration of myo­ globin during chill storage. A decrease in the concentration of myoglobin and an increase in the concentration of met­ myoglobin play a major role in the loss of redness in meat during chill storage [26]. The supplementation of NS seeds, RO leaves and their blend reduced drip loss in different muscles in Dorper lambs. How­ ever, the impact of dietary medicinal plants on cooking loss in mutton was muscle dependent. Dietary supplements re­ duced cooking loss in LD and ST muscles but had no effect on the cooking loss in SS muscle in Dorper lambs. The reduc­ tion in drip and cooking losses could be due to the presence of antioxidant compounds in the supplements, which reduced the oxidation of myofibrillar proteins during postmortem chill storage. The current finding is consistent with that of Yusuf [12] who observed that dietary supplementation of Androgra­ phis paniculata in goats reduced cooking loss in chevon. In contrast, dietary quercetin did not affect the drip and cooking losses in longissimus muscle of beef cattle [24]. Cooking and drip losses increased over postmortem chill storage. This ob­ servation could be due to the loss of the structural integrity of the myofibrils [25]. At rigor, the muscle pH nears the iso­ Dietary supplementation of medicinal plants reduced lipid oxidation in different muscles in Dorper lambs. This obser­ vation could be attributed to the presence of polyphenols in the medicinal plants, which exert anti-oxidative effect. Similar findings were observed in goats fed turmeric and Andrographis paniculata [5] and Moringa oleifera [7] leaves. However, dietary quercetin did not affect the TBARS values in longissimus mus­ cle in Holstein Friesian cattle [24]. Lipid oxidation increased over chill storage. This could be due to the loss of endogenous antioxidants in the meat samples. Similar observations were documented in beef [27] and chevon [1]. 1354    www.ajas.info Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 the concentration of C18:1n-9 and C18:3n-3 in the T4 lambs, which was not significantly different from those of lambs, fed other dietary treatments. CONCLUSION Dietary supplementation of NS seeds, RO leaves and their com­bination can be used to enhance water holding capacity, oxidative stability and tenderness of mutton. The muscle-de­ pendent changes in FA composition in response to dietary supplements, induced changes in the expression of lipogenic genes. These results provide insight into the mechanisms in­ volved in diet-induced changes in the muscle FA composition of Dorper lambs. The expression of SCD is physiologically important in the synthesis and metabolism of fat and plays a vital role in energy homeostasis [14]. Dietary NS seeds, RO leaves and their com­ bination did not affect the expression of SCD gene in LD and ST muscles in Dorper lambs. This observation suggests that the changes in the monounsaturated FAs content in the mus­ cles are of dietary origin. In addition, the similarity in IMF among the diets could be responsible for the non-significant differences in SCD expression. Similarly, dietary alfalfa hay or concentrate did not affect the IMF and SCD expression de­ spite changes in the FA profile of longissimus dorsi in lambs [14]. h ACKNOWLEDGMENTS This research was funded by the Ministry of Higher Education, Malaysia through Fundamental Research Grant Scheme (Pro­ ject code: 07-01-14-1436FR). The funder had no role in carrying out the experiment, writing of the manuscript and the decision to submit the manuscript for publication. CONFLICT OF INTEREST We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu­ script. The SREBF1 is a member of the basic helix-loop-helix-leu­ cine zipper family of transcription factors involved in adipocyte differentiation, biosynthesis of FAs and cholesterol [33] and plays an important role in energy homeostasis [34]. The expres­ sion of sterol regulatory element-binding protein 1 (SREBP1) in LD muscle of Dorper lambs did not differ among dietary treatments. Nonetheless, dietary supplementation of medicinal plants influenced the expression of SREBP1 in ST muscles in Dorper lambs. Dietary supplementation of NS seeds and RO leaves up regulated the expression of SREBP1 gene in ST mus­ cle in Dorper lambs compared with that of the control lambs. This observation could be attributed to the greater concen­ tration of C18:1n-9 and C18:3n-3 in the T3 and T2 lambs compared to the control lambs. The relative expression of SREBP1 in the ST muscles in Dorper lambs fed diet supple­ mented with blend of RO leaves and NS seeds did not differ from those fed other diets. This observation is consistent with The current obser­ vation is consistent with those of Anderson et al [31] and Bonnet et al [32] who observed that IMF deposition had a positive relationship with LPL gene expression in sheep.h AOAC International; 2007. 2. Bouvard V, Loomis D, Guyton KZ, et al. Carcinogenicity of consumption of red and processed meat. Lancet Oncol 2015; 16:1599-600. 17. Van Soest PV, Robertson J, Lewis B. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in rela­tion to animal nutrition. J Dairy Sci 1991;74:3583-97. 18. Makkar HP, Blümmel M, Borowy NK, Becker K. Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. J Sci Food Agric 1993;61: 161-5. 3. Jeong SH, Kang D, Lim MW, Kang CS, Sung HJ. Risk assess­ ment of growth hormones and antimicrobial residues in meat. Toxicol Res 2010;26:301-13. 4. 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Comparison of myofibrillar protein degradation, antioxidant profile, fatty acids, metmyoglobin reducing activity, physicochemical properties and sensory attributes of gluteus medius and infraspinatus mus­ cles in goats. J Anim Sci Technol 2016;58:23. 11. Allam SM, Abou-Ammou FF, Farghaly MS, Othman AA. Effect of some natural antioxidants on lamb performance. 1. Carcass characteristics of lambs fed partial full fat soybean with natural additives. Egyptian J Nutr Feed 2005;8:275-9. 27. Popova T, Marinova P, Vasileva V, Gorinov Y, Lidji, K. Oxidative changes in lipids and proteins in beef during storage. Arch Zootech 2009;3:30-8. 12. Yusuf AL. Evaluation of dietary supplementation of Andro­ graphis paniculata on growth performance and meat quality of Boer goats [PhD thesis]. Serdang, Malaysia: Universiti Putra Malaysia; 2014. 28. Weinstock PH, Levak-Frank S, Hudgins LC, et al. Lipoprotein lipase controls fatty acid entry into adipose tissue , but fat mass is preserved by endogenous synthesis in mice deficient in adipose tissue lipoprotein lipase. In: Proceedings of National Academy of Science, 1997; 1997 September 16; National Aca­ demy of Science; 1997;94:10261-6. 13. Dervishi E, Serrano C, Joy M, et al. The effect of feeding system in the expression of genes related with fat metabolism in semi­ tendinosus muscle in sheep. Meat Sci 2011;89:91-7. 14. González-Calvo L, Joy M, Blanco M, et al. Effect of vitamin E supplementation or alfalfa grazing on fatty acid composi­ tion and expression of genes related to lipid metabolism in lambs. J Anim Sci 2015;93:3044-54. 29. Zhao W, Hu S, Yu K, et al. Lipoprotein lipase, tissue expression and effects on genes related to fatty acid synthesis in goat mam­ mary epithelial cells. Int J Mol Sci 2014;15:22757-71. f 30. Sorisky A. REFERENCES 1. Adeyemi KD, Shittu RM, Sabow AB, Ebrahimi M, Sazili AQ. 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Official methods of analysis of the Association of Offi­cial Analytical Chemists. 18th ed. Washington DC, USA: 1356    www.ajas.info Odhaib et al (2018) Asian-Australas J Anim Sci 31:1345-1357 are influenced by growth and energy intake regimen. J Nutr Biochem 1996;7:610-6. 34. Bhuiyan MS, Yu SL, Jeon JT, et al. DNA polymorphisms in SREBF1 and FASN genes affect fatty acid composition in Korean cattle (Hanwoo). Asian-Australas J Anim Sci 2009;22: 765-73. 32. Bonnet M, Leroux C, Faulconnier Y, Martin P, Chilliard Y. Lipoprotein lipase activity and mRNA are up-regulated by refeeding in adipose tissue and cardiac muscle of sheep. J Nutr 2000;130:749-57. 35. Lee, HY, Choi BH, Lee JS, et al. Molecular characterization and chromosomal mapping of the porcine AMP-activated protein kinase α 2 (PRKAA 2) Gene. Asian-Australas J Anim Sci 2007; 20:615-21. 33. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. 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https://openalex.org/W4313226589
https://journals.openedition.org/pontourbe/pdf/13428
Portuguese
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A luta internacional contra a Aids no campo dos direitos humanos: uma análise do encontro “Stop Aids” na ONU, ou como se constroem os discursos globais sobre os direitos
Ponto Urbe
2,022
cc-by
5,844
Editora úcleo de Antropologia Urbana da Universidade de São Paul Núcleo de Antropologia Urbana da Universidade de São Paulo Edição electrónica URL: https://journals.openedition.org/pontourbe/13428 ISSN: 1981-3341 Ponto Urbe Revista do núcleo de antropologia urbana da USP  30 | 2022 Ponto Urbe 30 n.2 Ponto Urbe Revista do núcleo de antropologia urbana da USP  30 | 2022 Ponto Urbe 30 n.2 Ponto Urbe Revista do núcleo de antropologia urbana da USP  30 | 2022 Ponto Urbe 30 n.2 Ponto Urbe Revista do núcleo de antropologia urbana da USP  30 | 2022 Ponto Urbe 30 n.2 A luta internacional contra a Aids no campo dos direitos humanos: uma análise do encontro “Stop Aids” na ONU, ou como se constroem os discursos globais sobre os direitos The international struggle against AIDS in the field of human rights: an analysis of the “Stop AIDS” meeting at the UN, or how global discourses on human rights are constructed Paulo SC Neves e Laura Moutinho Paulo SC Neves e Laura Moutinho $OXWDLQWHUQDFLRQDOFRQWUDD$LGV QRFDPSRGRVGLUHLWRVKXPDQRV XPDDQ£OLVHGRHQFRQWURɝStopAidsɞ QD218RXFRPRVHFRQVWURHPRV GLVFXUVRVJOREDLVVREUHRVGLUHLWRV The international struggle against AIDS in the field of human rights: an analysis of the “Stop AIDS” meeting at the UN, or how global discourses on human rights are constructed Paulo SC Neves e Laura Moutinho Paulo SC Neves e Laura Moutinho NOTA DO EDITOR 9HUV¥RRULJLQDOUHFHELGDHP2ULJLQDO9HUVLRQ $FHLWD©¥R$FFHSWHG A luta internacional contra a Aids no campo dos direitos humanos: uma análise... Refêrencia eletrónica Paulo SC Neves e Laura Moutinho, «A luta internacional contra a Aids no campo dos direitos humanos: uma análise do encontro “Stop Aids” na ONU, ou como se constroem os discursos globais sobre os direitos», Ponto Urbe [Online], 30 | 2022, posto online no dia 28 dezembro 2022, consultado o 27 julho 2024. URL: http://journals.openedition.org/pontourbe/13428 Este documento foi criado de forma automática no dia 27 de julho de 2024. Apenas o texto pode ser utilizado sob licença CC BY 4.0. Outros elementos (ilustrações, anexos importados) são "Todos os direitos reservados", à exceção de indicação em contrário. 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W2056874893.txt
https://zenodo.org/records/1893491/files/article.pdf
de
Über spontane Arrosion und Ruptur der Carotis interna nach Jugularisunterbindung
European archives of oto-rhino-laryngology/European archives of oto-rhino-laryngology and head & neck
1,910
public-domain
3,659
XII. (Aus der Universit/itsklinik far 0hren-, Nasen- und Kehlkopfkrankheiten zu Giegen. Direktor Professor Dr. E. L e u t e r t . ) Uber spontanc Arrosion und Ruptur der Garotis interna nach Jugularisunterbindung. Yon Privatdozent Dr. F. Nuernberg~ I. Assistent der Ktinik. Der vorliegende Fall ist allein interessant wegen der spontanen Arrosion and Ruptar der Carotis interna und reehffertigt eine Publikation umsomehr, als Komplikationen gleicher Art naeh Jugularisunterbindung in der otologisehen Literatur meines Wissens nieht bekannt stud. Es handelte sich um einen i5jgthrigen jungen Mann, der bereits 1908 eine akute Otitis media auf demselben Ohr durchgemacht butte und Ende August wegen einer seit 10 Tagen bestehenden, sehr stark sezernierenden Otitis media sinistra in unsere Behandlung trat. Das rechte Ohr wies auch in funktioneller Beziehung normale VerhMtnisse auf. Aus der Anamnese war ersichtlich, dab er am ersten Tage vor der spontanen Perforation des Trommelfelles, Maximaltemperatur 3%5 gehabt, die Temperatnr sonst vor Eintritt in unsere Behandlung sich zwischen Maximaltemperatur 37,6 and 38,8 bewegt butte. Die Perzeption ftir Fttlstersprache war links aufgehoben~ das HSrvermOgen ftir tiefe, mittlere und hohe Tfne stark berabgesetzt. Die Therapie bestand in wiederholter Paracentese, Bettruhe and Applikation yon Ohreisbeatel. Die Maximaltemperatur am Tage der Aufnahme betrug 37,6~ am folgenden Tage 37,4 und stieg am dritten Tage auf 38,2. Da die Druckempfindlichkeit am Processus mastoideus erheblich zugenommen hatte, so zSgerte ich nicht~ die einfaChe AufmeiBelung zu machen, die ein freies Empyem and eine ausgedehnte Caries des Processus masteideus aufdeckte. Der Sulcus des Sinus war ca. 2 cm yon der Caries ergriffen and der Sinus wurde daher breit freigelegt. Er war in der Mitte zwischen unterem and oberem Knie etwas verfiirbt. Nach weiterer Freilegung des Sinus Bulbuswfixts, der hier normal blaue Farbe aufwies, wurde nach mehrmaliger Desinfektion mit H~O2 meine mit flacher, nieht ausgezogener Spitze versehene Punktionsnadel im gesund aussehenden Sinus eingestoBen un-d zun~chst ca. 2 ccm Blut entnommen, dann die ~Iadel tief nach dem Bulbus geftihrt, and 1 ccm Blur aufgesogen und so drei Agarplatten mit je I ccm Blut zur bakteriologischen Untersuchung gewonnen. Alle drei Platten blieben ebenso wie zwei aus der V. mediana beschickte Platten steril. Aus dem Eiter warden Streptokokken in Reinkultur geztichtet. Am nAchsten Tage stieg die Temperatur auf 39,6 (P. 130) als dessen Ursache eine Angina angenommen werden konnte. Aus dem Tonsillenbelage wurden ebenfalls Streptokokken in Reinkultur geztlchtet. Die Temperatur Uber spontane Arrosion und Ruptur der Carotis interna. 201 fiel am 2. Tage nach der Operation auf M+ 38,8 {P. 113), am 3. Tage p. o. auf 38,2 und betrug am 4. Tage 37,5. Die Tonsillen wiesen wieder normale Farbe auf, dagegen htistelte der Patient etwas und die Untersuchung der Lungen ergab eine ca. 5 Marksttick grol~e Zone im rechten Untertappen, die wohl fiir eine beginnende Lungenaffektion verdhchtig erschien, zurzeit abet nichts Charakteristisches bot. Wohl aber waren die Wnndr~nder etwas schmierlg belegt. Subiektiv ftihlte sich der Patient sehr wohL Am Mittag des folgenden Tages stieg die Temperatur pl~itzlich auf 3S,8. Patient klagte fiber beiderseitige Angenschmerzen. Von seiten der Augenklinik (Dr. G ~ r t n e r ) wurde normaler Augenbefund erhoben. Auch eine wiederholte eingehende Untersuchung des K0rpers des Patienten deckle kelne pathotogischen Ver~nderungen auf, welche aIs Ursache der Temperatursteigerung hhtten beschuldigt werden kfnnen. In der Nacht um 3 Uhr wurde T. 38,5 festgesiellt und fiel dann im Laufe der Nacht auf 38,6. Ebenso wie wit konnte auch der Consiliarius der Med. Klinik (Dr. H o h l w e g ) den normalen Befund der inneren Organe besthtigen. Mithin muI~te die Temperatursteigerung auf eine Sinusthrombose bezogen werden. Ich zSgerte daher nicht mit der weiteren Operation, legte den Sinus, in dessen Mitte zwei kleine linsengrol~e feste Granulationen aufsa~en, nach oben und unten welter fret, bis tief nnten nach dem Bulbus zu und ebenso nach oben etwa 21/s cm fiber dem oberen Knie der blaue Sinus im Gesichtsfelde erschien. Die dazwischen ]iegenden Partien sahen gelb-grau verf~rbt aus und ftihlten sich hart an. Die Punktion des Sinus bulbuswhrts fSrderte Blut zutage+ Auf den Kulturen wuchsen ca. 350 Kol. Streptokokken in Reinkultur, die starke Hhmolyse aufwiesen. ])as vor der Unterbirdung der V. jugularis gewonnene Blur blieb ebenso wie das aus der ¥. mediana entnommene steri]. Nach Exzision der huf~eren Sinuswand wurde der zum Tell obliterierte Sinus nach oben und unten bis zur Grenze des gesunden Sinus mit dem S c h w a r t z e s c h e n Trommelfellzirkumzissionsmesser, das fiir diese Zwecke stets bet uns verwendet wird, gespatten und die geriuge Blutung oben und unten durch Einfiihren eines Gazestreifens und weitere Tamponade leicht zum Stehen gebracht. Die Temperatur der nhchsten 8 Tage schwankte zwischen 38,0 rain. und 40,1 max. i)as Allgemeinbefinden des Patienten war im ganzen zufriedenstellend, die Nabrungsaufnahme immer ausgezeichnet Zwar klagte der Patient ab und zu fiber linksseitige Kopfschmerzen, zeigte motorische Unruhe, speziell Zuckungen in Hhnden und Ffil~en~ wies abet sonst keine meningitischen Symptome auf. Bereits am 2. Tage p. o. wurde der Tampon aus dem Bnlbus entternt, ohne daI~ eine l:;lutung erfolgte, und diese Sinus5ffnung wurde dann bis zum 29. Septbr. (20 Tage p. t[. ap.) welt often gelassen, um jederzeit die Bulbusoperation eventuell vornehmen zfi k0nnen. Der Tampon nach dem Torcular zu wurcle am 4. Tage p If. op etwas gelockert und da Bh~tung erfolgte, daneben ein zweiter Tamponstreifen eingeftihrt, die beide bis zum 9. Tage p. op. liegen blieben und dann mtihelos entfernt wurden. Die dabei eintretende minimale Blutung stand sofort durch leichte Kompression und durch Einfiihrung eines neuen Tampons, der spontan am iT. Septbr+ (13 Tage p. It. Op) sich abstiel~. Am 12. Oktober (7 Tage p. II. op.) stiefien sich die Jugularisunterbindnngsfhden ab. Die Jugulariswunde wurde stets lose mit Jodoformgaze tamponiert, sezernierte in den ersten Tagen ziemlich stark, dann lieI~ die Sekretion wesentlich nach, die Wunde granulierte gut~ zeigte aber in der Tiefe immer etwas schmierigen Belag. Die an diesem Tage vorgenommene HSrprttfung ergab: Fltistersprache wurde auf ca. 3/~ Meter gehSrt, die Perzeption ttir c+~ a~+ A~ fis4 war ganz minimal herabgesetzt. Bereits tags zuvor am 13. September klagte Patient fiber ab und zu auftretende Stirnkopfschmerzen, im Gegensatz zu frtiher~ wo er fiber linksseitige Kopfschmerzen klagt% die motorische Unruhe war erheblicher. Andere auf Meningitis deutende Symptome Iehlten: I)er Augenbefund war normal (Dr. K u f f i e r - A u g e n k l i n i k ) , Darreichung yon Lactophenin erzielte keine Herabsetzung der Temperatur. I-]erz und Lungen wiesen keine pathologischen Veritnderungen auf. In Anbetracht der anscheinenden Trostlosigkeit des Falles - der Patient war k6rperlich hochgradig herunterArchly f. 0hrenheilkunde. Bd, 81. 14 202 XII. F. NUERNBERG. glaubte ieh noeh einen Versuch mit Einspritzung yon gekommen Heilserum machen zu dfirfen. Ich spritzte innerhalb dreier Tage am I. und 3. Tage 2real je 10 cem M e n z e r s c h e s Heilserum ein. Die Temperatur an diesen drei Tagen betrug maximal 40,3 40, 39,5 (P. t42), am letzten Tage minimal 36,5: dabei leichter Schfittelfrost und stark eitrige Sekretion aus dem Bulbus und Antrum, Zur Belebung der Herzti~tigkeit wurde Strophantus gegebea. Am 17. September 12 Uhr a. m. typiseher Schfittelfrost. Temperatur 40,6, Pals 148: abends gegen 8 Uhr nochmals Schiittelfrost, Temperatur 40,9, Puls 148. Starke Mattigkeit und Unruhe des Patienten. Sekt. Pyramidon. Am 18. September frtth 35,7 {Pals 120). Patient klagte fiber starke Schmerzen an der Aul~enseite des rechten Obersehenkels in der H6he des Trochenter. Es tie[~ sicb bei starker Druckempfindtiehkeit an dieser Stelle eine ca. 5 Markstiick grol~e Zone abgrenzen, die pralle Spannung aafwies. Im Atherrausch, wobei der Patient sehr stark preBte und sicb heftig gegen die Narkose wehrte~ erfolgte die breite Spaltung eines c a I/4 Liters dtinnfltissigen stinkenden Eiter enthaltenden Abszesses, der welt unter den Glutaeus minimus sich erstreckte and durch eine GegenSffnung drainiert wurde. ]m Eiter warden Streptokokken in tleinkultur nachgewiesen. 51ach der Operation Temperatur 38,4 und wieder leichter Schfittelirost. Abends wieder •~9,7 (120). I)er Eintritt yon haufigen Durchihllen, die an den beiden folgenden Tagen anhielten und den Patienten kSrperlich recht mitnahmen, wurde wirkungsv011 durch Opium beeinflul~t, alas im allgemeinen wegen seiner Wirkung auf das Herz bei schweren Infektionen zu vermeiden ist, hier aber indiziert war, da der Patient sieh g.e-en Mor p.hiumeinspritzungen, wehrte. . Trional nsw. ebenso nicht nahm und wir somit zwel Wlrkungen des Opiums, dm Unruhe beki~mpfende und die Durchfalle wirkungsvolt beeinflussende, kombiniereu konnten. Die Temperaturen betragen an den folgenden drei Tagen bei relativ gutem Allgemeinbefinden und bei wie immer ausgezeiehneter bIahrangsaufhat, me (tonischer Wein yon Vialt 40,1 (P. 120), 38.9, 38,2 maximal. Am 22. September 07 Tage post Jug.-Unterbindung, 10 Tage post ,~bstoi3ung der Jugul.-Faden betrug die Temperatur 36.5 (P. 96). Patient hatte die ~Nacht sebr gut geschlafen, ffihtte sich aul~erst wohl, scherzte mit der Umgebung' und wurde wie fiblich auf der Trage nacb dem Operationszimmer zum Verbandwechsel getragem Der Verband wurde yon dem Diener abgeschnitten, and wahrend ich reich anschickte, die Desinfektion der Hande zu vollenden, wurde ich darauf aufmerksam gemacht, da~ etwas Blut aus der Halswunde sickerte. Bei naherer Inspizierung spritzte aus der Wunde in diinnem Strahle anscheinend aus einem oberflachlichen Gef~fle arterieIles Blut. In der Absicht mit einem P6an das Gefal~ zu fassen, zog ich die Wundri~nder etwas auseinander, als mir ein dicker Sprudel Blares am Gesicht vorbeischol~, -ohne Zweifel eine Carotisblutung. Ieh lie~ sofort digital weiter komprimieren, Chloroformnarkose einleiten, in fiiegender Hast desinfizierte ich reich und unterband mit notdiirftig desinfizierten ]nstrumenten im gesunden Gewebe die Carotis communis, ca. 3 cm unterhalb der Jugularisunterbindungs- und Carotis interna- Arrosionswunde, mit doppelter Seidentigatur. Zuvor mutate auch die Jugutaris externa, Vena thyreoidea media unterbunden and eine gro]~e die Carotis communis bedeckende, infiltrierte Driise exstirpiert werden. Die arterielte Btutung stand l)agegen bluteten im oberen Winkel der Jugulariswunde, deren hochgradige nekrotische Beschaffenheit man beim Auseinanderziehen der Wundrander erst sah, noch sehr stark zwei Venen. die in dem morschen Gewebe sich mit dem P~an nicht fassen lieflen, nach Umstechung aber ebenfalls standen. Alle Unterbindungsfaden wurden durch besondere Knotung markiert. Gleicbzeitig wurde darnaeb noch eine weitere breite Gegen6ffnung in der Glutaealgegend vorgenommen, die in der Tiefe noch eine weite Tasche des Abszesses er6ffnete. Von einer Unterbindung der Carotis interna oberhalb dcr Arrosionsstelle, die wegen einer eventuellen ~Naehblutung nach Einschaltung des Collateralkreislaufs wohl recht empfehlenswert gewesen ware, mu~te ieh absehen, da eine genaue 0rientierung in dem nekrotischen Gewebe ganz anm6glich war. Trotzdem der Pals w~brend der - Uber spontane Arrosion und Ruptur der Carotis interna. 203 Narkose und Operation relativ gut blieb, wurden dora Patienten 9 Spritzen Campher appliziert. Von einer Kochsalzinfusion sah ich ab. Sogleich nach dem Erwachen aus der Narkose bekam der Patient Kaffee, Bouillon und Gelatinewasser, sparer Fleischsaft mit der K l e i n schen Fleischpresse gewonnen. Kurze Zeit nach der Unterbindung fiel besonders der Farbenunterschied beider Gesichtsh/~lften auf. Die rechte Wange bedeckte brennende ROse, die linke tiefe B1hsse. Am Mittag fiel die wachsbleiche Farbe des Gesichtes aut; das linke Auge erschien eingesunken; die Stimme war nachmittags etwas heiser, wurde aber am Abend wieder klarer. Der Puls war sehr frequent, aber krh(tig (140). Die Temperatur betrug abends 39, L/~hmungser~cheinungen waren nicht eingetreten. Am n/~chsten Tage wurde der Verband gewechselt und man konnte in der Tiefe der Jugularisunterbindungswunde die ca. 1/2 linsengrol]e Rupturstelle der Carotis interna sehen, deren Rhnder gezackt und dunkel verfarbt waren und in deren Lumen Blutgerinnsel iagen. Auf Befragen erklfirte der Patient, keine Kopfschmerzen, kein Ohrensausen und keinen Schwindel zu haben. Zwar gab er an, etwas ,;Klingen" in beiden Ohren zu hOren, auch klagte er t~ber starke Scbluckbeschwerden. Nystagmus bestaud nieht. T. M. 37,6. lm Laufe des ~Xbends und der l~acht deli~ierte der Kranke, schlug naeh den Schwestern, war iiberhaupt recht ungeberdig. Die Augenuntersuchung 4Dr. K u f f l e r - A u g e n k l i n i k } ergab normalen Befund. Die nhchsten drei Tage verliefen obne l~esonderheiten. Es waren keine LAhmungserscheinung aufgetreten, er klagte fiber keinerlei Beschwerden. Nur fiel die auBerordentlich leichte Erregbarkeit des Patienten auf, der unter auderm sich mehrmals den Hiiftverband abriB. Ffinf Tage nach der Carotisunterbindung wurden die s~mtlichen Unterbiadungsf~den gel0st, nur die Carotisligatur wurde noch liegen gelassen, diese aber ebenfalls nach weiteren t t~nf Tagen entfernt. Eine ffinf Tage nach der Carotisunterbindung vorgenommene HOrpriifung ergab: Flfistersprache links ca. 1 Meter, e 1 W normal, c~L, A, a t wenig bis mittelstark herabgesetzt, fis 4 bei leisem F. K. A. Patient klagte nicht fiber Ohrensausen, batte weder subjektiv noch objektiv Scbwindel, auch bestand kein Nystagmus. Die Rekonvaleszenz machte nun gute Fortschritte. Die Temperatur, die nach der Carotisunterbindnng am hbend auf 40,0 gestiegen, bewegte sich an den folgenden i anf Tagen zwischen 36,5 und 38,2, betrug dann nur einmal 13 Tage nach tier Carotisunterbindung 38,1, als sich an der AuBenseite des rechten Unterschenkels eine kleine Metastase in der Muskulatur lokalisiert hatte, die aber spontan zuriickging, und blieb dann bis zur Entlassung (43 Tage nach der Carotisunterbindung) normal blOrpr~ifungen 18, 20, 28 Tage p. Carotisunterbindung ergaben: Fliistersprache wurde auf 4 bzw. auf 5 und 6 Meter gehOrt, die Perzeption ffir c t (Luft und Knochen), a ~, A, fis4 war normal W e b e r ~vurde naeh links lateralisiert. Eine genauere Bestimmung mit der ,,kontinuierlichen Tonreihe '~ naeh B e z o l d ergab : U. G. : r normal C2, 1 E~, jetzt C~, O. O. : r und 1 normal. Die Halswunden sind gesehlossen, die retroanrikul/~re Wunde ist noch nieht vOllig verheilt. Das Befinden ist andauernd ausgezeichnet. Beschwerden hat Patient hie mehr gehabt, nur spricht Patient etwas heiser. Es besteht linksseitige Posticusl~hmung, die aber bereits im Riickgange begriffen ist. Ftir die s p o n t a n e A r r o s i o n u n d R u p t u r d e r C a r o t i s i n t e r n a k o m m e n t h e o r e t i s c h d r e i M S g l i c h k e i t e n in B e t r a c h t . l . B e i d e r J u g u l a r i s u n t e r b i n d u n g a m 5. S e p t e m b e r ist d i e A d v e n t i t i a d e r C a r o t i s i n t e r n a d i r e k t 1/~diert o d e r a b e r die s t a r k e n Seidenf~den haben bei den Schluekbewegungen an der Adventitia g e r i e b e n u n d so o b e r f l / i e h l i e h e L ~ s i o n g e m a c h t . E s ist d a n n a n 14" 204 XII. F. NUERNBERG. der sp~teren Rupturstelle zu einer Oberfi~chliehen Entziindung gekommen, die yon aul~en naeh innen gewandert ist. 2. Es w~re denkbar, dug eine Ansehwemmung infektiSsen Materials an die Intima der Carotis interna und so eine Wanderuug der Entziindung yon innen naeh aufien erfolgt witre. 3. KSnnte dureh Kontakt mit dem infizierten Gewebe, insbesondere den infizierten Lymphdrtisen eine direkte Infizierung der Arterienwand erfolgt sein. Der entztindliche Prozcl~ hat die Arterienwand ergriffen and ist yon aul~en nach innen gewandert. Wir hKtten kS dann mit einer akuten.Arteriitis zu tun gehabt. Untersuehen wir nun die drei MSgliehkeiten. ad 1. Eine direkte Li~sion der Carotis interna bei der Jugularisunterbindung halte ich ftir ausgeschlossen. Wir sind j a allerdings nicht so in der gltieklichen Lage wie die Chirurgen, die meist aus prophylaktisehen Gr~inden, wie z. B. bei Oberkieferresektionen, die Carotis unterbinden und so im g'esunden Gewebe arbeiten. Unser Arbeitsfeld ist zum Teil bedeekt mit Drtisen, die dutch ihre VergrSl~erung und hyt)er~tmisehe Besebaffenheit sebon anzeigen, dais sie dm'eh die Sinus- bzw. Jugulariserkrankung mit infiziert sind. Wit mtissen oft unter grolSer Mtihe diese Drtisen, die gerade tiber und an der Abgangsstetle der V. fae. communis und V. j~g'ul, interna liegen~ mit exstirpieren, und dabei ist es meist der Fall~ dag das Gewebe und wohl aueh die Gefiil~seheide mehr als ihm dienlieh ist~ gequetseht wird. Dabei kommen wir ja abet nieht mit der Carotis in Konflikt. Ihre Lag'e ist doeh zu gesehiitzt und eher dtirfte es der Fall sein~ dab die dieht unter den Drilsen liegende V. jugularis u. ¥. faeiei eommunis, welehe 5fters pathologisehe Veri~nderungen ihrer Wandungen aufweisen, litdiert wlirden. Das passiert bei vorsiehtigem Arbeiten aber aueh nut ~tul~erst selten. Aueh bei der Unterbindung der Jugularis selbs~ dfirfte eine Liision der Carotis wohl ausgeseblossen sein. Unsere Unterbi~dungsnadelu habeu eine platt% stumpfe, abgerundete Spitze~ die ohne besonderen Druek, ohne Gewalt unter der frei pr~parierten Jugutaris durehgefiihrt werden. Die Carotis weieht bei dem sanften Druek, soweit man tiberhaupt yon Druek spreehen kaml, immer aus. Eher w~re kS sehon mSglieh, dal~ die dicken Seidenf/tden der Jugularisunterbindung bei den Sehluekbewegungen des Pa- Uber spontane Arrosion und Ruptur tier Carotis interna. 205 tienten an der Carotis des Patienten gerieben und oberfl~ehlishe Epitheldefekte gesehaffen h~ttten, an denen dann die Bakterien eins Angriffsfl~shs gshabt hatten. Aber aush diess Auffassung dtirfte mehr theoretiseh sein und entsprisht nisht den Erfahrungen, die man allgemein in der Chirurgie macht. Wit haben aufierdem ja oft woehenlang unsere Unterbindungsfaden an der Jugularis liegen~ ohne daft sic als FremdkSrper mechaniseh auf die Carotis und den Vagus wirken bzw. gewirkt h atten. ad. 2. Eine sichere Sttitze far dis Auffassung, daft eine Ansehwemmung infektiSsen Materiales an die lntima der Carotis interna und so elne Wanderung tier Entztindung yon innen nach aul~en erfolgt w~re, haben wit nieht. Vielleieht kSnnts man die Beobachtung daftir verwenden, dab der Patient 9 Tags vor der Ruptur iiber ,ab und zu auftretende ~ Stirnkopfsehmerzen geklag't hat und vermchrte motorisehe Unruhe zeigte. Es w~re mSg'lish, daft kleine Thrombenteilehen yon der Intima der Carotis in das IIirn verschleppt w~iren und im Gebiete der gleichseitigen A. tbssae Sytvii oder aueh in andern Hirnteilen kleine embolisehe Herde gebildet h~tten, die aber wegen ihrer Kleinheit sehwere Ver~inderungen, wie h~morrhagisehe gelbe und rote Erweichung nisht gezeigt haben. Dagegen w~re einzuwenden, dal~ diess Stirnkopfschmerzen und die motorische Unruhs ebensogut dutch eine serSse Meningitis oder zumindest dureh meningeale Reizerseheinungen h~tten ausgelSst werden kSnnen. Zudem kSnnen sie aueh wohl gelegentlieh hei Sinusthr0mbosen selbst durch den behinderten Blutabflu$ aus der Sch~tdelhShle ausgeISst werden, wenngleieh allerdings die Kopfsehmerzen in der Regel wohl halbseitig zu sein pflegen. ad 3. Eine direkte Infizierung der Arterienwand dutch Kontakt mit dem infizierten Gewebs der Jugulariswunde im allgemeinen und den infizierten Lymphdrtissn im besonderen ssheint mir yore pathologisch-anatomischen und klinischen Standpunkte aus am gegebensten zu sein. ~aeh der Ruptur der Carotis interna sah man erst, welch' hoehgradige nekrotisshe Beschaffenheit die Jugulariswunde in welter Ausdehnung hatte. Es war gewissermafien eine Unterminierung tier ganzen Parfie his zum Kisferwinkel erfolgt~ ohne dab man der Wunde yon angen die stattgehabte ZerstSruug in tier Tiefe ansah, eine Eigenart, wie sis septische Wunden so 206 XII. F. NUERNBERG. hitufig haben. Ob es nach der Infizierung und ZerstSrung der Adventitia und Media zu einer Aneurysmabildung gekommeu ist~ oder ob auch der nekrotische Proze{~ die Intima selbst ergriffen hatte mit dem beiderseitigen Endresultate der Ruptur, w~ire nur dadureh zu eutseheiden mSglieh gewesen, wenn an der Rupturstelle sieh reichlich Thrombenmassen hiitten naehweisen lassen. Das war nieht der Fall. Es ist aber wohl denkbar, dab bei der plStzliehen Ruptur und dem starken Blutdruek eventueli bestandene Thromben aus der Rupturstelle herausgesehleudert wurden, so dai~ die Frage often bleiben mul~, ob eine Aneurysmabildung stattgehabt hat oder nieht. Mag man nun diesen oder jenen Infektionsmodus annehmen, so ist es wei~erhinvielleichtniehCunwahrseheinlich, daft aueh die Serumbehandlung eine gewisse unterstiitzende Rolle bei der zerstSrenden Wirkun~ der Bakterien insofern gespielt hat, als dureh diese eine starke Phag'oeythose hervorg'erufen wurde, die im Verein mit der starken fermentativen Wirkung der Bakterien an der Jugulariswunde ebenso wie an der geschlossenen Glutiialpartie die Bakteriendepots zur Einsehmelzung braehte und das umgebende Gewebe destruktiv ver~nderte. Diese Auffasung diirfte wohl ftir die groi~e Ausdehnung der mctastatisehen Einschmelzun~ in der Glut~ialgegend zu reeht bestehen, denn sie erfolgte wenige Tage prompt naeh den Einspritzungen des Serums. Alsdann mifi~tejedoch eine besondere Irresistenz des Patienten ~egen das Serum angenommen werden, da nur 40 ccm injiziert wurden. Was nun die klinisehe Seite unserer Carotis communis-Ligatur anbelangt, so hat diese gltieklicherweise keine bemerkenswerten Folsen gezeitig L abgesehen yon der bereits im Riick~'ange begriffenen Postieusl~ihmun, die wohl auf Zerrung oder Quetsehung des N. reeurrens bezogen werden muff, trotzdem das jugendliehe Individuum dutch die sehwere Streptokokkeninfektion hoehgradig mitgenommen war, ein Umstand, der die Prognose auch bei jlingeren Individuen erheblieh trilbt, deren Gef~fisystem ja sonst den verfinderten ZirkulationsverhMtnissen leiehter Rechnung tr~igt, als dies bei ~ilteren Individuen der Fall ist. Wir h~itten Cerebrale Augen- und GehSr-StSrungen erwarren kSnnen0, und h~tten mit einer Naehblutung aus dem 1} L e b r am : a) Uber Arrosion der Carotis bei peritonsill~ren Abszessen. Z. f, O. 51. 1906. b) Uber StSrungen des GehSrorgans nach Unterbindung der Carotis. Z. f. O. 51. t905. Uber spontane Arrosion und Ruptur tier Carotis interna. 207 peripheren Ende naeh Wiederherstellung des Collateralkreislaufes reehnen miissen. Von Hirnsymptomen w~ren nur die Delirien und die PulserhShung zu verzeiehnen. Pathologische Augenver~nderungen fehlten und dem am ersten Tage naeh der Operation in beiden Ohren ab und zu aufgetretenem ,Klingen" ist eine Bedeutung wohl kaum beizumessen. Aueh die ~/aehblutung, die naeh Wiederherstellung des Collateralkreislaufes naeh der Carotisunterbindung aus dem peripheren Ende oft naeh Wochen noch auftreten kann, und bei unserem an und ftir sich reeht unrubigen Patienten bis zum Sehlufl der Halswunden zu beftirehten war, ist glticklieherweise nicht erfolgt. Es war ia unserem Falle ganz uu~nSglieh, das periphere Ende zu ligieren, d a das Gewebe an und tiber der Arrosionsstelle der Carotis interna itul~erst morseh war und ein Arbeiten darin nur mehr Sehaden als Nutzen gebraeht hatte. Die klinisehe Ausbeute im positiven Sinne war also gering, dagegen bewies die fast negative, daft in der Tat junge Indi. viduen mit intaktem, nieht arteriosklerotiseh veriindertem Geflifisystem, selbst so eingreifende Operationen, wie Carotis eommunisLigaturen, anstandslos vertragen.
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https://europepmc.org/articles/pmc5568304?pdf=render
English
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A large left atrial lipoma combined with coronary artery disease
Journal of cardiothoracic surgery
2,017
cc-by
1,690
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background: Primary benign tumors of the heart are extremely rare and usually difficult to diagnose for their asymptomatic signs Background: Primary benign tumors of the heart are extremely rare and usually difficult to diagnose for their asymptomatic signs. Case presentation: A 66-year-old woman was admitted for shortness of breath caused by a large left atrial lipoma combined with coronary artery disease. Next, we successfully performed simultaneous curative surgery for the large cardiac lipoma and coronary artery bypass grafting with a “starfish” and no cardiopulmonary bypass was used.The patient was discharged on the eighth postoperative day in a good condition, and has remained asymptomatic at the 5-month follow-up. Conclusions: Lipomas are rare and difficult to diagnose, while computed tomography and computed tomography angiography can give us very important clues. Surgery is necessary. We can introduce a “starfish”to the operationand the cardiopulmonary bypass is unnecessary for the left lipoma with coronary artery disease. Keywords: Lipoma, Cardiac benign tumor, Coronary artery bypass highly suspected that it was a lipoma on the basis of its CT manifestations. The ejection fraction was 62%. The electro- cardiogram was normal. Considering of her age, the coron- ary angiography was performed and the result revealed a 70% stenosis in the left anterior descending (LAD) branch. And we decided to perform simultaneous curative surgery for the cardiac lipoma and coronary artery bypass grafting (CABG). Background Cardiac lipomas are extremely rare, accounting for 8.4% of all primary tumors [1]and found at a frequency of only 0.001%–0.28% at autopsy [2]. They are frequently located in the left ventricle or right atrium. Left artrial lipomas are very rare with only 3 references in all of the epicardiac lipomas [3]. Clinically, this tumor is asymp- tomatic and found incidentally in the vast majority of cases [4]. Only large left atrial lipomas could alter atrial and ventricular functions and result in dyspnea, such as in our case. A median sternotomy and pericardial incision were performed undergeneral anesthesia. After we lifted up the heart with a “starfish”, a soft, yellow tumor which originated from the left atrium without any invasion to the pericardium was found (Fig. 2). Then we excised the basal part of the tumor carefully with an electrome and no cardiopulmonary bypass was used. The mass was re- moved completely and measured about 8 cm × 8 cm × 4 cm (Fig. 3). Next, we performed the anastomosis between the anterior descending branch and the left internal mammary artery. The procedure was smooth, and post- operative recovery was good. Liu et al. Journal of Cardiothoracic Surgery (2017) 12:71 DOI 10.1186/s13019-017-0633-1 Liu et al. Journal of Cardiothoracic Surgery (2017) 12:71 DOI 10.1186/s13019-017-0633-1 A large left atrial lipoma combined with coronary artery disease Yun Liu, Xiaomei Zheng, Yu Du, Zhicheng Zhu, Tiance Wang, Rihao Xu, Dan Li and Kexiang Liu* n Liu, Xiaomei Zheng, Yu Du, Zhicheng Zhu, Tiance Wang, Rihao Xu, Dan Li and Kexiang Liu* * Correspondence: kxliu64@hotmail.com The Second Affiliated Hospital of Jilin University, Changchun, China a good condition, and has remained asymptomatic at t * Correspondence: kxliu64@hotmail.com The Second Affiliated Hospital of Jilin University, Changchun, China © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Case presentation A 66-year-old female was admitted because of dyspnea. On admission, the temperature was normal, the pulse was 67 /min, the respiration rate was 20/min and the blood pres- sure was 108/68 mmHg. Laboratory examinations were nor- mal. Computed tomography (CT) showed that a large low density mass located behind the posterior wall of the left atrium. The left atrium was compressed. The mass showed a density similar to adipose tissue and was not enhanced on computed tomography angiography(CTA) (Fig. 1). We The histopathological examination revealed mature adi- pocytes confirming our suspicion of a limopa (Fig. 4). The patient was discharged on the eighth postoperative day in a good condition, and has remained asymptomatic at the * Correspondence: kxliu64@hotmail.com The Second Affiliated Hospital of Jilin University, Changchun, China Page 2 of 3 Liu et al. Journal of Cardiothoracic Surgery (2017) 12:71 Fig. 3 The mass was attached to the posterior pericardium beneath the left pulmonary artery with a stalk connected to the left atrium Fig. 1 CTA showed that a large low density mass located on the posterior wall of the left atrium. The left atrium was compressed. The tumor was not enhanced on CTA (1. Tumor,2. Left atrium) Fig. 1 CTA showed that a large low density mass located on the posterior wall of the left atrium. The left atrium was compressed. The tumor was not enhanced on CTA (1. Tumor,2. Left atrium) 5-month follow-up. Echocardiography (ECHO) detected no signs of recurrence. Discussion It’s usually difficult to diagnosis left arterial lipomas for its asymptomatic and low incidence. However, CT/CTA can give us very important clues to the diagnosis. When the CT showed a fat-like low density mass under the epi- cardium and was not enhanced on CTA, we should Fig. 3 The mass was attached to the posterior pericardium beneath the left pulmonary artery with a stalk connected to the left atrium consider it as a lipoma first. Cardiac lipomas are benign and slowly growing, but for large lipomas, surgical resec- tion is necessary to prevent tumor compression syndromes. Fig. 2 Intraoperative view of the tumor. A large lipoma was seen at thoracotomy with a starfish.(1.lipoma; 2.starfish) Fig. 2 Intraoperative view of the tumor. A large lipoma was seen at thoracotomy with a starfish.(1.lipoma; 2.starfish) As we know, in all of the three cases, cardiac surgeons in one case performed the resection of the tumor and repaired left atrium(LA) surface with an autologous peri- cardial patch under cardiopulmonary bypass. In the other two cases, cardiac surgeons removed the tumor Fig. 4 The histopathological examination revealed mature adipocytes Fig. 4 The histopathological examination revealed mature adipocytes Fig. 4 The histopathological examination revealed mature adipocytes Fig. 2 Intraoperative view of the tumor. A large lipoma was seen at thoracotomy with a starfish.(1.lipoma; 2.starfish) Liu et al. Journal of Cardiothoracic Surgery (2017) 12:71 Page 3 of 3 directly without cardiopulmonary bypass. In this case, we used a “starfish” to lift up the heart, which made the lipoma exposed well and removed easily. Also we under- went the off-pump coronary aortic bypass grafting (OPCABG). The result was good. So we consider that it’s unnecessary to perform the surgery under cardiopul- monary bypass for the left atrial lipoma pateints with coronary artery diseases. Abbreviations CABG: Coronary artery bypass grafting; CPB: Cardiopulmonary bypass; CT: Computed tomography; CTA: Computed tomography angiography; ECHO: Echocardiography; LA: Left atrium; LAD: Left anterior descending; OPCABG: Off-pump coronary aortic bypass grafting Funding Funding No funding sources No funding sources Availability of data and materials Data sharing not applicable to this article as no datasets were generated or analysed during the current study Conclusion Lipomas are rare and difficult to diagnose, while com- puted tomography and computed tomography angiog- raphy can give us very important clues. Surgery is necessary. We can introduce a “starfish” to the operation and the cardiopulmonary bypass is unnecessary for the left lipoma with coronary artery disease. Received: 24 March 2017 Accepted: 2 August 2017 • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Ethics approval and consent to participate Not applicable Ethics approval and consent to participate Not applicable Ethics approval and consent to participate Not applicable Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: Received: 24 March 2017 Accepted: 2 August 2017 Received: 24 March 2017 Accepted: 2 August 2017 Consent for publication Not applicable Competing interests Authors’ contributions KXL designed the study, performed the operation, and revised the manuscript critically. LY collected the date and wrote the article. XMZ and DY referred to the related literature. All authors read and approved the final manuscript. References 1. Khoueiry G. Nidal a, BiRafeh.Left atrial appendage lipoma: an unusual location of cardiac lipomas. Echocardiography. 2011;28:E91–3. location of cardiac lipomas. Echocardiography. 2011;28:E91–3. 2. Wilson S, Frederick J, Braunwald E. Primary tumors of the heart. In: Braunwald E, editor. Heart disease. A textbook of cardiovascular medicine. 5th ed. Philadelphia: Saunders; 1997. p. 1464–77. 3. Hayashi H, Hidaka F. A left ventricular lipoma diagnosed on three- dimensional electrocardiogramgated cardiac computed tomography. Heart Vessel. 2008;23:366–9. 4. Georges K, Nidal AR. A case of massive left atrial lipoma occupying pericardial space. Jpn Heart J. 2004;45:715–21.
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A Method to Find Longevity-Selected Positions in the Mammalian Proteome
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Introduction Despite a 10–100-fold difference in maximum lifespan (MLS), most known mammal species show similar phenotypes of aging [1]. This observation suggests that the genetic determinants of mammalian aging and lifespan may be relatively plastic. The classical evolutionary theory of antagonistic pleiotropy [2] posits that aging is an effect of the decrease in selection pressure that occurs after successful reproduction. Conversely, lifespan exten- sion has been shown to occur when selection pressure increases in later age [3]. Still relatively unexplored are the specific molecular mechanisms that determine differences in mammalian lifespan. Many mechanisms are possible and likely occur simultaneously, including changes in the sequence, structure, function, and expression of RNA and proteins. In contrast, we start from the perspective of protein structure, which emphasizes two concepts that may be useful to find interesting longevity-selected positions. First, positions within a protein are not interchangeable; whatever the estimated synon- ymous substitution rate, a single nonsynonymous substitution in a certain structural context can change a protein’s function. Second, not all nonsynonymous substitutions are equal; rather, a priori we should expect those amino acid substitutions that commonly change biochemical function to matter most for the function of a specific protein. Based on these observations, we present a simple regression-based method to find longevity- selected positions in orthologous protein families of mammals. Our method employs the phylogenetic generalized least squares framework (PGLS, equivalent to phylogenetic independent contrasts; [7]) to relate, for each position (column) of a protein alignment, the MLS of the species represented in the column to the biochemical divergence of their residues from the residue of a long-lived reference species. Two benefits of PGLS are that (1) it is straightforward to control for common gerontological con- founders, including species-specific mutation rate, body mass, and shared phylogeny [8], and (2) we can naturally fit a continuous variable such as MLS, removing the need to arbitrarily bin species. Here, we focus on changes in proteins caused by fixed substitutions. In this context, two recent studies [4,5] predicted a simple consequence of the evolutionary theory: we might expect that proteins necessary for long mammalian lifespan would have fewer substitutions, i.e. show more conservation, in long-lived versus short-lived species. Abstract Evolutionary theory suggests that the force of natural selection decreases with age. To explore the extent to which this prediction directly affects protein structure and function, we used multiple regression to find longevity-selected positions, defined as the columns of a sequence alignment conserved in long-lived but not short-lived mammal species. We analyzed 7,590 orthologous protein families in 33 mammalian species, accounting for body mass, phylogeny, and species-specific mutation rate. Overall, we found that the number of longevity-selected positions in the mammalian proteome is much higher than would be expected by chance. Further, these positions are enriched in domains of several proteins that interact with one another in inflammation and other aging-related processes, as well as in organismal development. We present as an example the kinase domain of anti-Mu¨llerian hormone type-2 receptor (AMHR2). AMHR2 inhibits ovarian follicle recruitment and growth, and a homology model of the kinase domain shows that its longevity-selected positions cluster near a SNP associated with delayed human menopause. Distinct from its canonical role in development, this region of AMHR2 may function to regulate the protein’s activity in a lifespan-specific manner. Citation: Semeiks J, Grishin NV (2012) A Method to Find Longevity-Selected Positions in the Mammalian Proteome. PLoS ONE 7(6): e38595. doi:10.1371/ journal.pone.0038595 shin NV (2012) A Method to Find Longevity-Selected Positions in the Mammalian Proteome. PLoS ONE 7(6): e38595. doi:10.1371/ Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received March 9, 2012; Accepted May 10, 2012; Published June 11, 2012 Received March 9, 2012; Accepted May 10, 2012; Published June 11, 2012 Copyright:  2012 Semeiks, Grishin. 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 work was supported in part by the National Institutes of Health (GM094575 to NVG) and the Welch Foundation (I-1505 to NVG). 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. Jeremy Semeiks1*, Nick V. Grishin2 1 Molecular Biophysics Program and Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 2 Department of Biochemistry and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America 1 Molecular Biophysics Program and Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dall 2 Department of Biochemistry and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Tex Abstract * E-mail: jeremy.semeiks@utsw.edu (1) species were binned as ‘‘long-lived’’ or ‘‘short-lived’’ based on MLS and (2) in keeping with the conventional framework, the total estimate of synonymous substitutions in a gene determined the threshold for whether any particular codon position in that gene was called longevity-selected. 2.1. Selection of Positions to Analyze y We analyzed a selected subset of the OrthoMaM database version 6, which comprises multiple sequence alignments (MSAs) of 11,746 protein ortholog families from 36 mammalian species [12]. Most of the sequences in the database were extracted from low-coverage genomes, so completeness and quality of alignments varied considerably. We first masked nonstandard isoforms and other divergent subsequences using a sliding window-based approach. Specifically, we excluded from further analysis any subsequence of at least 10 residues in which every 10-residue window had at least four residues each with less than 30% sequence identity to the rest of its column. We also excluded the three non-eutherian species due to high sequence divergence. Of the remaining data, we selected for fitting only columns that (1) included at least ten characters overall and (2) specifically included characters for both human and shrew. We refer to this subset as selected columns. To construct the randomized control dataset, for each species A except human, we swapped MLS, body mass, and phylogenetic label with those of one species B, which was randomly selected without replacement. All alignments remained unchanged, mean- ing that the same set of columns were fit in both the randomized control and real sets. Results and Discussion 3.1. Due to Overall Conservation, Most Positions in the Mammalian Proteome are Not Longevity-selected To identify specific positions (i.e., alignment columns) in the mammalian proteome that are conserved in long-lived but not in short-lived species, we fit a generalized multiple regression model to each position independently. Our overall approach followed from the observation that many of the positions we sought were distinguished by high correlation between (1) each species’ MLS and (2) functional similarity of each species’ residue to that of a long-lived reference species. We used human as the reference species, both because it was the longest-lived mammal whose sequence was available and because we had the best confidence in the accuracy of its sequence. Figure 1 shows a simplified conceptual example of our approach. Our method also accounted for each species’s body mass and overall mutation rate relative to human. We emphasize that we chose multiple regression not for rigorous statistical reasons, but only as a computational tool to help form new biological hypotheses. Longevity-Selected Protein Regions in Mammals Longevity-Selected Protein Regions in Mammals We use our method as a starting point to analyze longevity- selected positions, placing emphasis on their structural contexts. Our results concern both the proteome as a whole and a specific protein domain identified by our analysis, the kinase domain of anti-Mu¨llerian hormone type-2 receptor (AMHR2; kinase no- menclature per [9]). AMHR2 is a receptor protein serine/ threonine kinase in the TGF-beta Receptor Type II (TGFBR2) subfamily. The canonical role of AMHR2 is to inhibit the Mu¨llerian ducts during development of the male fetus, and mutations cause the rare disease persistent Mu¨llerian duct syndrome (PMDS; [10]). More recently, a role for AMHR2 in ovarian follicle development of the adult female has also been identified [11], and this noncanonical function may be relevant to our findings. expected number of amino acid substitutions over all fit columns. We used this tree to correct each BLOSUM80 score for the mutation rate of its respective species s relative to human by adding log10(ms/mref) to the score, where ms was the total tree length of species s and mref was the tree length of human. We restricted the range of each B80mut score to the standard range of B80 scores attainable by its human character. Each fit yielded both a longevity-selected slope, bMLS, and p- value, pMLS. We defined as a longevity-selected position any column with both bMLS.0 and pMLS,0.01. Conserved columns were assigned bMLS = 0 and pMLS = 1. For the large-scale analyses, in the human orthologs we predicted secondary structure with PSIPRED [18]; differences in composition were tested with Pearson’s x2 test. Protein domain definitions and other features were taken from Swiss-Prot [19] and mapped to OrthoMaM alignments by aligning each Swiss-Prot sequence to its human counterpart in OrthoMaM. Ontology enrichment analysis was performed using the Functional Annota- tion Clustering module of DAVID [20] with default parameters, including the human genome as background. We report only Benjamini-corrected p-values. For the rolling-window analysis of positions, we included only contiguous blocks of 10 selected positions. 2.2. Column Correction, Fitting, and Analysis We define fit columns as the subset of selected columns that have at least three characters different from the human reference character, and conserved columns as all other selected columns. We independently fit each column in the fit subset to a phylogenetic generalized linear model [7], as implemented in the R package caper, version 0.4 [13]. Briefly, this framework assumes a Brownian model of trait evolution and uses the method of generalized least squares to perform multiple regression with correction for global phylogenetic dependence, as indicated by the mammalian supertree. Specifically, for each column we fit the regression model. 2.3. Homology Modeling and Structural Analysis of AMHR2 We created a homology model of AMHR2 with Modeler [21], using as template the kinase domain of BMPR2 (PDB ID: 3G2F). Homology models made with SWISS-MODEL [22], or using as template the kinase domain of ACVR2B (PDB ID: 2QLU), yielded similar results. Positional conservation was calculated with AL2CO [23], using 3G2F as the structural model. PLoS ONE | www.plosone.org Introduction Thus, it may be possible to identify aging-related proteins (more specifically, families of orthologous proteins) by inferring and comparing some measure of such preferential substitutions, here called ‘‘longevity-selected posi- tions’’, among the several dozen mammal species whose proteomes are available. In particular, Jobson et al. [4] accom- plished this from the perspective of classical genetics, applying to a codon model a measure similar to the well-known dN/dS ratio (reviewed in [6]). Two relevant features of their method were that PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e38595 1 June 2012 | Volume 7 | Issue 6 | e38595 Longevity-Selected Protein Regions in Mammals doi:10.1371/journal.pone.0038595.g001 Rattus norvegicus − rat − 5 Mus musculus − mouse − 4 Dipodomys ordii − kangaroo rat − 10 Cavia porcellus − guinea pig − 12 Spermophilus tridecemlineatus − squirrel − 8 Ochotona princeps − pika − 7 Oryctolagus cuniculus − rabbit − 9 Gorilla gorilla − gorilla − 55 Homo sapiens − human − 90 Pan troglodytes − chimpanzee − 59 Pongo pygmaeus − orangutan − 59 Macaca mulatta − rhesus − 40 Callithrix jacchus − marmoset − 17 Tarsius syrichta − tarsier − 16 Otolemur garnettii − galago − 18 Microcebus murinus − mouse lemur − 18 Tupaia belangeri − treeshrew − 11 Bos taurus − cow − 20 Tursiops truncatus − dolphin − 52 Sus scrofa − pig − 27 Vicugna vicugna − vicuna − 32 Equus caballus − horse − 57 Canis lupus − dog − 24 Felis silvestris − cat − 30 Myotis lucifugus − little brown bat − 34 Pteropus vampyrus − flying fox − 21 Erinaceus europaeus − hedgehog − 12 Sorex araneus − shrew − 3 Dasypus novemcinctus − armadillo − 22 Choloepus hoffmanni − sloth − 37 Procavia capensis − hyrax − 15 Loxodonta africana − elephant − 65 Echinops telfairi − tenrec − MLS 19 y Figure 2. Phylogeny of species used in this study. Shown next to each species’ node are its binomial, common name, and MLS. Longevity-Selected Protein Regions in Mammals Number of positions and alignments selected, fit, and conserved, as defined in Sections 2.1 and 2.2. subset columns (6106) alignments include human 7.01 12,746a –selected 3.64 7,708a ––fit 0.73 7,590a ––conserved 2.91 118b aNumber of alignments that include at least one position in the indicated subset. bNumber of alignments that include only conserved positions. doi:10.1371/journal.pone.0038595.t001 subset columns (6106) alignments include human 7.01 12,746a –selected 3.64 7,708a ––fit 0.73 7,590a ––conserved 2.91 118b Longevity-Selected Protein Regions in Mammals doi:10.1371/journal.pone.0038595.g002 Rattus norvegicus − rat − 5 Mus musculus − mouse − 4 Dipodomys ordii − kangaroo rat − 10 Cavia porcellus − guinea pig − 12 Spermophilus tridecemlineatus − squirrel − 8 Ochotona princeps − pika − 7 Oryctolagus cuniculus − rabbit − 9 Gorilla gorilla − gorilla − 55 Homo sapiens − human − 90 Pan troglodytes − chimpanzee − 59 Pongo pygmaeus − orangutan − 59 Macaca mulatta − rhesus − 40 Callithrix jacchus − marmoset − 17 Tarsius syrichta − tarsier − 16 Otolemur garnettii − galago − 18 Microcebus murinus − mouse lemur − 18 Tupaia belangeri − treeshrew − 11 Bos taurus − cow − 20 Tursiops truncatus − dolphin − 52 Sus scrofa − pig − 27 Vicugna vicugna − vicuna − 32 Equus caballus − horse − 57 Canis lupus − dog − 24 Felis silvestris − cat − 30 Myotis lucifugus − little brown bat − 34 Pteropus vampyrus − flying fox − 21 Erinaceus europaeus − hedgehog − 12 Sorex araneus − shrew − 3 Dasypus novemcinctus − armadillo − 22 Choloepus hoffmanni − sloth − 37 Procavia capensis − hyrax − 15 Loxodonta africana − elephant − 65 Echinops telfairi − tenrec − MLS 19 y Figure 2. Phylogeny of species used in this study. Shown next to each species’ node are its binomial, common name, and MLS. doi:10.1371/journal.pone.0038595.g002 Figure 1. Conceptual example of our multiple regression method applied to a single column (Y465 of AMHR2; full result in Table S2). (left) Characters shown ordered by species MLS. For each non-human species, we calculate the similarity score (‘‘BLOSUM80’’) for the species’ amino acid character versus the human character (here Y); e.g., this score for Tursiops would be the similarity score for H versus Y, which is 2. (right) We then fit the MLS of all non- human species to their similarity scores; e.g., Tursiops’ contribution to this fit is the point (52, 2). Not shown are the steps to correct for mutation rate and shared phylogeny, and the simultaneous fit of body mass. For this column, the data provide relatively strong support for a nonzero slope in the fit of similarity to MLS, even given trends in mutation rate, phylogeny, and body mass, and so this position is assigned a relatively significant p-value (pMLS,0.01). doi:10.1371/journal.pone.0038595.g001 Figure 2. Phylogeny of species used in this study. Longevity-Selected Protein Regions in Mammals Shown next to each species’ node are its binomial, common name, and MLS. doi:10.1371/journal.pone.0038595.g002 selected and mass-selected, suggesting that pMLS,0.01 was a reasonable cutoff in general for our analyses. Among positions with positive MLS slope, we analyzed the distribution of p-values in order to determine whether proteome- wide trends existed with regard to longevity-selected positions (Figure 3). As a negative control, we also analyzed a matched set of positions whose MLS, body mass, and phylogenetic position had been randomly swapped (‘‘randomized control’’). If there were no overall relationship between MLS and amino acid conservation, then we would expect significant p-values to be no more common than nonsignificant p-values after accounting for shared phylogeny and body mass. This is indeed the case for the randomized control (Figure 3B). However, for the real data (Figure 3A), positions with more significant p-values are clearly overrepresented relative to those with less significant p-values. These results indicate that overall, longevity-selected positions in the mammalian proteome are much more likely than would be expected by chance. questionable quality, including highly divergent sequence at exon-intron boundaries and alternate isoforms. For this reason, we implemented several heuristic filters for selection. In particular, because rodents are over-represented among the short-lived species of OrthoMaM, we selected only columns containing a character for shrew (Sorex araneus), the shortest-lived non-rodent. (Sections 2.1 and 2.2 give precise definitions of the sets selected, fit, and conserved.) Using these criteria, we verified that most selected positions in the mammalian proteome (80%) are conserved across all species (Table 1; also found previously [4,5]). In particular, at least 10 of 261 genes in the GenAge database of aging-related genes [24] have protein products that show near- complete conservation in mammals ($90% ratio of conserved positions to total human ortholog length), for example beta- catenin (CTNNB1), valosin-containing protein (VCP), fibroblast growth factor receptor 1 (FGFR1), and lamin A (LMNA). Thus, if these genes contribute to differences in mammalian longevity, it is likely via some mechanism other than structural differences in their protein products. This finding is robust to several perturbations of the data (Figure S1), including use of chimp as the reference instead of human Table 1. Number of positions and alignments selected, fit, and conserved, as defined in Sections 2.1 and 2.2. Table 1. Number of positions and alignments selected, fit, and conserved, as defined in Sections 2.1 and 2.2. Table 1. Here, MLS are the maximum lifespans and mass the body masses of each species as reported in AnAge version 11 [14]. B80mut are the BLOSUM80 scores for each nonhuman ortholog character in the column versus the human ortholog character [15], corrected for mutation rate as described below. We used caper’s default parameter values, including fixed nonterminal branch length multiplier (l) of 1 for phylogenetic correction. As the input phylogeny, we used the mammalian supertree [16], which is ultrametric. Fitting completed after running for one day on a standard single processor (2.2 GHz, 16 GB RAM). In this manner, we fit selected columns among the proteomes of 33 species (Figure 2), with MLS ranging from 3 y (shrew) to 90 y (human). Initially, we attempted to fit the entire unfiltered OrthomMaM database. However, this effort yielded many obvious false-positives driven by low sample size and data of To control for each species’ overall mutation rate, we used an approach similar to that of Li and de Magalha˜es [5]. Specifically, to estimate these mutation rates we constructed by maximum likelihood (protml, [17]) a tree whose branch length was the June 2012 | Volume 7 | Issue 6 | e38595 June 2012 | Volume 7 | Issue 6 | e38595 2 Longevity-Selected Protein Regions in Mammals Longevity-Selected Protein Regions in Mammals Figure 1. Conceptual example of our multiple regression method applied to a single column (Y465 of AMHR2; full result in Table S2). (left) Characters shown ordered by species MLS. For each non-human species, we calculate the similarity score (‘‘BLOSUM80’’) for the species’ amino acid character versus the human character (here Y); e.g., this score for Tursiops would be the similarity score for H versus Y, which is 2. (right) We then fit the MLS of all non- human species to their similarity scores; e.g., Tursiops’ contribution to this fit is the point (52, 2). Not shown are the steps to correct for mutation rate and shared phylogeny, and the simultaneous fit of body mass. For this column, the data provide relatively strong support for a nonzero slope in the fit of similarity to MLS, even given trends in mutation rate, phylogeny, and body mass, and so this position is assigned a relatively significant p-value (pMLS,0.01). doi:10.1371/journal.pone.0038595.g001 selected and mass-selected, suggesting that pMLS,0.01 was a reasonable cutoff in general for our analyses. Among positions with positive MLS slope, we analyzed the distribution of p-values in order to determine whether proteome- wide trends existed with regard to longevity-selected positions Figure 1. Conceptual example of our multiple regression method applied to a single column (Y465 of AMHR2; full result in Table S2). (left) Characters shown ordered by species MLS. For each non-human species, we calculate the similarity score (‘‘BLOSUM80’’) for the species’ amino acid character versus the human character (here Y); e.g., this score for Tursiops would be the similarity score for H versus Y, which is 2. (right) We then fit the MLS of all non- human species to their similarity scores; e.g., Tursiops’ contribution to this fit is the point (52, 2). Not shown are the steps to correct for mutation rate and shared phylogeny, and the simultaneous fit of body mass. For this column, the data provide relatively strong support for a nonzero slope in the fit of similarity to MLS, even given trends in mutation rate, phylogeny, and body mass, and so this position is assigned a relatively significant p-value (pMLS,0.01). 3.2. Among Nonconserved Positions, Longevity-selected Positions Occur More often than Expected by Chance proteome as a whole (x2(2, n = 7,702) = 265.23, p,2.2e-16). This is easily explained by the observation that sequence in random coils tends to be less conserved than other sequence; thus, fit positions will tend to be overrepresented in these regions. p p g The second trend is that, relative to randomized longevity- selected positions, real longevity-selected positions are slightly enriched in a-helices at the expense of b-strands (x2(2, n = 7,702) = 18.36, p = 1.0e-4). The significance of this finding is unknown, but it is possible that an abundance of a-helices imparts extra stability to proteins and protein complexes, e.g. via coiled- coil interactions [27]. 3.3. Longevity-selected Positions are Enriched in Protein Domains with Known Roles in Inflammation, Development, and other Diverse Functions A plausible biological hypothesis to explain this overall plethora of longevity-selected positions is that the evolution of longer mammalian lifespan requires particular concerted patterns of substitutions throughout the proteome that subtly affect protein properties such as binding affinity, folding, and stability. Consistent with this hypothesis is that relative to mouse (MLS 4 y), the proteome of naked mole rat (a rodent with MLS ,30 y) is more resistant to urea-induced unfolding [25], suggesting increased protein stability in the longer-lived rodent. An analogous process requiring concerted patterns of substitution may be the convergent evolution of hyperthermostability in archaea and bacteria [26]. The majority of longevity-selected positions are located in regions of proteins that are unannotated and presumably unstructured. Since these regions are generally of unknown function at present, it is difficult to interpret the biochemical significance of substitutions within them. Thus, to find longevity- selected positions with the best likelihood of causing well- characterized changes to protein structure and function, we next narrowed our focus to known protein domains. Specifically, we compiled a list of all 129 domains that contain at least two longevity-selected positions. The 129 domains are contained in 114 proteins. Table S1 summarizes the proteins and domains, and Table S2 shows data for each longevity-selected position in the domains, including number of characters (i.e., species) fit and all slopes and p-values. Five genes for the 114 proteins shown in these tables are present in the GenAge database [24]: serine-protein kinase ATM, ATM; serine/threonine protein kinase ATR, ATR; breast cancer type 1 susceptibility protein, BRCA1; ATP- dependent DNA helicase Q4, RECQL4; and DNA-dependent protein kinase catalytic subunit, PRKDC. Functional annotation clustering revealed that several of the 114 proteins belong to functional classes that have been associated with aging (Table 3). We give three examples. First, leukemia inhibitory factor receptor To determine overall trends in longevity-selected positions with regard to structural features of the proteome, we created and searched databases of both predicted secondary structure and predicted disordered regions for the human proteome. Table 2 shows the secondary structure composition of the human genome as a whole, as well as in the longevity-selected positions of both the real fit data and the randomized control data. This table shows two trends. The first trend is that, in both real and randomized longevity-selected positions, random coils are overrepresented, at the expense of a-helices and b-strands, relative to the human Table 2. 3.2. Among Nonconserved Positions, Longevity-selected Positions Occur More often than Expected by Chance For each selected position, our fitting procedure yielded both a longevity-associated slope (bMLS) and associated p-value (pMLS), as well as corresponding measures for body mass (bmass and pmass). Only those positions with significantly positive slope were called longevity-selected (bMLS.0 and pMLS,0.01) or mass-selected (bmass.0 and pmass,0.01). There were no positions that were both longevity- PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e38595 June 2012 | Volume 7 | Issue 6 | e38595 3 Longevity-Selected Protein Regions in Mammals Figure 3. Density histograms of pMLS values. A. Real data. B. Randomized control. Each histogram shows the n = 734,741 fit positions with bMLS.0. See Section 3.2 for details. doi:10.1371/journal.pone.0038595.g003 Figure 3. Density histograms of pMLS values. A. Real data. B. Randomized control. Each histogram shows the n = 734,741 fit positions with bMLS.0. See Section 3.2 for details. doi:10.1371/journal.pone.0038595.g003 (Figure S1A) and use of BLOSUM62 scores instead of BLO- SUM80 scores (Figures S1D and S1E). We note that although BLOSUM62 is more commonly used than BLOSUM80, in this case BLOSUM80 is more appropriate because mammalian proteomes typically have 80–90% sequence identity. The result also holds for input in which all nonhuman primates except one (here, rhesus) are omitted (Figure S1B), indicating that it is not an artifact of primate over-representation in OrthoMaM. Almost all (7,689/7,723) of the positions called significant in Figure 3A have pMLS,0.05 in this control set, suggesting that its relative lack of very significant pMLS values is simply due to fewer data available for each position (n = 26 versus 32 species available to fit). Finally, as we would expect the result does not hold when dog, a shorter- lived species, is used as the reference instead of human (Figure S1C). proteome as a whole (x2(2, n = 7,702) = 265.23, p,2.2e-16). This is easily explained by the observation that sequence in random coils tends to be less conserved than other sequence; thus, fit positions will tend to be overrepresented in these regions. The second trend is that, relative to randomized longevity- selected positions, real longevity-selected positions are slightly enriched in a-helices at the expense of b-strands (x2(2, n = 7,702) = 18.36, p = 1.0e-4). The significance of this finding is unknown, but it is possible that an abundance of a-helices imparts extra stability to proteins and protein complexes, e.g. via coiled- coil interactions [27]. Longevity-Selected Protein Regions in Mammals specifically the kinase C-lobe and downstream C-terminal tail of AMHR2 are under lifespan-related selective pressure. (LIFR) and interleukin-6 receptor subunit beta (IL6ST) dimerize to form the receptor for leukemia inhibitory factor (LIF; not present in our results), whose signaling is upregulated with age in association with thymic atrophy [28]. Second, arachidonate 15- lipoxygenase (ALOX15) is an enzyme that is upregulated in aging rat brain [29]; it functions in production of inflammatory leukotrienes, and may also function nonenzymatically to upregu- late NFkB [30]. Third, several of these proteins function in blood coagulation, markers of which increase with age and may interact with markers of inflammation [31]. Detailed structural analysis of these domains remains to be performed. In sequence, five of the eight longevity-selected positions in the kinase domain of AMHR2 concentrate in two regions near the C- terminus of the domain (Figure 4). To determine the likely locations of our longevity-selected positions on the structure of AMHR2, we mapped them to the recently-solved structure of the kinase domain of bone morphogenetic protein type 2 receptor (BMPR2). This domain is the closest homolog to the kinase domain of AMHR2, with 40% sequence identity. Our mapping (Figure 5) shows that these five longevity-selected positions cluster at or near a common surface of the AMHR2 kinase C-lobe. Specifically, they are all located on two loops near the bottom- front face of the domain: the aG–aH loop (Y465, T469, and F473) and the C-terminal loop following aI (E513 and H515). All five side-chains at least partially face solvent. Table 3 also indicates that several of our hits are involved in development, and this highlights a limitation of our data set. It is well-known that developmental schedule and longevity have co- evolved in mammals [1,32]; thus, these positions may specifically be conserved due to their effect on development, or they may pleiotropically affect both development and adult longevity. We did not attempt to correct for developmental schedule when fitting the data. In our framework, such correction is possible in concept by adding to the regression model a third predictor variable, species age at maturity. However, in the present set of species, we face the problem of multicollinearity: age at maturity is too well-correlated with MLS for correction to be a realistic goal (R2 = 0.48 on n = 30 species for age at female maturity after phylogenetic correction). Longevity-Selected Protein Regions in Mammals Additionally, no maturity data are available for three important species in our set: Pteropus, Tarsius, and Tupaia. Thus, it is not possible to distinguish in general whether the positions found by our method are conserved due to their roles in development, adult longevity, or both. But most of these proteins, including our next example of AMHR2, do have verified roles in the adult organism and may plausibly affect longevity. The side-chain of Y465 forms an intra-loop hydrogen bond with R462, a conserved residue. Thus, in conjunction with P464, the length of the Y465 side-chain constrains the angle of a second conserved arginine, R463, which forms hydrogen bonds to residues on aF and the aF-aG loop. Y465 is conserved in 8/9 species with MLS of at least 30 y, but in 9/19 shorter-lived species it is instead H, F, C, N, or D, none of which (except possibly H) can hydrogen-bond with R463 at the same angle as can Y. We predict that these substitutions would abolish at least the hydrogen bond with R462, destabilizing the aG–aH loop. Although the aG–aH and C-terminal loops are both quite divergent overall and contain several positions that did not meet our significance criterion for longevity conservation, Y465 is the only noncon- served residue within them whose side-chain is predicted to interact with another residue of AMHR2. T469 represents a position that may be differentially phosphor- ylated, but it also highlights some current limitations of our method and data set. T469 is the only predicted phosphorylation site on the aG–aH loop (NetPhos score 0.692; [33]). However, Figure 4 shows that this residue is consistently serine or threonine in all species except shrew, where it is alanine. Substitution to a non-phosphorylatable residue would be of biological interest, but it is also possible that this position simply represents a genome assembly error in shrew. This position has pMLS = 0.006, making it the least significant position of the five. Our method could exclude it and many similar cases by adding more criteria to the position selection process, but at the cost of decreased sensitivity and increased complexity. We expect that the coming availability of high-coverage de novo mammalian genome assemblies will resolve many such cases (e.g., [34,35]). 3.3. Longevity-selected Positions are Enriched in Protein Domains with Known Roles in Inflammation, Development, and other Diverse Functions Predicted secondary structure in all positions of the human proteome and in two subsets of longevity-selected positions. a-helix b-strand random coil total human proteome 2,108,348 (29.95%) 956,422 (13.59%) 3,974,432 (56.46%) 7,039,202 (100%) real data 1,922 (24.95%) 736 (9.56%) 5,044 (65.49%) 7,702 (100%) randomized control 160 (23.26%) 62 (9.01%) 466 (67.73%) 688 (100%) doi:10.1371/journal.pone.0038595.t002 PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38595 ndary structure in all positions of the human proteome and in two subsets of longevity-selected positions ble 2. Predicted secondary structure in all positions of the human proteome and in two subsets of longev June 2012 | Volume 7 | Issue 6 | e38595 June 2012 | Volume 7 | Issue 6 | e38595 4 3.4. Longevity-selected Positions Cluster in the Kinase Domain and C-terminal Tail of AMHR2 The domain containing the greatest number of longevity- selected positions (n = 7) was the protein kinase domain of AMHR2, a protein introduced in Section 1. In human, this domain comprises residues 203–517. Downstream of this domain is the C-terminal tail of the protein, residues 518–573. This cysteine-rich tail is unique to the AMHR2 ortholog family and is predicted to lack secondary structure. On average, the residues in the region 463–573 that do not form secondary structure have pMLS values that are consistently in the top 2% of the 3.6 million selected positions (median pMLS,0.568 by sliding window analy- sis). But this trend does not hold for AMHR2 overall (median pMLS is 0.800 excluding secondary structure positions), suggesting that Table 3. Selected clusters, not mutually exclusive, of ontology terms enriched in top protein domains. Table 3. Selected clusters, not mutually exclusive, of ontology terms enriched in top protein domains. Table 3. Selected clusters, not mutually exclusive, of ontology terms enriched in top protein domains. functional class p examples extracellular region 5.1e-5 BTD, CP, LAMA2, LAMA3, PRSS12 cytokine-mediated signaling pathway 0.033 JAK1, IL31RA, IL6ST, LIFR, RIKP1, KIT protein tyrosine kinase activity 0.018 JAK1, ROS1, MST1R, NIN, OBSCN, KIT, TYK2 multicopper oxidase; copper ion binding 9.4e-3 AFP, CP, F8, HEPHL1 developmental process 0.041 AFP, AMHR2, ALOX15, CFTR, ATR, ATM, BRCA1, RECQL4 motor activity; myosin complex 0.015 KIF18B, KIF20B, KIF22, MYO5C, MYO7B, MYO18A complement and coagulation cascades; humoral immune response 0.061 F8, F11, CR2, C1R, CFD, LTF, CD83 serine-type endopeptidase activity 0.063 F11, C1R, CFD, KLK6, LTF, PRSS12 doi:10.1371/journal.pone.0038595.t003 PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e38595 June 2012 | Volume 7 | Issue 6 | e38595 5 Longevity-Selected Protein Regions in Mammals Figure 4. OrthoMaM alignment of the C-terminal regions of AMHR2 ortholog kinase domains. Orthologs are ordered by species MLS. The five longevity-selected positions in this region (Y465, T469, F473, E513, and H515) are highlighted in gray. ‘‘X’’ indicates regions that we masked due to excessive divergence (Section 2.1). Long regions of gaps are not necessarily real genome deletions, but are more likely to have been missed during genome assembly or annotation. doi:10.1371/journal.pone.0038595.g004 Most of the residues on the aG–aH loop face the solvent, suggesting that they may interact with another domain or protein. PLoS ONE | www.plosone.org 3.4. Longevity-selected Positions Cluster in the Kinase Domain and C-terminal Tail of AMHR2 The five longevity-selected positions discussed in the text are labeled; they are found on the aG–aH loop (Y465, T469, and F473) and the C-terminal loop following aI (E513 and H515). doi:10.1371/journal.pone.0038595.g005 follicles [11]. Follicle depletion is the cause of menopause [1], and follicular decline or menopause has been observed in most or all mammals studied, including whales [39,40], nonhuman primates [41], rodents, and others [1,42], although admittedly we lack data for most species in the wild. A reasonable deduction is that a species’s rate of follicle depletion scales inversely with its longevity. We speculate that differential regulation of AMHR2 in a lifespan- dependent manner could act as a mechanism that effects this scaling, increasing the probability that a female has used all her reproductive potential before her death. This hypothesis could be viewed as a case of the disposable soma theory of aging [43]. It might be tested by relating AMHR2 ortholog sequence to rate of follicular decline across several species. comprising the aF–aG loop, is conserved only in AMHR2 orthologs. Overall, these findings are consistent with the existence of a large interaction surface conserved in all AMHR2 orthologs whose area, and thus binding affinity, varies at the aG–aH loop in a species-specific manner. It is likely that this is a novel docking surface involved in the regulation of AMHR2; one possible regulatory binding partner is the C-terminal tail of AMHR2 itself, whose positions also have consistently low pMLS values relative to the proteome overall, as noted above. Since loop aG–aH flanks this conserved patch, and four of the five residues face solvent in our model, it is possible that overall these positions contribute to lifespan-specific binding affinity. We are unaware of reported mutations specifically in the two loops of AMHR2 that contain our longevity-selected positions. However, two prior lines of inquiry are consistent with the docking-surface hypothesis. First, Belville et al. [36] also mapped the AMHR2 kinase domain to a solved structure in order to investigate natural mutations found in PMDS. Although based on a structure of the more distantly-related ACVR2B instead of BMPR2, its details are similar to those of our model, including both overall tertiary structure and specific residue orientation. Of the seven mutated positions they analyzed, four were located in the C-lobe of the kinase domain. One, D491, lies in the conserved aH–aI loop and faces solvent; its mutation to H causes PMDS. 3.4. Longevity-selected Positions Cluster in the Kinase Domain and C-terminal Tail of AMHR2 If this is the case, then assuming that the gross function of AMHR2 is conserved within mammals, we would expect other residues on a common surface with the aG–aH loop to be conserved. We used positional conservation analysis to determine conservation of the surface residues of two alignments, (1) mammalian AMHR2 orthologs exclusively and (2) a representative set of mammalian orthologs in the TGFBR2 subfamily, including orthologs of TGFBR2, activin receptor type-2A and B (ACVR2A and ACVR2B), BMPR2, and AMHR2 (not shown). This analysis revealed two conserved solvent-facing patches flanking the region of our longevity-selected positions. One patch, mainly comprising the aH–aI loop, is conserved in all TGFBR2 subfamily members, confirming a previous observation [36]. The other patch, mainly F473 faces forward in our model. It is conserved hydrophobic (F or L) in all species except guinea pig, rat, mouse, and shrew, where it is S or C. Thus, F473 may be involved in a hydrophobic interaction with a binding partner. We have low confidence in the exact placement of the short C- terminal loop, because it is not conserved in BMPR2 and lacks contacts with the other elements of our model. However, both E513 and H515 on this loop are preferentially charged in long-lived species, consistent with differential binding affinity. E513 is conserved in all species except rat, mouse, and shrew, where it is V, G, and A. H515 is conserved positive (H or R) in 16/17 species with MLS at least 16 y, but it is charged (H, R, or D) in only 3/9 shorter-lived species. The preference for hydrophilic residues in long-lived species at these two positions specifically may suggest that flexibility of the C-terminal loop is a longevity-conserved property. June 2012 | Volume 7 | Issue 6 | e38595 6 Longevity-Selected Protein Regions in Mammals Figure 5. AMHR2 kinase domain mapped onto experimental structure of BMPR2 kinase domain. All eight longevity-selected positions found in this domain are shown as black sticks and are further described in Table S2. The five longevity-selected positions discussed in the text are labeled; they are found on the aG–aH loop (Y465, T469, and F473) and the C-terminal loop following aI (E513 and H515). doi:10.1371/journal.pone.0038595.g005 Figure 5. AMHR2 kinase domain mapped onto experimental structure of BMPR2 kinase domain. All eight longevity-selected positions found in this domain are shown as black sticks and are further described in Table S2. 3.4. Longevity-selected Positions Cluster in the Kinase Domain and C-terminal Tail of AMHR2 This mutation further supports the idea that the solvent-facing bottom of the C-lobe is critical for proper AMHR2 function. We also note the unusual cysteine conservation in the C- terminal tail, which is unique to AMHR2 orthologs. There are eight cysteine residues in this region of human AMHR2, all of which are relatively conserved. Multiple regression revealed a specific fit of the number of cysteines conserved to log10 MLS (pMLS = 0.002 and pmass = 0.082 after phylogenetic correction). It is possible that these residues bind zinc or another metal ion, thus imparting structure to this region, but the region does not match known zinc finger motifs. PLoS ONE | www.plosone.org 3.6. Conclusion Based on principles of protein structure, we have developed a simple, extensible, and gerontologically-oriented method to find longevity-selected positions in the mammalian proteome. Using this method we found that, surprisingly, longevity-selected positions are much more common in the mammalian proteome than would be expected based on a randomized control. We have also used our method to identify specific protein regions that deserve further study in the context of the comparative biology of aging and development, as well as specific aging-related proteins that are likely not lifespan-conserved due to their overall conservation. Topping the list of regions worth further study is the kinase domain and C-terminal tail of AMHR2, in which the longevity-selected residues lie on a common surface of unknown functional significance. Our results must be considered in light of the flaws we described in the method, the high error and omission rates of the mammalian proteome data that we used, and most of all the obvious limitations inherent in using the set of putative orthologs from a single individual’s genome to represent the proteome of an entire species. Given those caveats, we have found our method to be a reasonable starting point for comparative analysis of protein function. One apparent novelty of our method is that it allows analysis of individual positions in the proteome, not just entire proteins. In fact, this is not novel, as the method of Jobson et al. [4] also entails identification of specific longevity-selected positions, and the genes they called ‘‘longevity-selected’’ and ‘‘longevity-relaxed’’ were simply genes with statistical over- or under-abundances of such positions. Here, we have emphasized individual positions rather than entire proteins for three reasons. First, we think it plausible, as did Jobson et al., that a mark of a longevity-selected protein is an abundance of longevity-selected positions. Second, a focus on individual positions allows to more precisely determine arbitrary regions of a protein that may be longevity-selected, as exemplified by the C-terminus of AMHR2. In theory, such specific focus can suggest novel biochemical mechanisms. Third, since aging is a complex trait that is under weak selection, it is plausible that some major determinants are subtle general properties of the proteome itself (discussed in Section 3.2), rather than the explicit activity of a single protein or even a few functional collections of proteins. Longevity-Selected Protein Regions in Mammals protein kinase (PBK). As both myosin and ARHGEF16 are involved in cell migration [44], there may be lifespan-specific differences in this activity, or our findings may simply reflect its standard role in organismal development. Acknowledgments We thank Richard Jobson, Benoit Nabholz, and Nicolas Galtier for sharing data, Lisa Kinch and Jimin Pei for reading drafts of this report, and our reviewers for their helpful suggestions. 3.6. Conclusion Longevity-selected positions are the most obvious markers of such properties, and so may provide clues to identify them, in the same way as they may identify individual longevity-selected proteins. In short, we do not suggest that the positions that we call longevity- selected, in isolation, are major determinants of mammalian longevity. We only suggest that they may mark proteins or proteomic features that are such determinants, but are less obvious. Author Contributions Conceived and designed the experiments: JS NVG. Performed the experiments: JS. Analyzed the data: JS NVG. Contributed reagents/ materials/analysis tools: JS NVG. Wrote the paper: JS. Conceived and designed the experiments: JS NVG. Performed the experiments: JS. Analyzed the data: JS NVG. Contributed reagents/ materials/analysis tools: JS NVG. Wrote the paper: JS. p p 10. Imbeaud S, Faure E, Lamarre I, Mattei M-G, di Clemente N, et al. (1995) Insensitivity to anti-mullerian hormone due to a mutation in the human anti- mullerian hormone receptor. Nature Genet 11: 382–388. p 11. Durlinger ALL, Visser JA, Themmen APN (2002) Regulation of ovarian function: The role of anti-Mu¨llerian hormone. Reproduction 124: 601–609. 3.5. Comparison with Previous Methods Generally, we did not observe overlap between the longevity- selected proteins we identified and those identified in previous work [4,5]. But this is not surprising, because we differed in our assumptions, goals, and data sets (detailed in Sections 1 and 2.1). Most notably, we fit a smaller subset of high-quality protein alignments, focused on structured protein regions, and chose to ignore synonymous codon substitutions. Thus, we view our results as complementary, not conflicting. We do note that both our method and that of Li and de Magalha˜es [5] identified ‘‘myosin complex’’ as an ontology term enriched in longevity-selected proteins (Table 3). The two methods also agreed that two proteins were longevity selected, rho guanine nucleotide exchange factor 16 (ARHGEF16) and lymphokine-activated killer T-cell-originated Second, closer to the two loops on the bottom face of the domain is the residue E485, which is in aH and also faces solvent. In two independent population studies of Dutch women, the mutation E485Q was associated with menopause delayed by up to one year [37,38]. In addition to its canonical role in male fetal development, AMHR2 also plays a second role in adult reproductive function. It is expressed in granulosa cells of adult females, where it seems to act as a feedback inhibitor of follicle recruitment and growth by binding its ligand, AMH, which is secreted in a paracrine manner specifically by more mature June 2012 | Volume 7 | Issue 6 | e38595 7 1. Finch CE (1990) Longevity, Senescence, and the Genome. Chicago: University of Chicago Press. 922 p. 9. Knighton DR, Zheng J, TenEyck LF, Ashford VA, Xuong N-H, et al. (1991) Crystal structure of the catalytic subunit of cyclic adenosine monophosphate- dependent protein kinase. Science 253: 407–414. 2. Williams GC (1957) Pleiotropy, natural selection, and the evolution of senescence. Evolution 11: 398–411. 1. Finch CE (1990) Longevity, Senescence, and the Genome. Chicago: University of Chicago Press. 922 p. 2. Williams GC (1957) Pleiotropy, natural selection, and the evolution of senescence. Evolution 11: 398–411. 3. Rose MR (1984) Laboratory evolution of postponed senescence in Drosophila melanogaster. Evolution 38: 1004–1010. 4. Jobson RW, Nabholz B, Galtier N (2010) An evolutionary genome scan for longevity-related natural selection in mammals. Mol Biol Evol 27: 840–847. 5. Li Y, de Magalha˜es J (2011) Accelerated protein evolution analysis reveals genes and pathways associated with the evolution of mammalian longevity. AGE: 1– 14. doi:springerlink:10.1007/s11357-011-9361-y. 6. Yang Z, Bielawski JP (2000) Statistical methods for detecting molecular adaptation. Trends Ecol Evol 15: 496–503. 3. Rose MR (1984) Laboratory evolution of postponed senescence in Drosophila melanogaster. Evolution 38: 1004–1010. Table S1 The 107 protein domains that contain at least two longevity-selected positions. Table S2 Longevity-selected positions of the protein domains shown in Table S1. (XLS) Table S2 Longevity-selected positions of the protein domains shown in Table S1. 6. Yang Z, Bielawski JP (2000) Statistical methods for detecting molecular adaptation. Trends Ecol Evol 15: 496–503. 4. Jobson RW, Nabholz B, Galtier N (2010) An evolutionary genome scan for longevity-related natural selection in mammals. Mol Biol Evol 27: 840–847. 5. Li Y, de Magalha˜es J (2011) Accelerated protein evolution analysis reveals genes and pathways associated with the evolution of mammalian longevity. AGE: 1– 14. doi:springerlink:10.1007/s11357-011-9361-y. Supporting Information Figure S1 Density histograms of pMLS values yielded by fitting alternate input sets with the method described in Section 2.2. As in Figure 3, only fit positions with bMLS.0 are included. A. Chimp (Pan troglodytes) is the reference, and human is absent. B. Rhesus (Macaca mulatta) is the only primate fit. Human is the reference. C. Dog (Canis lupus) is the reference. D. Scores taken from BLOSUM62 instead of BLOSUM80. Real data. E. Randomized control data for (D). (EPS) For detecting longevity-selected protein positions, benefits of our method versus standard codon-level methods such as the codeml program of PAML [45] include emphasis on detection of significant biochemical changes that are likely to affect protein structure; straightforward single-position resolution, allowing to easily test hypotheses regarding arbitrary regions of proteins, as described above; simple control for species-specific mutation rate and shared phylogeny and fitting of body mass as an alternate hypothesis to MLS; avoidance of the need to arbitrarily bin species by MLS; and faster run time. We reiterate that our method in theory is compatible with any quantitative trait, as illustrated by our inclusion of both MLS and body mass, although in practice correlations between predictor variables in the data limit the application of this for the predictors of greatest interest, such as developmental schedule (Section 3.3). 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https://openalex.org/W1965481106
https://www.scielo.br/j/eagri/a/RPwkkhMdTt3QcWvXCbhxdMJ/?lang=pt&format=pdf
Portuguese
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Índice de capacidade do processo na avaliação da irrigação por aspersão
Engenharia agrícola
2,010
cc-by
3,469
PROCESS CAPACITY INDEX IN THE UNIFORMITY EVALUATION OF SPRINKLER IRRIGATION ABSTRACT: Analyzing the irrigation quality, besides evaluating its good operation, is a form of verifying the viability of its implantation and operation. As the distribution uniformity is one of the most used parameters for that evaluation, this work aimed to use techniques of quality engineering, by using the process capacity index (Cpl) to evaluate the uniformity of water distribution in overhead irrigation by sprinkler. The research was carried out at the Experimental Center of Agricultural Engineering, UNIOESTE, with 2 sprinklers Super10 model, NAANDAN, spaced 9m between themselves, during 25 irrigations with 1 h each. The Weather data were collected every 10 min. by a wireless weather station. It was found a medium CUC of 79.72% and a medium wind speed of 1.85 m s-1. It was applied quality control tests, elaborating SHEWHART control charts and it was calculated the process capacity index (Cpl). The obtained results allow us to affirm that the use of the process capacity index becomes a powerful tool to classify overhead irrigations in function of distribution uniformity. KEYWORDS: Quality control, wind speed, distribution uniformity. ÍNDICE DE CAPACIDADE DO PROCESSO NA AVALIAÇÃO DA IRRIGAÇÃO POR ASPERSÃO ANDRÉ L. JUSTI1, MARCIO A. VILAS BOAS2, SILVIO C. SAMPAIO2 RESUMO: Analisar a qualidade da irrigação, além de avaliar seu bom funcionamento, é uma forma de verificar a viabilidade de sua implantação e operação. Como a uniformidade de distribuição é um dos parâmetros mais utilizados para essa avaliação, este trabalho objetivou utilizar técnicas de engenharia de qualidade, usando o índice de capacidade do processo (Cpl) para avaliar a uniformidade de distribuição de água em um sistema de irrigação por aspersão convencional. Os ensaios foram conduzidos no Núcleo Experimental de Engenharia Agrícola, UNIOESTE, com dois aspersores modelo Super10, marca NAANDAN, espaçados 9 m entre si, durante 25 irrigações de 1 h cada. Os dados climáticos foram coletados a cada 10 min, por uma estação meteorológica sem fio. Encontraram-se um CUC médio de 79,72% e velocidade do vento média de 1,85 m s-1. Foram aplicados os testes de controle de qualidade, elaborando os gráficos de controle de Shewhart e calculado o índice de capacidade do processo (Cpl), sendo que os resultados obtidos permitem afirmar que a utilização do índice de capacidade do processo torna-se uma ferramenta poderosa para classificar sistemas de irrigação em função de sua uniformidade de distribuição. PALAVRAS-CHAVE: Controle de qualidade, velocidade do vento, uniformidade de distribuição. 1 Engo Agrícola, Doutorando Irrigação e Drenagem, UNESP, Botucatu - SP, Fone: (0XX14) 8129.7970, aljusti@fca.unesp.br 2 Professor, Área de Recursos Hídricos e Saneamento Ambiental, UNIOESTE/CASCAVEL/CCET/PGEAGRI/RHESA, Fone: (0XX45) 3220.3262, ssampaio@unioeste.br; vilasboas@unioeste.br p Recebido pelo Conselho Editorial em: 12-1-2009 Aprovado pelo Conselho Editorial em: 13-1-2010 INTRODUÇÃO A uniformidade de distribuição de sistemas de irrigação possui um papel importante na otimização do uso da água, com repercussões diretas na eficiência e na produção (CARRIÓN et al., 2001), e, geralmente, a uniformidade de distribuição é a principal maneira usada para determinar se um sistema de irrigação é aceitável ou não (BRENNAN, 2008). Em campo, a importância de uniformidade de irrigação e sua influência no rendimento e eficiência de aplicação são bem visíveis. São usados frequentemente vários parâmetros de desempenho para descrever a distribuição de água de irrigação no campo, sendo que um dos mais Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 Índice de capacidade do processo na avaliação da irrigação por aspersão 265 importantes é a uniformidade de distribuição (CLEMMENS & MOLDEN, 2007). Dentre os coeficientes de uniformidade, o mais utilizado é o Coeficiente de Uniformidade de Christiansen (CUC). importantes é a uniformidade de distribuição (CLEMMENS & MOLDEN, 2007). Dentre os coeficientes de uniformidade, o mais utilizado é o Coeficiente de Uniformidade de Christiansen (CUC). Diversos fatores influenciam na uniformidade de distribuição, desde os fatores de projeto até os climáticos, sendo que, na irrigação por aspersão, um dos mais importantes é a velocidade do vento. Segundo CARRIÓN et al.(2001), para avaliar os efeitos da velocidade do vento na uniformidade de distribuição da irrigação por aspersão, é necessário mensurar a distribuição de água em campo sob diferentes condições de vento, para então proceder ao cálculo do coeficiente de uniformidade. Como o CUC é um coeficiente para apenas um ensaio, para avaliar a uniformidade de distribuição ao longo de um ciclo de cultura ou do tempo, buscou-se utilizar o índice de capacidade do processo, aplicando um estudo de engenharia de qualidade, pois as técnicas de controle estatístico de qualidade foram desenvolvidas para avaliar a variabilidade ao longo do tempo (ciclo) do processo (MONTGOMERY, 2001). Buscou-se, com este trabalho, analisar a uniformidade de distribuição de água de um sistema de irrigação por aspersão, utilizando o índice de capacidade do processo. MATERIAL E MÉTODOS A tubulação da linha principal utilizou tubos de PVC com 50 m de comprimento, operando com três bombas associadas em série, marca THEBE, modelo M2B-12X, com potência de 0,5 cv, 3.500 rpm, com vazão máxima de 5,1 m3 h-1 e uma pressão máxima de 260 kPa. Foram realizados 25 ensaios, número de amostras recomendadas por MONTGOMERY (2001) para aplicação dos testes de controle de qualidade, com tempo de operação de 1 h, buscando manter em todos os ensaios as mesmas características de operação do sistema, ou seja, mesma pressão e tempo, fatores climáticos, que foram monitorados utilizando uma estação meteorológica sem fio (Wireless Weather Station) marca La Crosse, modelo WS-2310, sendo que as variações da velocidade do vento foram monitoradas em intervalos de 10 min. Foi utilizado, para avaliação da uniformidade de distribuição de água, o Coeficiente de Uniformidade de Christiansen (CUC), dado pela eq.(1):             − − = − = −  X n X X x CUC n i i 1 1 100 ue             − − = − = −  X n X X x CUC n i i 1 1 100 (1) que             − − = − = −  X n X X x CUC n i i 1 1 100 (1) que (1) em que, ue, CUC - Coeficiente de Uniformidade de Christiansen, %; i X - precipitação no coletor de ordem i, mm; i X - precipitação no coletor de ordem i, mm; − X - média aritmética das precipitações, mm, e n - número de coletores. n - número de coletores. Para o cálculo do índice de capacidade do processo (Cpl), dado pela eq.(2), quanto à média amostral, foi fixado como valor-alvo a lâmina média informada pelo fabricante, que para a pressão de serviço de 300 kPa é 4,9 mm h-1, e como limite inferior, a lâmina média do ensaio, cujo CUC foi 80,05%, por ser o mais próximo do aceitável pela norma, fixando assim a lâmina de 2,82 mm.h-1. (2) m que (2) (2) em que, q , LCL - limite inferior de especificação, mm LCL - limite inferior de especificação, mm - média amostral, mm, e - desvio-padrão amostral da variável em estudo. MATERIAL E MÉTODOS Os ensaios foram conduzidos no Núcleo Experimental de Engenharia Agrícola, NEEA, pertencente à Universidade Estadual do Oeste do Paraná - UNIOESTE, coordenadas 24°54’ de latitude sul e 53°31’ de longitude oeste, com altitude média de 750 m e clima subtropical temperado úmido, de acordo com a classificação climática de Köeppen. A precipitação média anual é de 1.971 mm e a temperatura média anual é de 19,6 °C. O conjunto avaliado constituiu-se de uma linha lateral com dois aspersores marca NAANDAN, modelo Super10, não compensante, bocal azul que, conforme o fabricante, para uma pressão de serviço de 300 kPa, possui vazão de 395 L h-1, gerando um diâmetro irrigado de 16 m e, para o espaçamento de 9 x 9 m, possui coeficiente de uniformidade de até 92%. Na Figura 1, apresenta-se o esquema do sistema montado em campo. Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 FIGURA 1. Esquema do sistema avaliado. Evaluated system layout. O modelo Super10 é um conjunto composto por um adaptador F de ½”, fixado em uma haste galvanizada de 8 mm diâmetro por 1,2 m de altura, ligado à lateral por um tubo de polietileno, com diâmetros externo e interno de 13 e 12 mm, respectivamente. FIGURA 1. Esquema do sistema avaliado. Evaluated system layout. FIGURA 1. Esquema do sistema avaliado. Evaluated system layout. O modelo Super10 é um conjunto composto por um adaptador F de ½”, fixado em uma haste galvanizada de 8 mm diâmetro por 1,2 m de altura, ligado à lateral por um tubo de polietileno, com diâmetros externo e interno de 13 e 12 mm, respectivamente. Eng Agríc Jaboticabal v 30 n 2 p 264-270 mar /abr 2010 O modelo Super10 é um conjunto composto por um adaptador F de ½”, fixado em uma haste galvanizada de 8 mm diâmetro por 1,2 m de altura, ligado à lateral por um tubo de polietileno, com diâmetros externo e interno de 13 e 12 mm, respectivamente. Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 André L. Justi, Marcio A. Vilas Boas, Silvio C. Sampaio 266 A linha lateral foi montada utilizando tubo de polietileno de 25 mm de diâmetro. MATERIAL E MÉTODOS Para a construção dos gráficos de controle estatístico de qualidade de Shewhart, foi necessário calcular os Limites Inferior e Superior de Especificação, obtidos pelas eqs.(3) e (4), respectivamente. (3) (4) (3) (3) (4) (4) em que, em que, q µ - média dos ensaios, %; q µ - média dos ensaios, %; n - número de ensaios, e - desvio-padrão amostral. RESULTADOS E DISCUSSÃO Na Tabela 1, apresenta-se a estatística descritiva, onde foram calculados a média, desvio- padrão, coeficiente de variação, valores de máximo e mínimo para o CUC, a velocidade do vento, umidade relativa do ar e temperatura. Foi realizado o teste de normalidade de Anderson-Darling, a Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 267 Índice de capacidade do processo na avaliação da irrigação por aspersão 5% de significância, onde todas as variáveis apresentaram distribuição normal. O valor médio encontrado de CUC para as 25 irrigações foi 79,72%, e a velocidade do vento médio, 1,85 m s-1. Ao longo do experimento, os valores máximos de CUC e velocidade do vento foram 89,45% e 2,97 m s-1, respectivamente. Os valores de desvio-padrão obtidos a partir dos dados foram 3,74 e 0,70% para CUC e velocidade do vento, respectivamente. Quanto ao coeficiente de variação para o CUC, foi 4,72%, e para a velocidade do vento, 37,98%. Quanto aos fatores umidade relativa e temperatura, foram encontrados valores entre 30 e 58% para umidade relativa e 27,30 e 41,80 oC, e valores médios de 46,04% e 32,59 oC. TABELA 1. Estatística descritiva dos dados coletados em campo (CUC, umidade relativa, temperatura e velocidade do vento). Exploratory analysis of data collected on field (CUC, relative humidity, temperature and wind speed). * Nível de 5% de significância. Análise CUC (%) Velocidade do vento (m s-1) Umidade Relativa (%) Temperatura (˚C) Norma para o CUC Média Desvio-Padrão Variância Coeficiente de Variação Mínimo Máximo Teste de Normalidade (Anderson-Darling) 80 79,72 3,74 14,05 4,72 72,25 89,45 Não se rejeita* -- 1,85 0,70 0,49 37,98 0,44 2,97 Não se rejeita* -- 46,04 8,19 67,12 17,80 30,00 58,00 Não se rejeita* -- 32,59 3,94 15,56 12,10 27,30 41,80 Não se rejeita* TABELA 1. Estatística descritiva dos dados coletados em campo (CUC, umidade relativa, temperatura e velocidade do vento). Exploratory analysis of data collected on field (CUC, relative humidity, temperature and wind speed). DECHMI et al. (2003) observaram em seu estudo do espaçamento na aplicação de água por aspersão no milho, que a uniformidade em 48% dos ensaios foi menor que o aceitável, sendo que os valores extremos de uniformidade corresponderam aos maiores e menores valores de vento. Salientaram, ainda, o que reforça os resultados obtidos neste experimento, que, para o sistema que avaliaram, grande parte da variabilidade, em torno de 90%, decorreu em função da velocidade do vento. RESULTADOS E DISCUSSÃO Houve tendência do CUC em diminuir com o aumento da velocidade do vento, sendo que a maior parte dos ensaios foi conduzida em condições de vento com velocidade acima de 1,5 m s-1. Essa tendência apresentada caracteriza uma relação entre CUC e velocidade do vento, pois como se percebeu neste estudo, um parâmetro aumenta em função do decréscimo de outro, sendo que, ao testar a regressão linear, encontrou-se um coeficiente de determinação R² = 83,6%. Percebe-se, pela Figura 2, que ocorreu uma concentração dos ensaios com valor de CUC acima de 80% entre as velocidades de 0,5 e 1,5 m s-1 e 11 irrigações resultaram em coeficientes de uniformidade abaixo de 80%, sendo que as mesmas resultaram da irrigação com velocidade do vento entre 2,0 e 3,0 m s -1. SOUZA et al. (2008), estudando o desempenho de um sistema de irrigação por aspersão convencional em vila rural, em função dos fatores climáticos, obteve relação entre CUC e velocidade do vento semelhante a este estudo, com valores de uniformidade também abaixo do mínimo aceitável de 80%, e obtendo um CUC médio de 77,9%. Sendo este um valor semelhante ao encontrado com este trabalho, pois o valor médio do CUC foi 79,48%, também ficando abaixo do mínimo para tal parâmetro, obtendo regressão linear com coeficiente de determinação R² = 74%. Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 268 André L. Justi, Marcio A. Vilas Boas, Silvio C. Sampaio FIGURA 2. Relação entre CUC e velocidade do vento. Relation between CUC and wind speed. FIGURA 2. Relação entre CUC e velocidade do vento. Relation between CUC and wind speed. FIGURA 2. Relação entre CUC e velocidade do vento. Relation between CU Resultado semelhante foi encontrado também por AZEVEDO et al. (2000), estudando a influência de fatores climáticos em sistemas por aspersão de alta pressão, que relataram que, quando a velocidade do vento aumentou de 1 para 7 m.s-1, o CUC caiu de 83 para 42%, ou seja, 50% menos de uniformidade com o aumento da velocidade do vento. Mesmo não operando com velocidade do vento alta, conforme o aumento desde fator, o CUC decaiu, chegando a 48% dos ensaios, ficando abaixo de 80%. Já PLAYÁN et al. RESULTADOS E DISCUSSÃO (2006), estudando a uniformidade de distribuição de água usando modelos balísticos, utilizando três modelos de aspersores, verificaram que, em todos os casos, com o aumento da velocidade do vento, a uniformidade foi afetada sensivelmente, sendo que, para a pressão de serviço de 300 kPa, ao testar a regressão linear, encontraram coeficiente de determinação R² de até 73%. Na Figura 3, podemos observar o comportamento do sistema de irrigação estudado ao longo dos 25 ensaios e verifica-se que um dos ensaios esteve acima do Limite Superior de Controle (UCL = 87,52%), e nenhum dos ensaios obteve CUC menor que o Limite Inferior de Controle (LCL = 71,45%). O restante dos ensaios permaneceu dentro dos limites, sendo que o valor médio foi 79,49%. Exceto o ensaio 8 (CUC = 89,45%), os demais ensaios estão sob controle, porém tendo grande variação em relação ao valor médio. Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 FIGURA 3. Controle de qualidade de Sewhart apresentando os limites superior e inferior de especificação, dados pelas eqs.(3) e (4), média e valor de referência para o CUC (80%). Quality control graph showing the upper and lower limits of specification, given by equations 3 and 4, average and reference value to CUC (80%). FIGURA 3. Controle de qualidade de Sewhart apresentando os limites superior e inferior de especificação, dados pelas eqs.(3) e (4), média e valor de referência para o CUC (80%). Quality control graph showing the upper and lower limits of specification, given by equations 3 and 4, average and reference value to CUC (80%). Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 Índice de capacidade do processo na avaliação da irrigação por aspersão 269 O sistema apresentou um ensaio com desempenho acima do limite superior, o que é considerado aceitável, pois foi encontrado o maior coeficiente de uniformidade, chegando próximo ao indicado pelo catálogo do fabricante, o qual afirma que, para a pressão de serviço usada no estudo, o emissor pode chegar a coeficientes de uniformidade de até 92%. Já a média foi inferior ao valor mínimo aceitável para o CUC (80%), fato esse ocorrido em função de fatores diversos, sendo a velocidade do vento o fator de maior influência em tal variação. CONCLUSÕES O sistema de irrigação estudado apresentou coeficiente de uniformidade de distribuição médio muito próximo do mínimo aceitável, tendo grande parte dos ensaios resultado em valores de CUC acima de 80%. Entre o CUC e a velocidade do vento, foi obtido um coeficiente de determinação para regressão linear de 83,6%, e entre o CUC e o índice de capacidade do processo, esse coeficiente foi 78%. O aumento do índice de capacidade do processo mostrou-se diretamente proporcional ao aumento da uniformidade de distribuição. O índice de capacidade do processo mostrou-se capaz de diagnosticar se a irrigação tem capacidade de se manter sob controle e constante, ou seja, se será capaz de manter níveis aceitáveis de uniformidade. RESULTADOS E DISCUSSÃO Na Tabela 2, apresentam-se os valores calculados da capacidade do processo (Cpl), ao longo dos 25 ensaios, indicando que, para um CUC entre 70 e 75%, o índice de capacidade do processo obtido foi 2,26; quando os valores de CUC estão entre 75 e 80%, o índice de capacidade do processo encontrado foi 2,97 e, quando o CUC foi maior que 80%, o índice de capacidade do processo foi 3,00. TABELA 2. Índice de capacidade do processo. Process capacity índex. CUC (%) fi Cpl Irrigação 70 - 75 75 - 80 > 80 3 9 13 2,26 2,97 3,00 Total 25 TABELA 2. Índice de capacidade do processo. Process capacity índex. Conforme a uniformidade de distribuição aumenta, o índice de capacidade também aumenta, ocorrendo uma relação entre tais variáveis expressa pela equação CUC (%) = 46,07 + 10,55 Cpl, com coeficiente de determinação R² = 78%. Quanto aos valores de Cpl, MONTGOMERY (2001) afirmou que, para considerar processos novos aceitáveis, o valor mínimo do índice de capacidade do processo (Cpl) deve ser 1,60, e o sistema estudado obteve índices acima de 2,00, sendo um sistema novo, em seu primeiro ano de uso. Resultados semelhantes quanto à viabilidade da utilização do controle de qualidade foram encontrados por CHEN et al. (2007), que afirmaram, após estudar a capacidade do processo para medidas unilaterais, que esse índice, utilizando gráficos de controle, pode ser usado não somente para monitorar a estabilidade do processo, mas também para monitorar a qualidade do mesmo, acompanhando índices de especificação e avaliando sua estabilidade. Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 REFERÊNCIAS Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 AZEVEDO, J.H.; BERNARDO, S.; RAMOS, M.M.; SEDIYAMA, G.C.; CECON, P.R. Influência de fatores climáticos e operacionais sobre a uniformidade de distribuição de água, em um sistema AZEVEDO, J.H.; BERNARDO, S.; RAMOS, M.M.; SEDIYAMA, G.C.; CECON, P.R. Influência de fatores climáticos e operacionais sobre a uniformidade de distribuição de água, em um sistema Eng. Agríc., Jaboticabal, v.30, n.2, p.264-270, mar./abr. 2010 270 André L. Justi, Marcio A. Vilas Boas, Silvio C. Sampaio de irrigação por aspersão de alta pressão. Revista Brasileira de Engenharia Agrícola e Ambiental, Campina Grande, v.4, n.2, p.152-158, 2000. de irrigação por aspersão de alta pressão. Revista Brasileira de Engenharia Agrícola e Ambiental, Campina Grande, v.4, n.2, p.152-158, 2000. de irrigação por aspersão de alta pressão. Revista Brasileira de Engenharia Agrícola e Ambiental, Campina Grande, v.4, n.2, p.152-158, 2000. BRENNAN, D. Factors affecting the economic benefits of sprinkler uniformity and their implications for irrigation water use. Irrigation Science, Heidelberg, v.26, n.2, p.109-119, 2008. CARRIÓN, P.; TARJUELO, J.M.; MONTERO, J. SIRIAS: A simulation model for sprinkler irrigation: I. A description of model. Irrigation Science, Heidelberg, v.20, n.2, p.73-84, 2001. CHEN, K.S.; HUANG, H.L.; HUANG, C.T. Control charts for one-sided capability indices. Quality & Quantity, Amsterdam, v.41, n.3, p.413-427, 2007. CLEMMENS, A.J.; MOLDEN, D.J. Water uses and productivity of irrigation systems. Irrigation Science, Heidelberg, v.25, n.3, p.247-261, 2007. DECHMI, F.; PLAYÁN, E.; CAVERO, J.; FACI, J.M.; MARTINEZ-COB, A. Wind effects on solid set sprinkler irrigation depth and yield of maize (Zea mays). Irrigation Science, Heidelberg, v.22, n.2, p.67-77, 2003. MONTGOMERY, D.C. Introduction to statistical quality control. 4th ed. New York: John Wiley & Sons, 2001. 500 p. & PLAYÁN, E.; ZAPATA, N.; FACI, J.M.; TOLOSA, D.; LACUEVA, J.L.; PELEGRÍN, J.; SALVADOR, R.; SÁNCHEZ, I.; LAFITA, A. Assessing sprinkler irrigation uniformity using a ballistic simulation model. Agricultural Water Management, Amsterdam, v.84, p.89-100, 2006. SOUZA, E.A.M.; SOUZA, P.C.; VILAS BOAS, M.A. Avaliação do desempenho de sistemas de irrigação por aspersão convencional fixo e gotejamento em vila rural. Irriga, Botucatu, v.11, n.1, p.47-62, 2008.
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The effect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bacteria and its antioxidant activity in the gastrointestinal digestion process
Food and health
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FOOD and HEALTH E-ISSN 2602-2834 Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article FOOD and HEALTH E-ISSN 2602-2834 Research Article The effect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bacteria and its antioxidant activity in the gastrointestinal digestion process Büşra KARKAR Cite this article as: Karkar, B. (2024). The effect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bacteria and its antioxidant activity in the gastrointestinal digestion process. Food and Health, 10(2), 104-114. https://doi.org/10.3153/FH24010 Cite this article as: Karkar, B. (2024). The effect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bact gastrointestinal digestion process. Food and Health, 10(2), 104-114. https://doi.org/10.3153/FH24010 Keywords: Pseudo-cereals, Amaranth, L. acidophilus, Probiotic bacteria, Antioxidant, Gastrointestinal FOOD and HEALTH E-ISSN 2602-2834 The effect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bacteria and its antioxidant activity in the gastrointestinal digestion process Büşra KARKAR Cite this article as: Karkar, B. (2024). The effect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bacteria and its antioxidant activity in the gastrointestinal digestion process. Food and Health, 10(2), 104-114. https://doi.org/10.3153/FH24010 University of Bursa Uludağ, Faculty of Science and Arts, Department of Chemistry, Bursa/Türkiye ORCID IDs of the authors: B.K. 0000-0001-6547-5558 Submitted: 17.07.2023 Revision requested: 07.11.2023 Last revision received: 18.11.2023 Accepted: 18.12.2023 Published online: 29.02.2024 ABSTRACT Pseudo-cereals are an excellent source of nutrients, rich in carbohydrates, dietary fiber, protein, lipids, phytochemicals, and minerals such as magnesium, zinc, copper, sodium, potassium, and calcium. The positive effects of gluten-free pseudo-cereals on the digestive system are an alterna- tive to natural cereals. Pseudo-cereals have prebiotic properties and strengthen digestion by posi- tively affecting the development of probiotic bacteria, especially Lactobacillus. Therefore, the ef- fect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bacteria, which helps digestion, was determined. First, solvent, acidic, and basic hydrolysis extractions of amaranth in eight different solvent media were performed, and total phenolic content and antioxidant activity values were determined. The total phenolic content values in the gastrointestinal digestion process were investigated by applying three different consumption methods, milling, boiling, and drying, to amaranth grains. L. acidophilus probiotic bacteria were activated with milled, dried, and boiled amaranth, and the increase in viability was examined. While the viability of L. acidophilus activa- ted with milled and dried amaranth increased by 9.47% and 7.46%, respectively, the viability of bacteria activated with boiled amaranth almost did not increase (0.60%). Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article FOOD and HEALTH E-ISSN 2602-2834 The effect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bacteria and its antioxidant activity in the gastrointestinal digestion process Büşra KARKAR Cite this article as: Karkar, B. (2024). The effect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bacteria and its antioxidant activity in the gastrointestinal digestion process. Food and Health, 10(2), 104-114. https://doi.org/10.3153/FH24010 University of Bursa Uludağ, Faculty of Science and Arts, Department of Chemistry, Bursa/Türkiye ORCID IDs of the authors: B.K. 0000-0001-6547-5558 Submitted: 17.07.2023 Revision requested: 07.11.2023 Last revision received: 18.11.2023 Accepted: 18.12.2023 Published online: 29.02.2024 ABSTRACT Pseudo-cereals are an excellent source of nutrients, rich in carbohydrates, dietary fiber, protein, lipids, phytochemicals, and minerals such as magnesium, zinc, copper, sodium, potassium, and calcium. The positive effects of gluten-free pseudo-cereals on the digestive system are an alterna- tive to natural cereals. Pseudo-cereals have prebiotic properties and strengthen digestion by posi- tively affecting the development of probiotic bacteria, especially Lactobacillus. Therefore, the ef- fect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bacteria, which helps digestion, was determined. First, solvent, acidic, and basic hydrolysis extractions of amaranth in eight different solvent media were performed, and total phenolic content and antioxidant activity values were determined. The total phenolic content values in the gastrointestinal digestion process were investigated by applying three different consumption methods, milling, boiling, and drying, to amaranth grains. L. acidophilus probiotic bacteria were activated with milled, dried, and boiled amaranth, and the increase in viability was examined. While the viability of L. acidophilus activa- ted with milled and dried amaranth increased by 9.47% and 7.46%, respectively, the viability of bacteria activated with boiled amaranth almost did not increase (0.60%). Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article FOOD and HEALTH E-ISSN 2602-2834 Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article Preparation of Amaranth (Amaranthus) Amaranth is a plant of the Amaranthaceae family, cultivated in Asia, Africa, and Central and South America (House et al., 2020; Olawoye & Gbadamosi, 2020). Amaranth has been consumed as a vegetable in the Americas for thousands of years. Nowadays, various parts of the plant are used for dif- ferent purposes; for example, the flowers are used to produce red dye, the seeds are used as grain for flour production and animal nutrition, and the leaves are used in food (López- Mejía et al., 2014). Various parts of the plant are rich in car- bohydrates (61.4%), dietary fibre (20.6%), protein (16.5%), vitamins, and minerals, which are highly beneficial for hu- man health (Petrova & Petrov, 2020; Shewry, 2016). These parts also contain components with antioxidant activity, such as flavonoids, phenolic acids, carotenoids, and tannins (Silva et al., 2021). Amaranth is approved as a superfood due to its rich content of beneficial components for human nutrition The amaranth used in the research was purchased commerci- ally. The amaranth, which will be used for spectroscopic analysis, was milled, and its dimensions were determined in the 14-30 mesh range. For in-vitro gastrointestinal digestion analysis, amaranth was prepared in three ways: grinding into flour, drying in an oven at 100°C for one h, and boiling at 100°C for 30 min. Introduction and health. The literature has revealed that amaranth can ef- fectively overcome nutrition-related health problems (Ruth et al., 2021). Pseudo-cereals are the seed grains of dicotyledonous plants of different plant families, such as Amaranthaceae and Che- nopodiaceae. This type of cereal is physically similar to na- tural cereal grains from monocotyledonous plants of the Poa- ceae or Gramineae family (Shewry, 2016). The most known pseudo-cereals are quinoa from Chenopodiaceae, chia from Lamiaceae, buckwheat from Polygonaceace, and amaranth from Amaranthaceae (Upasana & Yadav, 2022). The main aim of this study is to examine the effect of ama- ranth, which is a pseudo-cereal, on the activity of probiotic bacteria that help digestion and to determine its antioxidant activity. For this purpose, various extracts of amaranth were prepared, and total phenolic content and antioxidant capacity values were determined. In addition, amaranth was used as a prebiotic to increase the viability of Lactobacillus acidophi- lus probiotic bacteria, and its effect on bacterial viability was investigated. Also, with the preparation of consumable forms of amaranth, their digestion in the in-vitro gastrointestinal tract was investigated. Pseudo-cereal grains are a good source of nutrients. It conta- ins 50-70% of carbohydrates, 4-12% of dietary fibre, 7-16% of protein, 4-7% of lipid, and a high amount of micronutrients such as zinc, copper, manganese, potassium, sodium, cal- cium, and magnesium (Malleshi et al., 2020). Pseudo-cereal grains do not contain gluten, and they also contain low starch. Due to its high lipid and protein content, it is high in calories like natural cereals (Bekkering & Tian, 2019). Pseudo-cereals have antioxidant, antimicrobial, anti-inflammatory, and an- tihypertensive properties due to their rich phytochemical con- tent. In addition, pseudo-cereals show protective activities for bone and gastrointestinal system health (Upasana & Yadav, 2022). The positive activities of cereals on the health of the gastrointestinal tract have been known for many years. However, in recent years, many studies have supported the idea that pseudo-cereals can be an alternative to natural cere- als. The main emphasis of these studies is that pseudo-cereals contribute to the viability of probiotic bacteria, one of the most essential elements of the gastrointestinal system. Espe- cially pseudo-cereals are the substrate of Lactobacillus spe- cies from probiotic bacteria and have prebiotic activity (Ugural & Akyol, 2022). Chemical and Reagent Acetic acid, acetone, n-butanol, ethanol, ethyl acetate, hexane, hydrochloric acid, methanol, petroleum ether, and potassium persulfate were purchased from Merck (Darms- tadt, Germany). 2,2-azino-bis(3-ethylbenzothiazoline-6-sul- fonic acid) diammonium salt, 2,4,6-Tris(2-pyridyl)-s-tria- zine, copper(II) sulfate pentahydrate, Folin–Ciocalteau rea- gent, gallic acid (HPLC grade), pancreatin, pepsin, sodium acetate trihydrate, sodium carbonate, sodium hydroxide, po- tassium sodium tartrate, and Trolox (HPLC grade), were sup- ported from Sigma-Aldrich (St. Louis, MO., USA). Bile salt, potassium dihydrogen phosphate, and sodium chloride were purchased from Edukim, Isolab, and Tekkim, respectively. ABSTRACT University of Bursa Uludağ, Faculty of Science and Arts, Department of Chemistry, Bursa/Türkiye University of Bursa Uludağ, Faculty of Science and Arts, Department of Chemistry, Bursa/Türkiye ORCID IDs of the authors: B.K. 0000-0001-6547-5558 Pseudo-cereals are an excellent source of nutrients, rich in carbohydrates, dietary fiber, protein, lipids, phytochemicals, and minerals such as magnesium, zinc, copper, sodium, potassium, and calcium. The positive effects of gluten-free pseudo-cereals on the digestive system are an alterna- tive to natural cereals. Pseudo-cereals have prebiotic properties and strengthen digestion by posi- tively affecting the development of probiotic bacteria, especially Lactobacillus. Therefore, the ef- fect of amaranth, a pseudo-cereal, on the activity of L. acidophilus probiotic bacteria, which helps digestion, was determined. First, solvent, acidic, and basic hydrolysis extractions of amaranth in eight different solvent media were performed, and total phenolic content and antioxidant activity values were determined. The total phenolic content values in the gastrointestinal digestion process were investigated by applying three different consumption methods, milling, boiling, and drying, to amaranth grains. L. acidophilus probiotic bacteria were activated with milled, dried, and boiled amaranth, and the increase in viability was examined. While the viability of L. acidophilus activa- ted with milled and dried amaranth increased by 9.47% and 7.46%, respectively, the viability of bacteria activated with boiled amaranth almost did not increase (0.60%). ORCID IDs of the authors: B.K. 0000-0001-6547-5558 Submitted: 17.07.2023 Revision requested: 07.11.2023 Last revision received: 18.11.2023 Accepted: 18.12.2023 Published online: 29.02.2024 Correspondence: Büşra KARKAR E-mail: busrakarkar@uludag.edu.tr Available online at http://jfhs.scientificwebjournals.com 104 Total phenolic content (TPC) analysis The Folin-Ciocalteu method was used to determine the TPC of amaranth extracts (Aklan & Aybastıer, 2023; Güçlü et al., 2006; Karkar & Şahin, 2022; Singleton et al., 1999). Accor- ding to the method, 0.25 mL of amaranth extract was mixed with Lowry C (2.5 mL) (prepared by mixing Lowry A (0.4% NaOH and 2.0% Na2CO3) and Lowry B (1.0% NaKC4H4O6 and 0.5% CuSO4) in a ratio of 50:1) solution and 0.67 N Folin reagent (0.25 mL). The total volume was made up of 4.75 mL of distilled water. The samples were kept in a dark environ- ment for 30 min, and absorbance measurements were perfor- med at 750 nm using a UV-VIS spectrometer. The TPC of amaranth was determined as mg gallic acid equivalent (GAE)/ 100 g amaranth. The SGF medium (10 mL) was added to the milled, dried, and boiled amaranth samples (1 g), and gastric digestion was carried out for 2 h in a shaker incubator at 37°C and 100 rpm. After gastric digestion, the pH of the medium was adjusted to 7.0 with 0.2 N NaOH, and the SIF medium (10 mL) was ad- ded. For intestinal digestion, the medium was incubated for 2 h in a shaker incubator at 37°C and 100 rpm. During gastric and intestinal digestion, a 1 mL of sample was taken every 30 min from the digestive fluid, kept in an ice bath for 15 min, and centrifuged at 6000 rpm for 15 min. Then, the TPC of the samples (1 mL) taken from the media was determined, and the amounts of phenolic compounds released from the ama- ranth samples into the environment during gastrointestinal di- gestion were determined. In-vitro Gastrointestinal Digestion Analysis An analysis examined how the human digestive system bre- aks down amaranth. Two different simulated digestive fluids were created for gastrointestinal digestion. The simulated gastric fluid (SGF; pH 2.00) was prepared using sodium chlo- ride (0.2%; w/v) and porcine pepsin (1600 U/mL of final vo- lume), and the pH was adjusted to 2.0 ± 0.2 (0.2 N HCl). The simulated intestinal fluid (SIF; pH 7.00) was prepared using potassium dihydrogen phosphate (0.68%; w/v), bile salt (0.3%; w/v), and porcine pancreatin (800 U/mL of final vo- lume), and pH was adjusted to 7.0 ± 0.2 (0.2 N NaOH) (Tipigil, 2015). Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article Research Article Statistical Analysis ways: solvent extraction, acidic hydrolysis, and basic hyd- rolysis. For solvent extraction, 1 g of amaranth was extracted with 10 mL of solvent for 4 h at 45°C. For acidic hydrolysis extraction, amaranth (1 g) was extracted with solvent (10 mL) and 1 M HCl (0.1 mL) at 45°C for 4 h. At the same time, 1M NaOH was used instead of 1 M HCl for basic hydrolysis extraction. All the extracts were filtered through filter paper, and the extracts other than water, methanol, and ethanol were evaporated in an evaporator. The remaining residues were dissolved in methanol, and all extracts were kept at 4°C until analysis (Karkar & Şahin, 2022). The analysis data were statistically analysed using the MINI- TAB 17.0 (Minitab Inc., Stage College, PA) statistical prog- ram with Fit General Linear Model ANOVA (solvent, acidic, and basic hydrolysis extracts separately for each method (p < 0.01), and TPC, ABTS and FRAP methods separately for each extract (p < 0.01)). Analyses were performed in two repetitions. The antioxidant activity (AA) analysis The ABTS (Karkar & Şahin, 2022; Re et al., 1999) and FRAP (Benzie & Strain, 1996; Karkar & Şahin, 2022) methods de- termined the AA of amaranth. ABTS∗ radical solution used in the AA analysis by ABTS method was prepared by mixing 2.45 mM K2S2O8 with 7 mM ABTS solution. For the analysis, amaranth extract (0.1 mL), ethanol (3.9 mL), and ABTS• ra- dical solution (1 mL - 1:10 diluted with distilled water) were mixed, and the samples were kept for 6 min. The absorptions of the samples were measured with a UV-VIS Spectrophoto- meter at 734 nm, and the AA values of Amaranth were deter- mined as mg Trolox equivalent (TE)/100 g Amaranth. Extraction of Amaranth In order to determine the total phenolic content (TPC) and antioxidant activity (AA) of amaranth, ultrasonic extractions (160 W, 50 Hz) were performed in eight different solvents (water, methanol, ethanol, acetone, ethyl acetate, n-butanol, petroleum ether, and hexane) environments in three different 105 W: water, M: methanol, E: ethanol, A: acetone, EA: ethyl acetate, B: butanol, PE: petroleum ether, H: hexane, SE: solvent extraction, AH: acidic hydroly- sis, BH: basic hydrolysis, TPC: total phenolic content, AA: antioxidant activity; a–n: Lowercase superscripts indicate significant differences in TPC and AA values of amaranth under different extraction conditions for each method (p < 0.01). Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article were statistically analysed using the MINITAB 17.0 (Minitab Inc., Stage College, PA) statistical program with One-Way ANOVA (L. acidophilus bacterial viability for each amaranth sample p < 0.01)). der the same anaerobic conditions, and a stock bacterial cul- ture was prepared. L. acidophilus stock bacterial culture was activated by incubating at 37°C for 24 h under anaerobic con- ditions in MRS-Broth containing 1% (w/v) milled, dried, and boiled amaranth. Only L. acidophilus was activated under the same incubation conditions as the control group. The viability of the L. acidophilus strain was determined using the pour plate technique. Serial dilutions of activated bacterial strains in physiological saline were prepared and planted on sterile plates, and their growth in MRS-Agar medium was exami- ned. Plates were placed in anaerogenic jars with AnaeroGen Gas Packs and left for 72 h under anaerobic incubation at 37°C. Colony forming units (cfu) per mL of probiotic bacte- ria (log cfu/mL) were determined by counting the colonies formed after incubation. The effect of amaranth on the incre- ase in bacterial viability was determined by comparing the viability of ground, roasted, and boiled amaranth-containing bacteria with the control group. The results (two repetitions) ∗mean±standard deviation (two replicates) The Effect of Amaranth on Probiotic Bacterial Viability The effect of amaranth on the viability of probiotic bacteria was investigated using the prebiotic effect of amaranth. The analysis used three forms of amaranth - milled, dried, and bo- iled - while Lactobacillus acidophilus species from the Lac- tobacillaceae family was used as probiotic bacteria. L. aci- dophilus (DSM 20079) bacterial strain was activated by in- cubation at 37°C for 24 h under anaerobic conditions in 5 mL of De Man, Rogosa, and Sharpe (MRS) medium (sterilised at 121°C for 15 minutes). After incubation, the bacterial culture was sequentially activated in MRS-Broth medium twice un- According to the AA of the FRAP method, 0.1 mL of ama- ranth extract and 2.9 mL of FRAP reagent (prepared by mixing pH 3.6 acetate buffer, 20 mM FeCl3 solution, and 10 mM TPTZ solution (by 40 mM HCl) in a ratio 10:1:1) were mixed. The samples were kept in a dark environment for 30 min, and absorbance measurements were performed at 593 nm using a UV-VIS spectrometer. The AA of amaranth was determined as mg trolox equivalent (TE)/ 100 g amaranth. 106 Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article Table 2. P-values of the effect of solvent medium and extraction method on TPC and AA of amaranth p-value TPC ABTS FRAP Solvent medium < 0.01 < 0.01 < 0.01 Extraction method < 0.01 < 0.01 < 0.01 Solvent medium x Extraction method < 0.01 < 0.01 < 0.01 TPC: total phenolic content, AA: antioxidant activity AA of amaranth was found in the highest basic hydrolysis extract of water (176.53 ±0.89 mg TE/100g amaranth) with the ABTS method and the lowest in the acidic hydrolysis extract of ethanol (7.45 ±0.14 mg TE/100g amaranth), while the highest in the ethanol extract (231.97 ±12.85 mg TE/100 g amaranth) with the FRAP method and the lowest in the aci- dic hydrolysis extract of water (15.84 ±0.12 mg TE/100g amaranth). According to Sarker et al. (2020), in a study con- ducted with different genotypes of amaranth, the AA range was found to be between 16.71 ±0.06 and 49.64 ±0.04 µg/g DW by the ABTS method. The effects of solvent media and extraction method separa- tely on TPC and AA values of amaranth at a 99% confidence level were investigated by ANOVA analysis (Table 3). When the TPC values of amaranth extracts were examined, it was found that the TPC of water extracts was the highest, while the TPC of petroleum ether extract was the lowest. As the solvent polarity decreased, the TPC of amaranth decreased. Compared to extraction methods, amaranth’s TPC is gene- rally highest in acidic hydrolysis and lowest in solvent extrac- tion (Table 3). While the TPC of the aqueous basic hydrolysis extract of amaranth was found to be 722.11 ±18.68 mg GAE/100g amaranth in the highest amount, it was determined as 34.70 ±0.40 mg GAE/100g amaranth in the lowest amount of ethyl acetate solvent extract (Table 1). According to Peiretti et al. (2017), the TPC of the methanolic extract of amaranth seeds was determined as 4.35 ±0.19 mg/g. Sandoval-Sicairos et al. (2020) determined the TPC of unp- rocessed amaranth seed as 23.3 ±1.2 mg/100g DW and the TPC of germinated amaranth seed as 27.3 ±1.8 mg/100g DW. In another study, the total phenolic content of amaranth flour after fermentation with water was 2.55 ±0.20 mg GAE/g (Yeşil & Levent, 2022). Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 According to Sarker, Oba, & Daramy (2020), in a study conducted with different genotypes of ama- ranth, the TPC range was between 78.22 ±0.35 and 228.66 ±0.42 µg/g DW. The most commonly used solvents in the extraction of phe- nolic compounds are water, ethanol, methanol, acetone, and their acidic/non-acidic water mixtures. Studies on this subject have reported that ethanol and methanol solvents are more effective than other solvents in extracting phenolic compo- unds. However, TPC and AA values of amaranth samples were generally determined to be higher in extracts obtained with water solvent than in methanol and ethanol solvents. Phenolic compounds do not show similar AA in hydrophilic and hydrophobic solvent environments. In the AA measure- ments, solvent type and polarity affect the hydrogen atom and electron transfer. Most of the phenolic compounds respon- sible for antioxidant properties are hydrophilic. Therefore, hydrogen atom bonding in polar solvents causes significant changes in the H-atom donor activities of phenolic compo- unds and affects the measured AA. Although there is a sol- vent effect in frequently used AA determination methods, the type and properties of the solvent affect each method diffe- rently. Therefore, each method gives different AA results even in the same solvent environment. When the effects were examined, the AA values of amaranth were found to be high in the water extract with the ABTS method, while they were increased in the ethanol extract with the FRAP method. ABTS and FRAP methods can be applied to hydrophilic and lipophilic phenolic compounds. However, while the ABTS method is in neutral conditions, the FRAP method is in acidic conditions (Boeing et al., 2014; Çelik, 2011; Karaaslan et al., 2018; Turkmen et al., 2006). When the AA results of amaranth are compared according to solvent mediums, the highest AA was observed in water sol- vent mediums in the ABTS method, while the lowest was in petroleum ether solvent mediums (Table 3). It was observed that the AA value increased with the ABTS method as the solvent polarity increased. In the FRAP method, while the highest AA was detected in the ethanol solvent medium, the lowest was observed in the methanol solvent medium (Table 3). It was observed that the AA value was higher in the me- dium polarity solvent with the FRAP method. Total Phenolic Content and Antioxidant Activity of Amaranth Total Phenolic Content and Antioxidant Activity of Amaranth The TPC and AA values of different amaranth extracts are given in Table 1. The effect of solvent medium, extraction method, and solvent medium x extraction method interaction on the TPC and AA results of amaranth extracts was statisti- cally significant at a 99% confidence level according to ANOVA analysis (Table 2). The TPC and AA values of ama- ranth were seen to vary at different solvent mediums and extraction methods. e 1. The TPC (mg GAE/100 g amaranth) and AA (mg TE/100 g amaranth) of amaranth Table 1. The TPC (mg GAE/100 g amaranth) and AA (mg TE/100 g amaranth) of amaranth Solvent TPC ABTS FRAP W SE 581.80 ±18.40c 148.01 ±1.37b 32.28 ±0.33ı,j AH 606.70 ±18.98b 97.92 ±1.36c 15.84 ±0.12j BH 722.11 ±18.68a 176.53 ±0.89a 60.70 ±2.34g,h,ı M SE 70.17 ±4.29h,ı 47.30 ±1.27e 37.08 ±2.17ı,j AH 134.90 ±5.37e 46.60 ±1.24e 40.43 ±1.14h,ı,j BH 88.25 ±2.88g 79.73 ±0.79d 138.89 ±3.59d,e E SE 113.25 ±5.04f 11.89 ±0.87m 231.97 ±12.85a AH 364.11 ±0.70d 7.45 ±0.14n 207.65 ±4.10a,b BH 66.62 ±0.92h,ı,j 34.52 ±0.79g 159.08 ±17.76c,d A SE 41.65 ±0.43l,m,n 19.00 ±1.11j 152.88 ±0.40c,d AH 59.10 ±3.66ı,j,k 25.36 ±0.60h 131.22 ±27.56d,e BH 40.31 ±0.42l,m,n 16.81 ±1.30j,k,l 130.38 ±42.72d,e EA SE 34.70 ±0.40n 14.51 ±0.90l,m 159.86 ±5.61c,d AH 52.54 ±1.27k,l 20.14 ±1.10ı,j 196.72 ±6.31b BH 47.49 ±2.88k,l,m,n 22.86 ±0.42h,ı 180.25 ±5.02b,c B SE 53.66 ±0.66j,k,l 24.58 ±1.57h 112.06 ±0.84e,f AH 77.79 ±1.67g,h 39.47 ±2.83f 35.98 ±0.86ı,j BH 60.01 ±2.63ı,j,k 35.48 ±3.71g 88.95 ±1.43f,g PE SE 36.71 ±3.30m,n 15.34 ±0.71k,l,m 160.68 ±8.65c,d AH 45.75 ±1.36k,l,m,n 15.15 ±0.91k,l,m 72.19 ±3.90g,h BH 42.38 ±2.33l,m,n 18.54 ±1.58j,k 151.59 ±17.19c,d H SE 48.81 ±1.21k,l,m 14.23 ±0.22l,m 149.83 ±9.47c,d AH 66.86 ±4.93h,I,j 17.92 ±0.11j,k,l 84.79 ±3.86f,g BH 47.03 ±4.82k,l,m,n 16.80 ±0.46j,k,l 148.91 ±7.56c,d ∗mean±standard deviation (two replicates) M: methanol, E: ethanol, A: acetone, EA: ethyl acetate, B: butanol, PE: petroleum ether, H: hexane, SE: solvent extraction, AH: acidi asic hydrolysis TPC: total phenolic content AA: antioxidant activity; a–n: Lowercase superscripts indicate significant differences in 107 Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article Table 3. The effects of solvent media and extraction method on TPC and AA of amaranth Solvent Medium N TPC (mg/100g) ABTS (mg/100g) FRAP (mg/100g) W 6 646.46a 140.57a 36.28e M 6 96.76c 57.88b 72.13d E 6 181.33b 17.95e,f 199.57a A 6 47.02e,f 20.39d 138.16c EA 6 44.91f 19.17d,e 178.94b B 6 63.82d 33.18c 79.00d PE 6 41.76f 16.21f 128.15c H 6 54.23e 16.23f 127.84c Extraction Method N TPC (mg/100g) ABTS (mg/100g) FRAP (mg/100g) SE 16 123.90c 36.81b 129.58a AH 16 176,44a 33.66c 98.11b BH 16 140,77b 50.13a 132.34a W: water, M: methanol, E: ethanol, A: acetone, EA: ethyl acetate, B: butanol, PE: petroleum ether, H: hexane, SE: solvent extraction, AH: acidic hydroly- sis, BH: basic hydrolysis, TPC: total phenolic content, AA: antioxidant activity; a–f: Lowercase superscripts indicate significant differences in TPC and AA values of amaranth under different solvent mediums and extraction methods for each method (p < 0.01). ble 3. The effects of solvent media and extraction method on TPC and AA of amaranth W: water, M: methanol, E: ethanol, A: acetone, EA: ethyl acetate, B: butanol, PE: petroleum ether, H: hexane, SE: solvent extraction, AH: acidic hydroly- sis, BH: basic hydrolysis, TPC: total phenolic content, AA: antioxidant activity; a–f: Lowercase superscripts indicate significant differences in TPC and AA values of amaranth under different solvent mediums and extraction methods for each method (p < 0.01). According to the results of the LSD (Least Significance Dif- ference) test performed to determine the difference between extracts in terms of TPC, ABTS, and FRAP values, it is seen that all samples are in different groups (p<0.01). The correla- tion of TPC, ABTS, and FRAP of amaranth obtained under different extraction conditions with various solvents was de- termined using the MINITAB 17.0 (Minitab Inc., Stage Col- lege, PA) statistical program with Basic statistical analysis. The inter-method correlation coefficient stated in the 99% confidence interval is given in Table 4. The structure of phe- nolic compounds and the application conditions of AA met- hods cause different antioxidant activities obtained from dif- ferent solvent environments. The TPC method is carried out in basic conditions, the ABTS method in neutral conditions, and the FRAP method in acidic conditions. The different app- lication conditions of the methods lead to differences in the antioxidant activity values of extracts prepared with solvents of different polarities. Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 This situation also affects the correla- tion between methods. A positive correlation was observed between TPC and ABTS, while a negative correlation was observed between FRAP/ABTS and FRAP/TPC. varies between 1.5 and 2.5. For the gastric medium, the di- gestion time and pH of SGF were chosen to be 120 min and 2.00 ±0.20, respectively. After gastric digestion, the samples were taken directly into the intestinal medium, and intestinal digestion was performed for 120 min. The TPC values of the samples taken from the gastric and intestinal mediums every 30 min were determined and examined against digestion time. The TPC of milled, dried, and boiled amaranth after in-vitro gastrointestinal digestion is given in Figure 1. The initial TPC of milled amaranth was 4.52 ±0.22 mg GAE/g sample, while the initial TPC of dried and boiled amaranth decreased (3.45 ±0.13 and 2.38 ±0.17 mg GAE/g sample, respectively). When amaranth was directly heat treated, a decrease in TPC value was observed compared to milled amaranth. Likewise, the TPC value of boiled amaranth in an aqueous medium decre- ased more than that of dried amaranth. Due to the complex structure of phenolic substances, some phenolic compounds are inactivated due to heat treatment, while others can be- come free. As a result, some phenolics in dried amaranth ap- pear to be inactivated after heat treatment. In boiled ama- ranth, while some of the phenolics were inactivated after heat treatment, it was accepted that some were extracted by pas- sing into the aqueous medium. Therefore, the initial TPC va- lue of untreated amaranth (milled) was higher than that of tre- ated (dried and boiled) amaranth samples. Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 When the anti- oxidant activities of amaranth were compared according to the extraction methods, it was determined that the basic hyd- rolysis results were the highest and the acidic hydrolysis re- sults were the lowest in both analysis methods (Table 3). The 108 The Effect of Amaranth on Probiotic Bacterial Viability of dried amaranth increased by 9.61% to 3.79 ±0.20 mg GAE/g sample, and the TPC of boiled amaranth was increa- sed by 13.93% to 2.71 ±0.07 mg GAE/g sample. The ama- ranth samples were passed directly into the intestinal environ- ment after gastric digestion, and an increase in TPC was ob- served in all amaranth samples. After intestinal digestion, the TPC of milled amaranth was increased by 114.62% to 9.69 ±0.48 mg GAE/g sample, while the TPC of dried and boiled amaranth was increased by 21.02% (4.18 ±0.35 mg GAE/g sample) and 27.96% (3.05 ±0.20 mg GAE/g sample). The Effect of Amaranth on Probiotic Bacterial Viability Amaranth (prebiotic) with high carbohydrate and dietary fibre content was used to increase the viability of L. acidop- hilus probiotic bacteria, which is naturally found in the hu- man microbiota and widely used in the dairy industry. The viability results of L. acidophilus probiotic bacteria are given in Table 5. The bacterial viability of L. acidophilus without amaranth as a control was 7.18 ±0.01 log cfu/mL. In order to determine the prebiotic effect of amaranth on L. acidophilus probiotic bacteria, amaranth was added while the bacteria were activated, milled, dried, and boiled. It was observed that the viability of probiotic bacteria activated with amaranth inc- reased. Compared to the control, a log increase of 0.68 units was seen in the viability of L. acidophilus bacteria activated with milled amaranth. Likewise, an increase of log 0.54 and log 0.05 units was detected in the bacterial viability of L. aci- dophilus activated with dried and boiled amaranth, respecti- vely. While milled amaranth increased bacterial viability by 9.47%, dried amaranth increased by 7.46%, and boiled ama- ranth increased by 0.60%. Based on the statistical analysis, it was observed that the viability of bacteria activated by milled and dried amaranth increased significantly compared to the control (initial) L. acidophilus probiotic bacteria. However, no significant increase in viability was observed in bacteria activated by boiled amaranth. Phenolic compounds in food materials are generally linked to chemical bonds by carbohydrates, proteins, and dietary fibres (de Araújo et al., 2021; Jakobek, 2015). Phenolic compounds become more resistant to in-vitro gastrointestinal conditions as they are present in foods in glycosylated form (de Araújo et al., 2021; Pavan et al., 2014). Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article In-vitro Gastrointestinal Analysis In in-vitro gastrointestinal analyses, digestion time and pH of the medium in simulated gastric fluid differ between researc- hers. Digestion in the simulated gastric medium is considered 90 min in some research, while 120 min in some research. It has been observed that the average pH range of gastric fluid After gastric digestion, the TPC of milled amaranth was inc- reased by 46.03% to 6.59 ±0.18 mg GAE/g sample, the TPC 109 Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article Table 4. Correlation of TPC, ABTS, and FRAP of amaranth TPC ABTS ABTS R2 0.853 p-value <0.01 FRAP R2 -0.426 -0.578 p-value <0.01 <0.01 TPC: total phenolic content Table 5. The effect of amaranth on the viability of L. acidophilus probiotic bacteri Table 5. The effect of amaranth on the viability of L. acidophilus probiotic bacteria Viability of bacteria∗ (log cfu/mL) Increase in vitality (%) Control 7.18 ±0.01c LA-MA 7.86 ±0.03a 9.47 LA-DA 7.72 ±0.09b 7.46 LA-BA 7.23 ±0.02c 0.60 ∗mean ± standard deviation (two replicate) Control: L. acidophilus, LA-MA: L. acidophilus-1% milled amaranth, LA-DA: L. acidophilus-1% dried Amaranth, LA-BA: L. acidophilus-1% boiled amaranth; a-c: Lowercase superscripts indicate differences in L. acidophilus bacterial viability for each amaranth sample. LA-BA 7.23 ±0.02 0.60 ∗mean ± standard deviation (two replicate) Control: L. acidophilus, LA-MA: L. acidophilus-1% milled amaranth, LA-DA: L. acidophilus-1% dried Amaranth, LA-BA: L. acidophilus-1% boiled amaranth; a-c: Lowercase superscripts indicate differences in L. acidophilus bacterial viability for each amaranth sample. Kockova et al. (2013) conducted a study to investigate the effect of various pseudocereals on the viability of Lactobacil- lus rhamnosus GG probiotic bacteria. The researchers incu- bated the bacteria with different grains and flours, including rye flour, rye grain, barley flour, whole barley flour, amaranth flour, amaranth grain, buckwheat flour, whole buckwheat flour, whole oat flour, and millet grain at a temperature of 37°C for 18 h. They then examined the increase in bacterial viability after fermentation with each of these substrates. The viability increased after fermentation with rye flour (log 2.37), rye grain (log 2.73), barley flour (log 1.99), whole bar- ley flour (log 2.06), amaranth flour (log 3.57), amaranth grain (log 2.60), buckwheat flour (log 2.31), whole buckwheat flour (log 2.33), whole oat flour (log 1.95), and millet grain (log 2.61) (Kocková et al., 2013). The Effect of Amaranth on Probiotic Bacterial Viability At the same time, as a result of chemical reactions with pH and enzyme changes during in- vitro gastrointestinal digestion, the TPC value and antioxi- dant properties of the food material may change (Dantas et al., 2019; de Araújo et al., 2021). The increase in TPC values of various forms of amaranth (milled, dried, and boiled) after in-vitro gastrointestinal digestion indicates that phenolic compounds were released more into the medium during di- gestion. At the same time, this increase shows that phenolics are hydrolysed into different forms, such as glycosides, in acidic and basic environments. Figure 1. Gastrointestinal digestion of milled, dried, and boiled amaranth Figure 1. Gastrointestinal digestion of milled, dried, and boiled amaranth 110 Disclosure: - Güçlü, K., Altun, M., Özyürek, M., Karademir, S.E., & Apak, R. (2006). Antioxidant capacity of fresh, sun- and sul- phited-dried Malatya apricot (Prunus armeniaca) assayed by CUPRAC, ABTS/TEAC and folin methods. International Journal of Food Science and Technology, 41, 76–85. https://doi.org/10.1111/j.1365-2621.2006.01347.x Food and Health 10(2), 104-114 (2024) • https://doi.org/10.3153/FH24010 Research Article Conclusion Pseudo-grains, which protect the health of in-vitro gastroin- testinal digestion, positively affect the vitality of probiotic bacteria, are a good source of nutrients, and are an alternative to natural grains. This study determined the TPC and AA va- lues of the extracts of amaranth, which is a pseudo-grain pre- pared in various solvent environments. In general, it was ob- served that TPC and AA values of amaranth increased with increasing solvent polarity. The change in TPC values of amaranth consumed today using different cooking techniques was investigated after in-vitro gastrointestinal digestion. The- refore, the increase in TPC values of milled, dried, and boiled amaranth after in-vitro gastrointestinal digestion was highest in milled amaranth and lowest in boiled amaranth. In addi- tion, these forms of amaranth on the viability of L. acidophi- lus probiotic bacteria, which positively affects in-vitro gast- rointestinal digestion, were investigated. As a result, it was observed that the prebiotic effect of boiled amaranth decrea- sed due to the decrease in nutritional values, and it did not affect the increase in the number of live bacteria. However, the viability of L. acidophilus probiotic bacteria activated with milled amaranth increased by 9.47%. In this case, it is predicted that consuming amaranth, which has a very high nutritional value and a prebiotic effect, will be more benefi- cial for human health without applying heat treatment. Different processing and cooking methods applied to ama- ranth grains affect the nutritional composition of amaranth. Studies were shown that the nutritional content of amaranth, such as protein, fat, carbohydrate, dietary fiber, magnesium, iron, calcium, and phenolic compounds, decreases by boiling and cooking with various methods (Ugural & Akyol, 2022). When bacterial viability increases are examined, boiled ama- ranth's effect on bacterial viability is quite low. Accordingly, boiling amaranth significantly reduces its prebiotic proper- ties. Considering the literature studies, boiling amaranth re- duces its nutritional content (especially carbohydrates and di- etary fibre) much more than drying cooking. 111 Compliance with Ethical Standards ölçümleri. İstanbul Üniversitesi, Fen Bilimleri Enstitüsü, (Doktora Tezi), 193 sayfa. Conflict of interests: The author(s) declares that for this article, they have no actual, potential, or perceived conflict of interest. Dantas, A.M., Mafaldo, I. ., Oliveira, P. . de L., Lima, M. dos S., Magnani, M., & Borges, G. da S. . (2019). Bioac- cessibility of phenolic compounds in native and exotic frozen pulps explored in Brazil using a digestion model coupled with a simulated intestinal barrier. Food Chemistry, 274, 202–214. https://doi.org/10.1016/j.foodchem.2018.08.099 Ethics committee approval: Authors declare that this study in- cludes no experiments with human or animal subjects. Ethics com- mittee approval is not required for this study. Data availability: Data will be made available on request. Data availability: Data will be made available on request. Funding: This work was not funding-supported. Funding: This work was not funding-supported. de Araújo, F.F., de Paulo Farias, D., Neri-Numa, I.A., Dias-Audibert, F.L., Delafiori, J., de Souza, F.G., … Pas- tore, G.M. (2021). Gastrointestinal bioaccessibility and bio- activity of phenolic compounds from araçá-boi fruit. 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Pseudocereals, (April). https://doi.org/10.5772/intechopen.103708 Ugural, A., & Akyol, A. (2022). Can pseudocereals modu- late microbiota by functioning as probiotics or prebiotics? Critical Reviews in Food Science and Nutrition, 62(7), 1725– 1739. Yeşil, S., & Levent, H. (2022). The influence of fermented buckwheat, quinoa and amaranth flour on gluten-free bread quality. LWT - Food Science and Technology, 160. https://doi.org/10.1016/j.lwt.2022.113301 Yeşil, S., & Levent, H. (2022). The influence of fermented buckwheat, quinoa and amaranth flour on gluten-free bread quality. LWT - Food Science and Technology, 160. https://doi.org/10.1016/j.lwt.2022.113301 https://doi.org/10.1080/10408398.2020.1846493 https://doi.org/10.1080/10408398.2020.1846493 https://doi.org/10.1080/10408398.2020.1846493 Upasana, & Yadav, L. (2022). Pseudocereals: A Novel Path Upasana, & Yadav, L. (2022). Pseudocereals: A Novel Path 114
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When less is more: shortening the Lpp protein leads to increased vancomycin resistance in Escherichia coli
Journal of antibiotics
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The Journal of Antibiotics (2023) 76:746–750 https://doi.org/10.1038/s41429-023-00658-3 BRIEF COMMUNICATION The Journal of Antibiotics (2023) 76:746–750 https://doi.org/10.1038/s41429-023-00658-3 BRIEF COMMUNICATION The Journal of Antibiotics (2023) 76:746–750 https://doi.org/10.1038/s41429-023-00658-3 BRIEF COMMUNICATION Abstract Vancomycin is a naturally occurring cell-wall-targeting glycopeptide antibiotic. Due to the low potency of this antibiotic against Gram-negative pathogens, such as Escherichia coli, there is a limited knowledge about interactions between vancomycin and this group of bacteria. Here, we show that an in-frame 63 bp deletion of the lpp gene caused a fourfold increase in vancomycin resistance in E. coli. The resulting protein, LppΔ21, is 21 amino acids shorter than the wild-type Lpp, a helical structural lipoprotein that controls the width of the periplasmic space through its length. The mutant remains susceptible to synergistic growth inhibition by combination of furazolidone and vancomycin; with furazolidone decreasing the vancomycin MIC by eightfold. These findings have clinical relevance, given that the vancomycin concentration required to select the lpp mutation is reachable during typical vancomycin oral administration for treating Clostridioides difficile infections. Combination therapy with furazolidone, however, is likely to prevent emergence and outgrowth of the lpp-mutated Gram-negative coliforms, avoiding exacerbation of the patient’s condition during the treatment. Introduction accumulated regarding the interaction between vancomycin and this bacterial group, whereas this is not the case for Gram- negative bacteria. The outer membrane of Gram-negative bacteria is highly impermeable to large molecules (≥600 Da) [4], which effectively restricts vancomycin (~1449 Da) from reaching its target in the periplasm, thwarting its antibacterial activity. Nonetheless, recent efforts to develop vancomycin- based therapies against Gram-negative pathogens, such as synergistic drug combinations [5] and outer-membrane per- meabilising vancomycin analogues [6–8] necessitate investi- gation into the potential mechanism of vancomycin resistance in Gram-negative bacteria. Vancomycin, a naturally occurring glycopeptide antibiotic, exerts an antibacterial effect by binding to the N-terminal D-Ala-D-Ala of the pentapeptide stem of the peptidoglycan precursors and the growing peptidoglycan. This inhibits the peptidoglycan synthesis and causes a synthesis/degradation imbalance, resulting in cellular lysis [1]. Vancomycin is conventionally used as the first line therapy to treat nosoco- mial infections caused by methicillin-resistant Staphylococcus aureus and other Gram-positive β-lactam-resistant bacteria by intravenous infusion [2] or to treat severe C. difficile infection by oral administration [3]. Given the clinical importance of vancomycin for treatment of Gram-positive infections, it is not surprising that a vast number of studies have been To understand the basis of E. coli resistance to vanco- mycin, we isolated mutants having an increased resistance to vancomycin as compared to the parental strain and used whole-genome shotgun sequencing and comparative genome analysis to identify underlying genetic mutations. * Vuong Van Hung Le van.le@bio.ku.dk * Jasna Rakonjac J.Rakonjac@massey.ac.nz * Vuong Van Hung Le van.le@bio.ku.dk * Jasna Rakonjac J.Rakonjac@massey.ac.nz When less is more: shortening the Lpp protein leads to increased vancomycin resistance in Escherichia coli Hannah Wykes1 ●Vuong Van Hung Le 1,2 ●Catrina Olivera1 ●Jasna Rakonjac 1 Received: 15 July 2023 / Revised: 1 September 2023 / Accepted: 11 September 2023 / Published online: 25 September 2023 © The Author(s) 2023. This article is published with open access 1 School of Natural Sciences, Massey University, Palmerston North, New Zealand 2 Section of Microbiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark Isolating the vancomycin-resistant mutants Vancomycin-resistant mutants of the E. coli parental strain BW25113 [9] were selected on CAMH agar containing van- comycin at 1024 μg ml−1. Briefly, 100 μl of each of twenty independent overnight cultures was added to 2.5 ml of molten 0.5% CAMH agar (at ~47 °C), vortexed, then poured onto the selective plates. Resistant mutant colonies were observed after 48 h incubation at 37 °C and sub-streaked on non-selective plates (without vancomycin) and incubated overnight at 37 °C. The colonies grown on this non-selective agar were used to make stock cultures for later analyses. Antimicrobial susceptibility assays Vancomycin (GoldBio) minimum inhibitory concentrations (MICs) of E. coli strains were determined using broth microdilution and agar dilution methods according to Clinical and Laboratory Standards Institute guidelines [10]. Searching for the LppΔ21 mutation in clinical isolates The amino-acid sequence encoding the LppΔ21 variant was searched against the NCBI non-redundant protein sequence database using the blastp webserver v2.14.0+ with default parameters [13]. Growth conditions 1 School of Natural Sciences, Massey University, Palmerston North, New Zealand 2 Section of Microbiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark E. coli growth media used in this study was Cation-Adjusted Mueller Hinton (CAMH from BD BBLTM) in liquid broth or solid agar plates made at 1% agar (Pure Science Ltd.). When less is more: shortening the Lpp protein leads to increased vancomycin resistance in Escherichiay 747 All strains were grown at 37 °C with shaking at 200 rpm for liquid cultures. (Massey University, Palmerston North, New Zealand). DNA libraries were prepared using the Illumina DNA Prep kit and sequenced on the Illumina MiSeq™2 × 250-base paired-end v2 platform. The sequencing data is available at the National Centre for Biotechnology Information (NCBI) GenBank under the BioProject PRJNA854676. The bioinformatic workflow to map sequencing reads to the BW25113 refer- ence genome (accession number CP009273.1) and call mutations was performed as previously described [12]. All strains were grown at 37 °C with shaking at 200 rpm for liquid cultures. (Massey University, Palmerston North, New Zealand). DNA libraries were prepared using the Illumina DNA Prep kit and sequenced on the Illumina MiSeq™2 × 250-base paired-end v2 platform. The sequencing data is available at the National Centre for Biotechnology Information (NCBI) GenBank under the BioProject PRJNA854676. The bioinformatic workflow to map sequencing reads to the BW25113 refer- ence genome (accession number CP009273.1) and call mutations was performed as previously described [12]. Modelling the structure of Lpp and LppΔ21 proteins The structure of the mature Lpp has been solved [14]. The LppΔ21 mutant was modelled in trimeric complexes using ColabFold v1.5.2 set at default parameters [15] and visua- lised using ChimeraX v1.5 [16]. Results and discussion To isolate vancomycin-resistant mutants, 20 independent overnight cultures of E. coli (strain BW25113) were each plated on a vancomycin selective plate at 1024 μg ml−1. Only one vancomycin-resistant mutant was observed and isolated from the totality of 20 plates. This mutant displayed a van- comycin MIC of 1024 μg ml−1, which was fourfold higher than that of the parental strain (256 μg ml−1) in liquid broth microdilution assays (Fig. 1a). Whole genome analysis revealed that the mutant had a 63 bp deletion mutation in the lpp gene (coordinates 1751783–1751845 in the reference genome BW25113) which led to an in-frame deletion of 21 amino acids in the Lpp protein (Fig. 1b). Fractional inhibitory concentration index (FICI) was calculated using the following equation: FICI ¼ MICFZ combination ð Þ MICFZ alone ð Þ þ MICVAN combination ð Þ MICVAN alone ð Þ Where MICFZ(combination) MICVAN(combination) are the MICs for furazolidone and vancomycin when used in combination and MICFZ(alone) MICVAN(alone) are the MICs for furazolidone and vancomycin when used alone. The lowest FICI values were used to determine interactions: FICI ≤0.5 indicates synergy, FICI > 4 indicates antagonism, and 0.5 < FICI ≤4 indicates additivity [11]. Lpp is a highly abundant lipoprotein that folds into a tri- meric coiled coil structure (Fig. 1c) [14]. It is present in two forms, transmembrane (free) and periplasmic (bound) [17]. The transmembrane form is exposed on the bacterial surface and has an unknown function, while the periplasmic form anchors its lipidated N-terminus in the periplasm-facing phospholipid leaflet of the outer membrane and covalently links the C-terminal Lys residue to the peptidoglycan (Fig. 1c). This provides the only covalent link between the outer membrane and the peptidoglycan, controlling the width and therefore the overall volume (size) of the periplasmic space [18] and contributing towards the structural integrity and function of the cell envelope [19]. This has been shown Growth inhibition checkerboard assays Growth inhibition checkerboard assays were done to examine the interaction between furazolidone and vancomycin in CAMH broth in a 384-well microplate format as previously described with some modifications [5]. Briefly, twofold serial dilutions of furazolidone and vancomycin were used. Each well contained 5 × 105 cfu ml−1, 1% DMSO, and antibiotics in a final volume of 50 μl. The microplates were incubated at 37 °C and the OD600 measured after 18 h using a MultiskanTM GO Microplate Spectrophotometer. Each treatment was per- formed in triplicate. Comparative genome analysis Genomic DNA was extracted from overnight cultures using the DNeasy UltraClean Microbial Kit (Qiagen) and submitted for whole genome sequencing at Massey Genome Service H. Wykes et al. 748 Fig. 1 Mutation in lpp confers resistance to vancomycin. a MIC of the lppΔ21 mutant and parental strain. MIC experiments were conducted using three replicates and yielded identical results. b The Lpp protein sequence; the 21 deleted residues in the lppΔ21 mutant are highlighted in red. The N-terminal cysteine (orange) of the mature Lpp protein is lipidated and anchored to the inner leaflet of the outer membrane. The C-terminal lysine (purple) makes a covalent bond to the peptidoglycan. c Schematic illustration of a Gram-negative envelope in which the Lpp protein connects the outer membrane to peptidoglycan, dictating the periplasmic space. The trimeric complex of the wild-type Lpp and the LppΔ21 mutant was modelled using ColabFold. The 21 residues deleted from the wild-type Lpp to make the LppΔ21 mutant are coloured in red. WT wild-type Fig. 1 Mutation in lpp confers resistance to vancomycin. a MIC of the lppΔ21 mutant and parental strain. MIC experiments were conducted using three replicates and yielded identical results. b The Lpp protein sequence; the 21 deleted residues in the lppΔ21 mutant are highlighted in red. The N-terminal cysteine (orange) of the mature Lpp protein is lipidated and anchored to the inner leaflet of the outer membrane. The C-terminal lysine (purple) makes a covalent bond to the peptidoglycan. c Schematic illustration of a Gram-negative envelope in which the Lpp protein connects the outer membrane to peptidoglycan, dictating the periplasmic space. The trimeric complex of the wild-type Lpp and the LppΔ21 mutant was modelled using ColabFold. The 21 residues deleted from the wild-type Lpp to make the LppΔ21 mutant are coloured in red. WT wild-type through reported properties in E. coli expressing an in-frame lpp insertion mutant containing an additional 21 amino acids (Lpp+21 variant), that was found to have a 3–4 nm longer inter- to outer-membrane distance [18, 20]. Interestingly, a combination of electron cryotomography and molecular dynamics simulations of the cells expressing this mutant showed that the Lpp+21 is tilted rather than perpendicular to the membrane surface, minimising the increase in the thick- ness of the periplasmic space [20]. Comparative genome analysis Furthermore, non- essential genes involved in binding the outer membrane to the peptidoglycan (at a set distance) become essential in this lpp mutant, presumably by minimising the increase in thickness of the periplasmic space due to a longer Lpp. These findings demonstrate that increasing the thickness of the periplasm above a limit of about 3–4 nm is lethal to E. coli. In addition, it has been shown that increasing the length of Lpp softened the cell-envelope (decreased stiffness) and rendered E. coli more susceptible to vancomycin [18]. By contrast, the Δlpp mutant (i.e. total deletion of the lpp coding sequence) and the lppΔK58 mutant (i.e. deletion of the C-terminal lysine responsible for the peptidoglycan ancho- rage) were found to have little effect on the vancomycin sensitivity as compared to the wildtype [18]. In our study, we showed a reciprocal effect of the shorter Lpp length on van- comycin susceptibility, where decreasing the Lpp protein length by 21 residues caused a fourfold increase in vanco- mycin resistance. We speculate that the lppΔ21 mutation may decrease the distance between the outer membrane and the peptidoglycan, decreasing the periplasmic space and thereby increasing the mechanical strength of the cell envelope, resulting in a higher peptidoglycan impairment threshold, and consequently a higher vancomycin MIC required to induce cell death (Fig. 1c). The decreased periplasmic space may also limit the access of vancomycin to its peptidoglycan target by further restricting the already very limited ability of vancomycin to accumulate in the periplasmic space. Alternatively, cells that possess the lppΔ21 variant may have an unusual envelope archi- tecture and, to release envelope stress, produce outer membrane vesicles that can act as a decoy to sequester vancomycin from bacterial cells. Future work looking into the underlying molecular mechanism is warranted. through reported properties in E. coli expressing an in-frame lpp insertion mutant containing an additional 21 amino acids (Lpp+21 variant), that was found to have a 3–4 nm longer inter- to outer-membrane distance [18, 20]. Interestingly, a combination of electron cryotomography and molecular dynamics simulations of the cells expressing this mutant showed that the Lpp+21 is tilted rather than perpendicular to the membrane surface, minimising the increase in the thick- ness of the periplasmic space [20]. Comparative genome analysis The isobologram curves were generated from the growth inhibition checkerboard assay for the parental strain and vancomycin mutant, performed as described in the “Materials and Methods” section. Each data point indicates the MIC of the drugs, either alone or in combination. Each treatment was per- formed in triplicate; each replicate yielded identical MICs for indivi- dual antibacterials and the combinations Fig. 2 Properties of the lppΔ21 mutant. a Bacterial density in over- night cultures is unchanged in the lppΔ21 mutant as compared to the parent strain. The optical density at 600 nm of the culture after 18 h incubation at 37 °C. Twelve replicates of cultures were used. n.s., non- significant, Student’s t test (p = 0.13, n = 12). b Synergistic interaction between vancomycin and furazolidone in the lppΔ21 mutant (top graph) and the parent strain (bottom graph). The isobologram curves were generated from the growth inhibition checkerboard assay for the parental strain and vancomycin mutant, performed as described in the “Materials and Methods” section. Each data point indicates the MIC of the drugs, either alone or in combination. Each treatment was per- formed in triplicate; each replicate yielded identical MICs for indivi- dual antibacterials and the combinations were generated from the growth inhibition checkerboard assay for the parental strain and vancomycin mutant, performed as described in the “Materials and Methods” section. Each data point indicates the MIC of the drugs, either alone or in combination. Each treatment was per- formed in triplicate; each replicate yielded identical MICs for indivi- dual antibacterials and the combinations Fig. 2 Properties of the lppΔ21 mutant. a Bacterial density in over- night cultures is unchanged in the lppΔ21 mutant as compared to the parent strain. The optical density at 600 nm of the culture after 18 h incubation at 37 °C. Twelve replicates of cultures were used. n.s., non- significant, Student’s t test (p = 0.13, n = 12). b Synergistic interaction between vancomycin and furazolidone in the lppΔ21 mutant (top graph) and the parent strain (bottom graph). The isobologram curves for severe C. difficile infection may select for vancomycin resistance in gut resident Gram-negative bacteria, includ- ing the reported lppΔ21 Shiga-toxin-producing E. coli isolate. Comparative genome analysis Furthermore, non- essential genes involved in binding the outer membrane to the peptidoglycan (at a set distance) become essential in this lpp mutant, presumably by minimising the increase in thickness of the periplasmic space due to a longer Lpp. These findings demonstrate that increasing the thickness of the periplasm above a limit of about 3–4 nm is lethal to E. coli. through reported properties in E. coli expressing an in-frame lpp insertion mutant containing an additional 21 amino acids (Lpp+21 variant), that was found to have a 3–4 nm longer inter- to outer-membrane distance [18, 20]. Interestingly, a combination of electron cryotomography and molecular dynamics simulations of the cells expressing this mutant showed that the Lpp+21 is tilted rather than perpendicular to the membrane surface, minimising the increase in the thick- ness of the periplasmic space [20]. Furthermore, non- essential genes involved in binding the outer membrane to the peptidoglycan (at a set distance) become essential in this lpp mutant, presumably by minimising the increase in thickness of the periplasmic space due to a longer Lpp. These findings demonstrate that increasing the thickness of the periplasm above a limit of about 3–4 nm is lethal to E. coli. In addition, it has been shown that increasing the length of Lpp softened the cell-envelope (decreased stiffness) and rendered E. coli more susceptible to vancomycin [18]. By contrast, the Δlpp mutant (i.e. total deletion of the lpp coding sequence) and the lppΔK58 mutant (i.e. deletion of the C-terminal lysine responsible for the peptidoglycan ancho- rage) were found to have little effect on the vancomycin We next asked if the laboratory-selected lppΔ21 muta- tion is clinically relevant. First, there was no significant When less is more: shortening the Lpp protein leads to increased vancomycin resistance in Escherichiay 749 Fig. 2 Properties of the lppΔ21 mutant. a Bacterial density in over- night cultures is unchanged in the lppΔ21 mutant as compared to the parent strain. The optical density at 600 nm of the culture after 18 h incubation at 37 °C. Twelve replicates of cultures were used. n.s., non- significant, Student’s t test (p = 0.13, n = 12). b Synergistic interaction between vancomycin and furazolidone in the lppΔ21 mutant (top graph) and the parent strain (bottom graph). Comparative genome analysis Vancomycin selection for such a severely patho- genic strain that is shed into the clinical setting and the environment is clearly not desirable in the long term, as it will enrich the environment for this severely pathogenic strain thriving in the gut of vancomycin-treated patients, where nearly all bacteria are wiped out. difference between the OD600 of the mutant and parental strains after 18 h of incubation in CAMH broth at 37 °C (Fig. 2a), indicating that the lpp mutation does not sig- nificantly impact the E. coli growth. In other words, even in the absence of vancomycin selection, the lppΔ21 variant is unlikely to be outcompeted by the parental strain. Second, searching the LppΔ21 amino-acid sequence against the NCBI protein database retrieved one hit belonging to a pathogenic Shiga-toxin producing E. coli strain isolated from the USA, designated PNUSAE059255 (Accession number AAVXHG010000001.1). Finding of such a mutant in the database indicates that the vancomycin resistance conferring LppΔ21 variant can be readily selected upon vancomycin exposure. Third, oral vancomycin administra- tion at the dose of 125 or 250–500 mg four times per day is recommended to treat severe or very severe C. difficile infections, respectively [3]. With the former dosing, the vancomycin concentrations in the faecal samples, as a proxy for colonic levels, have been reported to reach the range of 50–2000 μg ml−1. Furthermore, with a higher dosing regime (500 mg), the faecal vancomycin con- centration was consistently higher than 2000 μg ml−1 [3]. Opposite to the conventional thought that vancomycin alone does not affect Gram-negative bacteria, it is plau- sible to argue that the current oral vancomycin treatment difference between the OD600 of the mutant and parental strains after 18 h of incubation in CAMH broth at 37 °C (Fig. 2a), indicating that the lpp mutation does not sig- nificantly impact the E. coli growth. In other words, even in the absence of vancomycin selection, the lppΔ21 variant is unlikely to be outcompeted by the parental strain. Second, searching the LppΔ21 amino-acid sequence against the NCBI protein database retrieved one hit belonging to a pathogenic Shiga-toxin producing E. coli strain isolated from the USA, designated PNUSAE059255 (Accession number AAVXHG010000001.1). Finding of such a mutant in the database indicates that the vancomycin resistance conferring LppΔ21 variant can be readily selected upon vancomycin exposure. Comparative genome analysis Third, oral vancomycin administra- tion at the dose of 125 or 250–500 mg four times per day is recommended to treat severe or very severe C. difficile infections, respectively [3]. With the former dosing, the vancomycin concentrations in the faecal samples, as a proxy for colonic levels, have been reported to reach the range of 50–2000 μg ml−1. Furthermore, with a higher dosing regime (500 mg), the faecal vancomycin con- centration was consistently higher than 2000 μg ml−1 [3]. Opposite to the conventional thought that vancomycin alone does not affect Gram-negative bacteria, it is plau- sible to argue that the current oral vancomycin treatment The lppΔ21 mutant isolated in this work was further tested for the reported susceptibility to the synergistic effect between vancomycin and furazolidone, a synthetic nitrofuran anti- biotic [5]. The synergy was maintained, shown by an unal- tered FICI of 0.25 compared to the parental strain (Fig. 2b). This FICI implies vancomycin susceptibility in the lppΔ21 mutant, with the MIC for vancomycin dropping from 1024 to 128 μg ml−1 in the presence of 0.25 μg ml−1 furazolidone or to 64 μg ml−1 in the presence of 0.5 μg ml−1 furazolidone (Fig. 2b). This shows that a combined furazolidone- vancomycin treatment regime potentially prevents selection for and/or eradicates the lppΔ21 mutant which is otherwise impossible with vancomycin monotherapy. This finding suggests that a furazolidone-vancomycin combination treat- ment for C. difficile infection could be a potential option to prevent emergence of vancomycin resistance in E. coli within 750 H. Wykes et al. the patients’ gut. Since the Lpp homologue can be found in other Gram-negative bacteria and the synergy between furazolidone-vancomycin has been demonstrated previously [5], we speculate that the effect of this combinatorial regime on vancomycin resistance suppression can be generalised to other Gram-negatives, not just limited to E. coli, in the gut environment. the patients’ gut. Since the Lpp homologue can be found in other Gram-negative bacteria and the synergy between furazolidone-vancomycin has been demonstrated previously 2. Álvarez R, Cortés LEL, Molina J, Cisneros JM, Pachón J. Optimizing the clinical use of vancomycin. Antimicrob Agents Chemother. 2016;60:2601–9. 3. Gonzales M, Pepin J, Frost EH, Carrier JC, Sirard S, Fortier L-C, et al. Faecal pharmacokinetics of orally administered vancomycin in patients with suspected Clostridium difficile infection. BMC Infect Dis. 2010;10:363. 4. Delcour AH. Outer membrane permeability and antibiotic resis- tance. Biochim et Biophys Acta (BBA) - Proteins Proteom. Comparative genome analysis 2009; 1794:808–16. In conclusion, we report here a 63-nucleotide in-frame deletion mutation in the lpp gene, conferring resistance to vancomycin in E. coli. The concentration of vancomycin we used to select the lppΔ21 mutant is reachable during oral vancomycin treatment for C. difficile infection. Furthermore, the lppΔ21 mutation was also found in a clinical Shiga-toxin producing E. coli isolate, confirming the clinical relevance of the mutation we have isolated. Overall, for those efforts to develop oral vancomycin-based therapies [6–8], we suggest inclusion of the E. coli lppΔ21 mutant to the drug testing panel to evaluate the potential of the developed therapies to escape the known vancomycin resistance in Gram-negative pathogens. 5. Olivera C, Le VVH, Davenport C, Rakonjac J. In vitro synergy of 5-nitrofurans, vancomycin and sodium deoxycholate against Gram-negative pathogens. J Med Microbiol. 2021;70:001304. 6. Yarlagadda V, Manjunath GB, Sarkar P, Akkapeddi P, Para- manandham K, Shome BR, et al. Glycopeptide antibiotic to overcome the intrinsic resistance of Gram-negative bacteria. ACS Infect Dis. 2016;2:132–9. 7. van Groesen E, Slingerland CJ, Innocenti P, Mihajlovic M, Masereeuw R, Martin NI. Vancomyxins: Vancomycin-polymyxin nonapeptide conjugates that retain anti-Gram-positive activity with enhanced potency against Gram-negative strains. ACS Infect Dis. 2021;7:2746–54. 8. Antonoplis A, Zang X, Wegner T, Wender PA, Cegelski L. Vancomycin–arginine conjugate inhibits growth of carbapenem- resistant E. coli and targets cell-wall synthesis. ACS Chem Biol. 2019;14:2065–70. Acknowledgements This work was supported by a Massey University- MBIE PSAF II grant MU001985 and a generous donation by Anne and Bryce Carmine as well as the Massey University School of Natural Sciences. HW was supported by the Graduate Women Manawatū Charitable Trust and the William Georgetti Scholarship. VVHL at his current affiliation to the University of Copenhagen was supported by a Villum Experiment Grant. 9. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. Construction of Escherichia coli K‐12 in‐frame, single‐ gene knockout mutants: the Keio collection. Mol Syst Biol. 2006;2:2006.0008. 10. Cockerill FR, Wikler MA, Alder J, Dudley MN, Eliopoulos GM, Ferraro MJ, et al. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard—Ninth Edition. Clinical and Laboratory Standards Institute; 2012 11. Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother. 2003;52:1. Funding Open Access funding enabled and organized by CAUL and its Member Institutions. 12. Le VVH, Davies IG, Moon CD, Wheeler D, Biggs PJ, Rakonjac J. Comparative genome analysis Novel 5-nitrofuran-activating reductase in Escherichia coli. Antimicrob Agents Chemother. 2019;63:e00868–19. Conflict of interest The authors declare no competing interests. Conflict of interest The authors declare no competing interests. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 14. Shu W, Liu J, Ji H, Lu M. Core structure of the outer membrane lipoprotein from Escherichia coli at 1.9 å resolution. J Mol Biol. 2000;299:1101–12. 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/. 15. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nat Methods. 2022;19:679–82. 16. Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 2021;30:70–82. 17. Cowles CE, Li Y, Semmelhack MF, Cristea IM, Silhavy TJ. The free and bound forms of Lpp occupy distinct subcellular locations in Escherichia coli. Mol Microbiol. 2011;79:1168–81. 18. Mathelié-Guinlet M, Asmar AT, Collet J-F, Dufrêne YF. Lipo- protein Lpp regulates the mechanical properties of the E. coli cell envelope. Nat Commun. 2020;11:1–11. 19. Asmar AT, Ferreira JL, Cohen EJ, Cho S-H, Beeby M, Hughes KT, et al. Communication across the bacterial cell envelope depends on the size of the periplasm. PLoS Biol. 2017;15:e2004303. 1. Kahne D, Leimkuhler C, Lu W, Walsh C. Glycopeptide and lipoglycopeptide antibiotics. Chem Rev. 2005;105:425–48. Compliance with ethical standards 13. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new gen- eration of protein database search programs. Nucleic Acids Res. 1997;25:3389–402. References 20. Mandela E, Stubenrauch CJ, Ryoo D, Hwang H, Cohen EJ, Torres VL, et al. Adaptation of the periplasm to maintain spatial con- straints essential for cell envelope processes and cell viability. eLife 2022;11:e73516. 1. Kahne D, Leimkuhler C, Lu W, Walsh C. Glycopeptide and lipoglycopeptide antibiotics. Chem Rev. 2005;105:425–48.
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Strategi Pemasaran Untuk Meningkatkan Jumlah Nasabah Pada Produk Pembiayaan KPR di Bank BTN KC Jember
Journal of Indonesian Social Society
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Strategi Pemasaran Untuk Meningkatkan Jumlah Nasabah Pada Produk Pembiayaan KPR di Bank BTN KC Jember Adeliya Gita Silviana Putri1*, Rendy Andika Putra2, M.F.Hidayatullah3 1,2,3 Perbankan Syariah, Universitas Kiai Haji Achmad Siddiq Jember 1Adeliyagita25@email.com, 2Andikaputrarendy77@email.com, 3mf.hidayatullah@uinkhas.ac.id 1,2,3 Perbankan Syariah, Universitas Kiai Haji Achmad Siddiq Jember 1Adeliyagita25@email.com, 2Andikaputrarendy77@email.com, 3mf.hidayatullah@uinkhas.ac.id Journal Of Indonesian Social Society Volume 2 ; Nomor 1 ; Februari 2024 ; Page 17-21 Doi : https://doi.org/10.59435/jiss.v2i1.217 Web : https://jurnal.padangtekno.com/index.php/jiss Journal Of Indonesian Social Society Volume 2 ; Nomor 1 ; Februari 2024 ; Page 17-21 Doi : https://doi.org/10.59435/jiss.v2i1.217 Web : https://jurnal.padangtekno.com/index.php/jiss E-ISSN : 2985-5705 P-ISSN : 2985-8771 Abstrak Article History: Received Jan 11th, 2024 Revised Jan 15th, 2024 Accepted Jan 30th, 2024 Dunia perbankan, mengalami perkembangan pesat, oleh karena itu persaingan produk- produk pada perbankan tida dapat dihindarkan dan semakin ketat. Salah satunya yaitu pada produk Kredit Pemilikan Rumah (KPR). Perbankan harus memiliki strategi pemasaran efektif dan efesien, sehingga mendukung kegiatan operasional perusahaan untuk menguasai segmen pasar, serta pula memiliki daya tarik untuk memasarkan produk KPR agar dapat menarik nasabah atau konsumen serta bersaing dengan perbankann lainnya. Penelitian ini bertujuan untuk mengetahui penerapan taktik dan strategi pemasaran Bank Tabungan Negara Kantor Cabang Jember dalam meninngkatkan jumlah nasabah pada produk pembiayaan KPR. Metodologi yang digunakan dalam penelitian ini menggunakan pendekatan kualitatif yaitu dengan melakukan pengumpulan data dengan metode observasi, wawancara dengan karyawan bagian marketing Bank Tabungan Negara Kantor Cabang Jember yang bertanggungjawab atas pemasaran produk KPR sehingga mendapatkan data yang akurat pada proses penelitian yang diteliti.Melalui wawancara dan observasi dapat diketahui bahwa strategi pemasaran yang diterapkan oleh Bank BTN KC Jember dalam memasarkan produk Kredit Pemilikan Rumah (KPR) adalah dengan menggunakan segmenting, targeting,positioning (STP) dan marketing mix 4P (product,Price,Promotion,Place). Kata Kunci : Strategi, Marketing, KPR, STP, Marketing Mix Kata Kunci : Strategi, Marketing, KPR, STP, Marketing Mix CC Attribution-ShareAlike 4.0 License. Abstract The banking world, experienced a very rapid development. That way, competition between banking products is inevitable and increasingly fierce. One of them is the Home Ownership Credit (KPR) product. Banks must have an effective and efficient marketing strategy, so as to support the company's operational activities to dominate market segments, and also have the attractiveness to market mortgage products in order to attract consumers and compete with other banks. This study aims to determine the application of tactics and marketing strategies of Bank Tabungan Negara Cilegon Branch Office in increasing the number of customers in mortgage financing products. The methodology used in this study uses a qualitative approach, namely by conducting literature research, which is a data collection technique using written sources. Field research, namely techniques with data collection by observation methods, interviews with employees of the Marketing Department of Bank Tabungan Negara Cilegon Branch Office who are responsible for marketing mortgage products so as to obtain accurate data in the research process studied. Through interviews and observations, it can be seen that the marketing strategy carried out by Bank Tabungan Negara Cilegon Branch Office in marketing Home Ownership Credit (KPR) products is to use Segmenting, Targeting, Positioning (STP) and also Marketing Mix 3P (Product, Price, Promotion). Keyword : Strategy,marketing,KPR,STP,Marketing Mix Keyword : Strategy,marketing,KPR,STP,Marketing Mix Adeliya Gita Silviana Putri | Page 17 PENDAHULUAN Masih ada masalah signifikan dalam memenuhi permintaan perumahan swasta di Indonesia. Selain kebutuhan hidup seperti sandang, pangan, pendidikan, dan kesehatan,perumahan juga penting bagi kesejahteraan penghuninya. Pertumbuhan populasi dan ruang bangunan yang langka mempersulit upaya untuk memecahkan masalah ini. Kredit Kepemilikan Rumah (KPR) merupakan salah satu produk layanan peerumahan yang dikembangkan oleh industry perbankan untuk menjawab kebutuhan tersebut. Bank BTN merupakan salah satu lembaga keuangan terkemuka di Indonesia. PT. Bank Tabungan Negara (Persero) Tbk adalah Badan Usaha Milik Negara (BUMN) yang bergerak di bidang perbankan. BTN berkomitmen menjadi bank yang melayani dan mendukung pembiayaan sektor perumahan melalui tiga produk utama, perbankan perseorangan, bisnis, dan syariah. Strategi pemasaran adalah rencana yang menggambarkan harapan perusahaan atas dampak kegiatan pemasaran yang berbeda terhadap permintaan produk di pasar sasaran yang berbeda.Perlu adanya strategi pemasaran untuk masing- masing bank karena adanya persaingan diantara mereka dan bergamnya produk yang ditawarkan oleh masing-masing bank sesuai dengan kebutuhan masyarakat. Tujuan studi ini adalah untuk mempelajari seberapa sukses strategi dan pendekatan pemasaran bank dalam menarik nasabah yang tertarik dengan opsi pembiayaan Kredit Pemilikan Rumah Rumah atau hipoteknya. Metode yang digunakan adalah metode kualitatif, dikumpulkan dengan infromasi baik dari sumber primer ataupun skunder. METODE Penelitian ini menggunakan pendekatan kualitatif.Penelitian kualitatif Penelitian kualitatif bertujuan untuk lebih memahami individu dengan mempertimbangkan pengalaman dan perspektif orang lain(Salim, 2012) . Metode penelitian kualitatif digunakan untuk melakukan evaluasi menyeluruh terhadap lingkungan objek penelitian (Abbdussamad, 2021) .Penelitian kualitatif deskriptif dilakukan dengan cara mengumpulkan data dalam bentuk naratif daripada data numerik, digunakan untuk memecahkan masalah dalam penelitian ini. Data lapangan disusun, diklasifikasikan, dianalisis, dan dirangkum untuk menggambarkan strategi pemasaran produk KPR di Bank BTN Kantor Cabang Jember. Unit analisis penelitian ini adalah karyawan pada devisi marketing dan yang menangani produk pembiayaan KPR pada PT. Bank Tabungan Negara Kantor Cabang Jember. Dalam penelitian ini pengumpulan data diperoleh dengan teknik bservasi, wawancara,dan dokumentasi. Adeliya Gita Silviana Putri | Page 17 CC Attribution-ShareAlike 4.0 License. Journal Of Indonesian Social Society Volume 2 ; Nomor 1 ; Februari 2024 ; Page 17-21 Doi : https://doi.org/10.59435/jiss.v2i1.217 Web : https://jurnal.padangtekno.com/index.php/jiss E-ISSN : 2985-5705 P-ISSN : 2985-8771 Adeliya Gita Silviana Putri | Page 18 Penerapan Segmenting, Targeting dan Positioning KPR yang dilakukan oleh PT. Bank Tab (Persero) Kantor Cabang Jember Penerapan Segmenting, Targeting dan Positioning KPR yang dilakukan oleh PT. Bank Tabungan Negara (Persero) Kantor Cabang Jember Secara teoritis, ini adalag segmentasi,penargetan dan pemosisian. Untuk memposisikan penawaran uniknya, perusahaan pertama-tama mengidentifikasi sejumlah kebutuhan dan kelompok pasar, membidik hal-hal yang dapat dipenuhi dengan cara yang luar biasa, dan kemudian mengejar kelompok tersebut. Segmentasi, Targeting, dan positioning produk pembiayaan KPR di Bank BTN KC Jember. p y 1. Segmenting Segmentasi pasar PT Bank Tabungan Negara KC Jember secara umumnya yaitu pada nasabah atau konsumen yang ingin memiliki tempat tinggal yang layak. Pasar pada Bank BTN tersegmentasi secara unik di Kantor Cabang Jember dengan beberapa kriteria seperti: a. Segmentasi Geografis Segmentasi pasar PT Bank Tabungan Negara (persero) lebih memfokuskan pada wilayah Jember, Lumajang,Situbondo, dan Bondowoso dan tidak menutup memungkinkan dari wilayah lain untuk yang ingin mengambil pembiayaan produk KPR Subsidi atau KPR Non Subsidi. b. Segmentasi Demografis Ditujukan kepada seluruh kalangan masyarakat baik perorangan serta lembaga yang sebagian penghasilannya sudah mulai di alokasikan untuk berinnvestasi. KPR BTN didominasi oleh masyarakat dengan kisaran usia 21-40 tahun. Batas maksimal umur diterapkan oleh bank adalah 65 tahun saat cicilan KPR lunas, jika melewati batas tersebut kemungkinan besar akan menolak. c. Segmentasi Psikografis Dalam mendapatkan hasil yang terbaik juga perusahaan melakukan metode pendekatan yang berbeda-beda tergantung kepada calon konsumennya dengan karakter konsumen. d. Segmentasi Perilaku Segmen yang dituju adalah msyarakat yang gemar menabung, berinvestasi dan memiliki pengetahuan mengenai keunggulan pembiayan Produk KPR BTN. Targeting Dakam penarapan hal memenuhi karakteristik target pasar ini juga mengingat Bank BTN memiliki syarat-syarat yang harus dipenuhi oleh calon nasabah BTN, diantaranya: a geting Dakam penarapan hal memenuhi karakteristik target pasar ini juga mengingat Bank BTN memiliki syarat-syarat yang harus dipenuhi oleh calon nasabah BTN, diantaranya: a. Persyaratan pemohon WNI minimal usia 21 tahun atau sudah menikah dengan maks umur 65 tahun pada saat jatuh tempo kredit, Maksimal penghasilan tidak kawin RP 6.000.000 (khusus Papua& Papua Barat Rp 7.500.000),Maksimal penghasilan kawin Rp 8.000.000(khusus Papua&Papua Barat Rp 10.000.000),Pemohon dan pasangannya belum memiliki rumah, belum pernah menerima subsidi perumahan dari pemerintah,memiliki NPWP dan SPT PPh orang pribadi, NIK terdaftar di dukcapil. b. Persyaratan Dokumen KTP,KK NPWP,Buku atau akta nikah bagi yang telah menikah atau surat/akta cerai bagi yang telah bercerai,slip gaji 3 bulan terakhir, pas foto pemohon,SK pegawai tetap. P iti i g Positioning memungkinkan perusahaan untuk menciptakan ekuitas merk suatu identitas produk atau layanan.. HASIL DAN PEMBAHASAN Lembaga keuangan yang fokus mencari keuntungan perlu melalukan kegiatan pemasaran.Setiap perusahaan berupaya untuk mempertahankan dan meningkatkan tingkat keuntungan karena hal tersebut sangat penting bagi kelangsungan dan perluasan suatu perusahaan.Opsi ini memungkinkan bisnis untuk mempertahankan dan meningkatkan pendapatan melalui layanan pelanggan yang cermat dan dominasi industry. Strategi pemasaran yang canggih dapat mempertahankan posisi perusahaan di pasar ,karena strategi pemasaran adalah kunci kesuksesan bisnis. Dari hasil wawancara serta observasi yang telah dilakukan oleh penulis menunjukkan bahwa produk pembiayaan KPR di bank BTN Kantor Cabang Jember menggunakan startegi pemasaran, targeting, dan positioning. Selain itu, pembuatan strategi penasaran menekankan kebutuhan 4P (produk, harga, tempat, dan promosi) untuk memikat dan mempertahankan pelanggan. Gambar 1:Wawancara dengan devisi marketing Gambar 1:Wawancara dengan devisi marketing Adeliya Gita Silviana Putri | Page 18 CC Attribution-ShareAlike 4.0 License. CC Attribution-ShareAlike 4.0 License. Journal Of Indonesian Social Society Journal Of Indonesian Social Society E-ISSN : 2985-5705 P-ISSN : 2985-8771 E-ISSN : 2985-5705 P-ISSN : 2985-8771 y Volume 2 ; Nomor 1 ; Februari 2024 ; Page 17-21 Doi : https://doi.org/10.59435/jiss.v2i1.217 Web : https://jurnal.padangtekno.com/index.php/jiss KESIMPULAN Berdasarkan hasil penelitian dan pembahasan ,bahwa starategi pemasaran dalam meningkatkan jumlah nasabah pada produk pembiayaan KPR di Bank BTN Jember dan dikenal memiliki system pemasaran yang efektif berkat mekanisme strategi pemasaran yang diterapkan untuk produk KPR BTN bersubsidi atau non subsidi. Diantaranya menerapkan 4P bauran pemasaran (Product, Price, Place dan Promotion) serta memperhatikan segmentasi, targeting, dan positioning. d. Promotion d. Promotion Dalam hal promosi untuk memasarkan produk KPR BTN yaitu melakukan pemanfaatan media sosial (website,instagram),media cetak (Brosur, Baliho, Majalah), Sales promotion (Merchandising untuk meningkatkan apresiasi dari masyarakat). UCAPAN TERIMA KASIH Ucapan terimakasih kami sampaikan kepada PT Bank Tabungan Negara KC Jember yang telah memberikan izin dan kesempatan untuk melakukan pengabdian di lembaganya.Terima kasih kami ucapkan kepada pimpinan dan LPPM Universitas Kiai Haji Achmad Siddiq Jember. Kami juga berterimakasih kepada seluruh responden yang telah memberikan data dan informasi yang sangat bermanfaat bagi keberhasilan pengabdian ini dengan judul “Strategi Pemasaran untuk meningkatkan jumlah nasabah pada produk pembiayaan KPR”Selain itu, disampaikan terimakasih kepada pihak-pihak yang telah banyak membantu sehingga kegiatan pengabdian ini dapat berjalan dengan lancer dan dapat bermanfaat bagi pembaca. Penerapan Segmenting, Targeting dan Positioning KPR yang dilakukan oleh PT. Bank Tab (Persero) Kantor Cabang Jember Place Dalam hal ini lokasi bank BTN Kantor Cabang Jember terletak di JL.Jendral Ahmad Yani, Kepatihan, Patrang, Kabupaten Jember,Jawa Timur 68118, dimana lokasi tersebut berada di tengah kota Jember sehingga sangat cocok dan juga strategis untuk memudahkan kegiatan operasional perusahaan. Sehingga dapat disimpulkan bahwa lokasi bank BTN ini sangat strategis yang akan mempengaruhi kesuksesan bisnis yang dijalankan. Selain iu konsisi yang tepat juga berperan penting pada produk yang ditawarkan. Dalam hal ini lokasi bank BTN Kantor Cabang Jember terletak di JL.Jendral Ahmad Yani, Kepatihan, Patrang, Kabupaten Jember,Jawa Timur 68118, dimana lokasi tersebut berada di tengah kota Jember sehingga sangat cocok dan juga strategis untuk memudahkan kegiatan operasional perusahaan. Sehingga dapat disimpulkan bahwa lokasi bank BTN ini sangat strategis yang akan mempengaruhi kesuksesan bisnis yang dijalankan. Selain iu konsisi yang tepat juga berperan penting pada produk yang ditawarkan. d. Promotion Dalam hal promosi untuk memasarkan produk KPR BTN yaitu melakukan pemanfaatan media sosial (website,instagram),media cetak (Brosur, Baliho, Majalah), Sales promotion (Merchandising untuk meningkatkan apresiasi dari masyarakat). Penerapan Segmenting, Targeting dan Positioning KPR yang dilakukan oleh PT. Bank Tab (Persero) Kantor Cabang Jember Tingginya permintaan konsumen yang membuktikan bahwa PT Bank Tabungan Negara memiliki reputasi yang unik di mata masyarakat Indonesia. PT Bank Tabungan Negara telah mendapatkan kepercayaan masyarakat dalam kesanggupan dan kualitas layannya dalam menyediakan pembiayaan produk KPR serta memiliki Tag line “Sahabat Keluarga Indonesia”. g enerapan Marketing Mix (4p:Product,Price,Place,Promotion) yang dilakukan PT Bank Tabungan Persero) Kantor Cabang Jember g Penerapan Marketing Mix (4p:Product,Price,Place,Promotion) yang dilakukan PT Bank Tabunga (P ) K C b J b Negara (Persero) Kantor Cabang Jember Baura pemasaran di BTN KC Jember dalam mengeksploitasi pangsa pasar yang lebih memfokuskan pada 4P Product, Price, Place, Promotion) yakni: a Product a. Product Produk terbaik PT Bank Tabungan Negara KC Jember adalah Kredit Pemilikan Rumah. Bank BTN mendorong akuisisi nasabah melalui penggunaan produk pembiayaan KPR. Bank BTN menawarkan KPR dan produk pembiayaan lainnya yang bermanfaat bagi nasabahnya. Keunggulan spesifikasi produk KPR Subsidi yaitu uang muka ringan 1% suku bunga tetap 5% ,jangka waktu hingga 20 tahun, dan subsidi bantuan khusus uang muka sebesar 4 juta. g j b. Price Penetapan harga merupakan hal penting, perusahaan akan melakukan hal ini dengan penuh pertimbangan karena penetapan harga akan dapat mempengaruhi pendapatan total dan biaya, harga merupakan faktor utama penentu posisi dan harus ditetapkan sesuai dengan target pasar, bauran produk, dan jasa serta persaingan Adeliya Gita Silviana Putri | Page 19 Adeliya Gita Silviana Putri | Page 19 CC Attribution-ShareAlike 4.0 License. Journal Of Indonesian Social Society Volume 2 ; Nomor 1 ; Februari 2024 ; Page 17-21 Doi : https://doi.org/10.59435/jiss.v2i1.217 Web : https://jurnal.padangtekno.com/index.php/jiss E-ISSN : 2985-5705 P-ISSN : 2985-8771 Tabel 1. Tabel perbandingan suku bunga KPR dengan kompetitor lain. No Bank Suku Bunga Dasar Kredit (SBDK) 1 BTN 5,46% 2 BNI 6,75% 3 PANIN 8,75% 4 BCA 6,88% 5 MANDIRI 6,75% E-ISSN : 2985-5705 P-ISSN : 2985-8771 Tabel 1. Tabel perbandingan suku bunga KPR dengan kompetitor lain. No Bank Suku Bunga Dasar Kredit (SBDK) 1 BTN 5,46% 2 BNI 6,75% 3 PANIN 8,75% 4 BCA 6,88% 5 MANDIRI 6,75% Pada tabel diatas menujukkan bahwa suku bunga KPR pada bank BTN KC Jember sebesar 5,46% yang lebih kecil dibanding dengan kompetitor lain.Hal ini menjadi keunggulan produk pembiayaan KPR BTN KC Jember dan dapat menjadi faktor utama dalam menarik nasabah karena selisish suku bunga Bank BTN yang kecil dibandingkan dengan kompetitor lainnya. c. Journal Of Indonesian Social Society Volume 2 ; Nomor 1 ; Februari 2024 ; Page 17-21 Doi : https://doi.org/10.59435/jiss.v2i1.217 Web : https://jurnal.padangtekno.com/index.php/jiss E-ISSN : 2985-5705 P-ISSN : 2985-8771 Produk KPR BNI KRIYA. Angewandte Chemie International Edition, 6(11), 951–952., 1(3), 10–27. Masturoh. (2019). Strategi Pemasaran Pembiayaan Kredit Pemilikan Rumah (KPR) Syariah Di Bank Tabungan Negara Syariah Kantor Cabang Pembantu Cipuat Tangerang Selatan. In Skripsi Fakultas Agama Islam Universitas Muhammadiyah Jakarta. Salim. (2012). METODOLOGI PENELITIAN KUALITATIF.pdf. Citapustaka Media. Wijaya, H., & Sirine, H. (2016). Strategi Segmenting, Targeting, Positioning Serta Strategi Harga Pada Perusahaan Kecap Blekok Di Cilacap. Ajie, 1(3), 175–190. https://doi.org/10.20885/ajie.vol1.iss3.art2 CC Attribution-ShareAlike 4.0 License. DAFTAR PUSTAKA Strategi Pemasaran Pembiayaan Kredit Pemilikan Rumah (KPR) Syariah Di Bank Tabungan Negara Syariah Kantor Cabang Pembantu Cipuat Tangerang Selatan. In Skripsi Fakultas Agama Islam Universitas Muhammadiyah Jakarta. Salim. (2012). METODOLOGI PENELITIAN KUALITATIF.pdf. Citapustaka Media. Wijaya, H., & Sirine, H. (2016). Strategi Segmenting, Targeting, Positioning Serta Strategi Harga Pada Perusahaan Kecap Blekok Di Cilacap. Ajie, 1(3), 175–190. https://doi.org/10.20885/ajie.vol1.iss3.art2 E-ISSN : 2985-5705 P-ISSN : 2985-8771 E-ISSN : 2985-5705 P-ISSN : 2985-8771 CC Attribution-ShareAlike 4.0 License. Adeliya Gita Silviana Putri | Page 21 Adeliya Gita Silviana Putri | Page 21 DAFTAR PUSTAKA Abbdussamad. (2021). Metodologi Penelitian Kualitatif (Syakir Media Press (ed.)). Aditya, M. A. (2014). Strategi Pemasaran Pembiayaan KPR Syariah Bersubsidi Bagi Masyarakat Berpenghasilan Rendah Di BTN Syariah. Tesis, 1–140. y , Alusyi, A. K. (2010). Dengan Skim Syariah Konsentrasi Perbankan Syariah Program Studi Muamalat ( Ekonomi Islam ) 2010 M / 1431H Analisis Kebijakan Kepemilikan Rumah ( Kpr ) Bersubsidi Dengan Skim Syariah Program Studi Muamalat ( Ekonomi l Alusyi, A. K. (2010). Dengan Skim Syariah Konsentrasi Perbankan Syariah Program Studi Muamalat ( Ekonomi Islam ) 2010 M / 1431H Analisis Kebijakan Kepemilikan Rumah ( Kpr ) Bersubsidi Dengan Skim Syariah Program Studi Muamalat ( Ekonomi Islam ). Anjelisa, Mananeke, L., & Rogi, M. (2018). Analisis Pengaruh Strategi Segmentasi, Targeting Dan Positioning (Stp) Terhadap Keputusan Pembelian Produk Bp-Smart Protection Di Ajb Bumiputera 1912 Cabang Sam Ratulangi Manado. Jurnal EMBA: Jurnal Riset Ekonomi, Manajemen, Bisnis Dan Akuntansi, 6(4), 4075. https://ejournal.unsrat.ac.id/index.php/emba/article/download/21970/21671 Fajar Nugraha Permana, & Asmai Ishak. (2023). Strategi Pemasaran Untuk Upaya Meningkatkan Jumlah Nasabah Pada Produk Pembiayaan KPR Di PT Bank Tabungan Negara Kantor Cabang Cilegon. Jurnal Publikasi Sistem Informasi Dan Manajemen Bisnis, 2(3), 167–176. https://doi.org/10.55606/jupsim.v2i3.2017 Fatmasari, N. (2013). Analisis Sistem Pembiayaan KPR Bank Konvensional dan Pembiayaan KPRS Bank Syariah (Studi Kasus Bank BTN dengan Bank Muamalat). Jurnal Akuntansi AKUNESA, 1(3), 1–25. Adeliya Gita Silviana Putri | Page 20 CC Attribution-ShareAlike 4.0 License. Karamoy, S. W. (2018). Strategi Segmentasi, Targeting Dan Positioning Pengaruhnya Terhadap Keputusan Konsumen Menggunakan Adeliya Gita Silviana Putri | Page 20 CC Attribution-ShareAlike 4.0 License. CC Attribution-ShareAlike 4.0 License. Journal Of Indonesian Social Society Volume 2 ; Nomor 1 ; Februari 2024 ; Page 17-21 Doi : https://doi.org/10.59435/jiss.v2i1.217 Web : https://jurnal.padangtekno.com/index.php/jiss E-ISSN : 2985-5705 P-ISSN : 2985-8771 Produk KPR BNI KRIYA. Angewandte Chemie International Edition, 6(11), 951–952., 1(3), 10–27. Masturoh. (2019). Strategi Pemasaran Pembiayaan Kredit Pemilikan Rumah (KPR) Syariah Di Bank Tabungan Negara Syariah Kantor Cabang Pembantu Cipuat Tangerang Selatan. In Skripsi Fakultas Agama Islam Universitas Muhammadiyah Jakarta. Salim. (2012). METODOLOGI PENELITIAN KUALITATIF.pdf. Citapustaka Media. Wijaya, H., & Sirine, H. (2016). Strategi Segmenting, Targeting, Positioning Serta Strategi Harga Pada Perusahaan Kecap Blekok Di Cilacap. Ajie, 1(3), 175–190. https://doi.org/10.20885/ajie.vol1.iss3.art2 Journal Of Indonesian Social Society Volume 2 ; Nomor 1 ; Februari 2024 ; Page 17-21 Doi : https://doi.org/10.59435/jiss.v2i1.217 Web : https://jurnal.padangtekno.com/index.php/jiss E-ISSN : 2985-5705 P-ISSN : 2985-8771 Produk KPR BNI KRIYA. Angewandte Chemie International Edition, 6(11), 951–952., 1(3), 10–27. Masturoh. (2019).
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Automated evaluation of cardiac contractile dynamics and aging prediction using machine learning in a Drosophila model
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Automated evaluation of cardiac contractile dynamics and aging prediction using machine learning in a Drosophila model dynamics and aging prediction using machine learning in a Drosophila model Aniket Pant  Georgia Institute of Technology Yash Melkani  University of California, Berkeley Girish Melkani  University of Alabama Birmingham, Heersink School of Medicine https://orcid.org/0000-0003-0353- 4633 Article Keywords: Machine learning, medical segmentation, cardiovascular disease, Drosophila heart model, aging prediction Posted Date: March 21st, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2635745/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: There is NO Competing Interest. Version of Record: A version of this preprint was published at Communications Biology on June 7th, 2024. See the published version at https://doi.org/10.1038/s42003-024-06371-7. learning in a Drosophila model Aniket Pant  Georgia Institute of Technology Yash Melkani  University of California, Berkeley Girish Melkani  University of Alabama Birmingham, Heersink School of Medicine https://orcid.org/0000-0003-0353- 4633 Article Keywords: Machine learning, medical segmentation, cardiovascular disease, Drosophila heart model, aging prediction Posted Date: March 21st, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2635745/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: There is NO Competing Interest. Version of Record: A version of this preprint was published at Communications Biology on June 7th, 2024. See the published version at https://doi.org/10.1038/s42003-024-06371-7. Aniket Pant  Georgia Institute of Technology Yash Melkani  University of California, Berkeley Girish Melkani Aniket Pant  Georgia Institute of Technology Yash Melkani Article Additional Declarations: There is NO Competing Interest. Version of Record: A version of this preprint was published at Communications Biology on June 7th, 2024. See the published version at https://doi.org/10.1038/s42003-024-06371-7. 4Corresponding address: Department of Pathology, Division of Molecular and Cellular Pathology, School of Medicine, University of Alabama at Birmingham, AL 35205, USA. Tel.: 1-205-996-0591; Fax: 1-205-934-7447; E-mail: girishmelkani@uabmc.edu * These authors contributed equally; Aniket Pant, Yash Melkani Automated evaluation of cardiac contractile dynamics and aging prediction using machine learning in a Drosophila model Aniket Pant1,2*, Yash Melkani1,3*, Girish C. Melkani1,4 1Department of Pathology, Division of Molecular and Cellular Pathology, Heersink School of Medicine, University of Alabama at Birmingham, AL 35205, USA 2Department of Materials Science and Engineering, Georgia Institute of Technology, GA 30332, USA 3Engineering Physics Department, College of Engineering, University of California, Berkeley, CA 94720, USA 1Department of Pathology, Division of Molecular and Cellular Pathology, Heersink School of Medicine, University of Alabama at Birmingham, AL 35205, USA 2Department of Materials Science and Engineering, Georgia Institute of Technology, GA 30332, USA 3Engineering Physics Department, College of Engineering, University of California, Berkeley, CA 94720, USA 4Corresponding address: Department of Pathology, Division of Molecular and Cellular Pathology, School of Medicine, University of Alabama at Birmingham, AL 35205, USA. Tel.: 1-205-996-0591; Fax: 1-205-934-7447; E-mail: girishmelkani@uabmc.edu * These authors contributed equally; Aniket Pant, Yash Melkani 1 1 Significance Significance • Current analysis of Drosophila cardiac recordings is capable of limited cardiac physiological parameters and are error-prone and time-consuming. • Current analysis of Drosophila cardiac recordings is capable of limited cardiac physiological parameters and are error-prone and time-consuming. • Current analysis of Drosophila cardiac recordings is capable of limited cardiac physiological parameters and are error-prone and time-consuming. • We present the first deep-learning pipeline for high-fidelity automatic modeling of Drosophila contractile dynamics. • We present the first deep-learning pipeline for high-fidelity automatic modeling of Drosophila contractile dynamics. • We present methods for automatically calculating all relevant parameters for diagnosing cardiac performance in aging model. • Using the machine and deep learning age-classification approach, we can predict aging hearts with an accuracy of 83.3% (AUC 0.90) and 77.1% (AUC 0.85), respectively. Abstract The Drosophila model has proven tremendously powerful for understanding pathophysiological bases of several human disorders including aging and cardiovascular disease. Relevant high- speed imaging and high-throughput lab assays generate large volumes of high-resolution videos, necessitating next-generation methods for rapid analysis. We present a platform for deep learning-assisted segmentation applied to optical microscopy of Drosophila hearts and the first to quantify cardiac physiological parameters during aging. An experimental test dataset is used to validate a Drosophila aging model. We then use two novel methods to predict fly aging: deep- learning video classification and machine-learning classification via cardiac parameters. Both models suggest excellent performance, with an accuracy of 83.3% (AUC 0.90) and 77.1% (AUC 0.85), respectively. Furthermore, we report beat-level dynamics for predicting the prevalence of cardiac arrhythmia. The presented approaches can expedite future cardiac assays for modeling human diseases in Drosophila and can be extended to numerous animal/human cardiac assays under multiple conditions. Introduction Cardiovascular disease (CVD) continues to be among the leading causes of death and disability in the United States and a major public health burden. Despite other risk factors, aging is one of the major risk factors for advancing CVD. As the modern human population is increasingly adapting to lifestyles that mimic shift work further exacerbates CVD risks1. The Drosophila melanogaster (fruit flies) model system will allow us to comprehensively examine the potential cardiac benefit of a simple lifestyle modification and identify the relevant molecular and physiological changes. Gaining pathophysiological insights is essential for designing therapies and in vivo models including Drosophila models and modern technologies including machine learning are powerful tools for understanding several aspects of human pathophysiology, including aging and CVD. Despite some morphological and functional differences, Drosophila and human hearts conserved pathways appear to govern form and function2–9. Several genetic and non-genetic risks for heart diseases in humans also increase disease risks in Drosophila during aging. Moreover, mutant flies carrying genetic variants that are associated with a higher risk for CVD result in a similar outcome in the fly3,4,6. Furthermore, flies like humans living in industrial societies, consume some of their daily caloric intake at night and nutritional challenges that compromise cardiac function in humans have similar effects in flies10–12. p Progress in the high-speed recording of optical cardiac imagery has led to remarkable development in the application of Drosophila, zebrafish, and embryonic mouse heart for cardiac modeling13. For example, current canonical methods using Semi-Automatic Optical Heart Analysis (SOHA) software offers methods for measuring relevant cardiac parameters during aging and under multiple disease conditions13. However, the SOHA software requires manual selection of points of interest in diastole and systole phases, allowing the software to track changes in heart morphology. Moreover, Dong et. al demonstrated the use of 3D convolutional architectures for segmentation of Drosophila heart images studied via optical coherence microscopy (OCM) setups14. The authors reported robust segmentation (92% IoU) of OCM videos, with capabilities for calculating EDD, ESD, heart area and heart rate. Similarly, Lee et. al demonstrate Drosophila heart-beat counting using segmentation of optical coherence tomography (OCT) recordings15. They demonstrate similar segmentation of OCM videos and also apply PCA procedure for heart morphology reconstruction. Work by Klassen et al. enabled in vivo imaging of Drosophila aging models with a fluorescence imaging and conventional image-processing techniques for high- resolution, unanesthetized beating patterns16. Keywords ing, medical segmentation, cardiovascular disease, Drosophila heart model, aging Machine learning, medical segmentation, cardiovascular disease, Drosophila heart model, aging prediction Machine learning, medical segmentation, cardiovascular disease, Drosophila heart model, aging prediction 2 2 Introduction Using a conventional computer vision approach, the authors provide methods for automatic segmentation and parameter (chamber diameter, fractional shortening, systolic interval, cardiac output, and heart wall velocity) calculations. We find much discussion of successful automatic analysis techniques for use with optical coherence microscopy but find a lack of literature surrounding similar techniques in standard high-resolution optical microscopy setups, likely due to added complexity in visualized heart morphology. Through literature review, we find that a limited amount of cardiac physiological data has been analyzed using machine learning from morphological data collected with OCT or OCM techniques, with quantification of all relevant cardiac parameters. Moreover, heart analyses using machine learning techniques in the literature have not tested rigorous Drosophila aging or disease models. Furthermore, there is a lack of automated methods for analysis of high-speed Drosophila cardiac optical recordings in general optical microscopy setups. Modern video volumes outgrow the use of manual analysis techniques, necessitating automatic analysis methods. Our method differs from others in that it allows for automatic analysis of optical cardiac recordings at high spatial and temporal resolutions. Furthermore, we are the first to provide all cardiac statistics via a deep learning assisted pipeline. We attempt a solution with a well-known deep learning-assisted medical image segmentation architecture, the UNet. Recent developments in machine learning have enabled state-of-the-art dense image segmentation platforms, such as the development of full convolutional networks (FCNs)17, SegNets18, and UNets19–22. Historically, UNet architectures 3 have been applied for segmentation cell nuclei, spleen, and liver segmentation23,24, COVID-19 prognosis25, and more. Human heart analysis has been a central focus in medical segmentation efforts26–28. In 2020, Ouyang et. al26 demonstrated the use of convolutional architectures for segmentation and beat-level analysis of human echocardiograms. They demonstrate real-time prediction of ejection fraction with comparable or less variance than that of human predictions. In this work, we draw inspiration from similar applications of medical image segmentation and apply UN t hit t t t ti d b t l l l i f D hil di di To augment human analysis of Drosophila cardiac recordings, we apply techniques in medical segmentation for analysis of high-speed optical recordings. We calculate multiple diagnostic cardiac parameters for assessing cardiac function from videos alone. A 2D attention- based UNet architecture is applied for frame-level dense segmentation of cardiac recordings. Using only the generated annotations, we calculate beat-level cardiac parameters and beating patterns. Introduction Cardiac parameters are calculated on a per-beat resolution. We used 𝑛𝑛 = 54 hearts for model training and validated an experimental aging model with 𝑛𝑛 = 177 hearts. This deep- learning approach to Drosophila cardiac analysis facilitates robust analysis of Drosophila cardiac physiology and enables access to future downstream analysis techniques. We investigate the use of both Drosophila cardiac statistics and cardiac videos for fly age prediction as an example downstream task. We determine that conventional machine learning and deep learning methods can be applied for predicting fly age, suggesting that morphological features evident in video recording can be an effective predictor of an aging phenotype. Overview of presented machine learning pipeline Overview of presented machine learning pipeline Analysis of Drosophila high-speed cardiac recordings requires high-throughput and accurate measurement techniques. Current methods involve tedious analyses with large amounts of personnel hours, necessitating automatic next-generation methods. In this work, we propose three machine learning enabled pipelines that is well-suited for rapid analysis of Drosophila cardiac recordings. Depicted in Fig. 1, we establish two unique tasks: (1) heart wall detection via semantic segmentation and (2) age prediction. In the presented segmentation pipeline (Fig. 1), a user begins by uploading high-speed cardiac recordings to a central workstation via a file-transfer protocol. After this, the user selects videos for segmentation and analysis and provides them to our segmentation model, which is made accessible via a PyTorch interface29. Then, using our segmentation model, heart wall regions are tagged on a frame-by-frame basis, allowing us to easily calculate average heart diameter and area per-frame. After results have been generated per frame, users may vary network parameters such as selecting a region-of-interest in diameter calculations and a model confidence threshold. Supplementary Fig. 1 (.mp4) demonstrates a video-format output of our network results for a given heart, for five seconds. Once a satisfactory segmentation is acquired, users may export time-resolved morphological data. Finally, using the exported morphological data, we make available relevant cardiac statistics for assessing function and rhythmicity. Example Python notebooks are provided for segmenting and analyzing heart videos with easy to use, interactive user-interfaces. Two separate pipelines are presented for age prediction. One such pipeline involves using segmentation-calculated cardiac statistics for age-prediction. Age-prediction via cardiac statistics is powered via a logistic classification model. Such a logistic classification approach enables transparent reporting of model interpretation via black-box methods such as calculation of SHAPly scores. Furthermore, we present a fully deep learning-based pipeline for age classification. Through use of raw video frames, we determine that convolutional neural networks can predict fly age with high accuracy and specificity. Additional detail for both methods is provided in the Method section. 4 Representative output from our deep learning pipeline Representative output from our deep learning pipeline Representative output from our deep learning pipeline Fig. 2 demonstrates a representative output from our deep learning pipeline. This includes frame- by-frame heart wall output, visualized beating patterns, time-resolved morphological data, and calculated cardiac statistics on a representative level. Heart morphology is captured on a per- frame basis, denoted by overlaid red layers (Fig. Experimental validation of Drosophila aging model Experimental validation of Drosophila aging model p p g g A study investigating cardiac aging phenotypes was performed on a cohort population. We demonstrate that our model can capture aging phenotypes presented in existing literature with a fraction of the effort required from canonical analysis techniques (SOHA). We calculate a full suite of cardiac statistics including diastolic diameter (Fig. 3a), systolic diameter (Fig. 3b), fractional shortening (Fig. 3c), heart rate (Fig. 3d), and arrythmia index (Fig. 3e). Furthermore, we provide select temporal statistics including heart period, diastolic intervals, and systolic intervals in the SI (Supplementary Fig. 2). Fig. 4 presents model-detected contractile dynamics, including aging phenotypes in spatial and temporal modes. Supplementary Fig. 3 depicts heart fractional shortening and heart period, as a function of gender and age at the beat-level. Overview of presented machine learning pipeline 2a), as indicated in the methods section. Qualitative inspection suggests excellent agreement between detected heart regions and ground truth through all stages of the cardiac cycle. Fig. 2b, d, f present representative model outputs for a 1-week male heart. Alternatively, Fig. 2c, e, g present representative model outputs for a 5- week male heart. We trace the annotation over time at a single vertical slice, creating a mechanical mode (M-Mode) image, demonstrated in Fig. 2b, c. Again, one may note excellent agreement between annotated and ground truth MMode, indicating excellent capture of the heart wall dynamics. Furthermore, periods of diastolic and systolic intervals are automatically labeled with red and green vertical lines. Fig. 2d, e depicts time-resolved beating patterns constructed via measurement of heart diameter through segmented heart walls. Average diameter is calculated by measuring each vertical pixel-slice distance in our frame-by-frame heart walls. This is further discussed in the Methods section. The time-resolved beating patterns clearly demonstrate differences in contractility and arrhythmia, making neural-network captured aging phenotypes clear on a representative basis. These are further quantified in tabulated cardiac statistics for a single heart (𝑛𝑛= 1), made available in Fig. 2f, g. These aging phenotypes are presented for a larger-scale study in Figs. 3, 4. Deep learning recovers aging phenotypes including contractile dynamics in a Drosophila cardiac model We find that expected directional trends in cardiac function and arrythmia are captured successfully across aging groups and genders (Fig. 3). Spatial (cardiac morphology) associated statistics are first discussed. We demonstrated a statistically significant decrease in diastolic diameter in male aging groups and no change in diastolic diameter across female aging groups, with agreement between experimental machine-learning (ML) and SOHA groups. Systolic diameter demonstrates little variance across aging groups with agreement between ML SOHA groups, except for a weak statistically significant difference in the ML female aging group (𝑝𝑝= 0.049). Furthermore, we observe a strong statistically significant decrease of cardiac function (fractional shortening) across both male and female aging (𝑝𝑝 < 0.001). 𝑅𝑅2 measurements between ML and SOHA data are high for diastolic and systolic diameter measurements, at 𝑅𝑅2 = 0.76 and 𝑅𝑅2 = 0.69, respectively. However, 𝑅𝑅2 is low for fractional shortening measurements at 𝑅𝑅2 = 0.32. We demonstrate a strong reduction in cardiac function with age across both male and female groups in ML and SOHA studies, as expected. This agrees with morphological parameters quantified in Fig. 4, with a statistically significant reduction in stroke volume (Fig. 4a) and cardiac output (Fig. 4b). Methods for calculating all discussed parameters are made available in the Methods section. 5 Next, we discuss temporal associated cardiac statistics. We find that our model can accurately capture changes in heart rate, with expected significant differences across aging groups in both ML and SOHA datapoints. Similar agreement is presented in studied heart period, diastolic and systolic interval data across ML and SOHA groups. The model captures an accurate evolution of cardiac arrythmia in female aging data in both ML and SOHA groups but exhibits an unexpected significant difference in male aging groups. 𝑅𝑅2 measurements between ML and SOHA data are high for temporal statistics including heart rate, heart period, diastolic interval, and systolic intervals, at 𝑅𝑅2 = 0.88, 𝑅𝑅2 = 0.91, 𝑅𝑅2 = 0.89, and 𝑅𝑅2 = 0.63, respectively. However, 𝑅𝑅2 is low for arrythmia index measurements at 𝑅𝑅2 = 0.46. We observe a small increase in cardiac arrythmia with aging in both ML and SOHA groups. While heart period and derivative temporal parameters demonstrate statistically significant shifts with aging, changes in rhythmicity and contractile latency are not exhibited with aging (Figs. 3, 4). Latency is quantified in Fig. Deep learning recovers aging phenotypes including contractile dynamics in a Drosophila cardiac model 4c, d, concluding that no significant difference in latency to peak contraction and latency between peak contraction to peak relaxation is expressed in our aging model across aging in gender-separated data. This signals agreement with previously presented results in measured arrythmia shifts with aging. We also demonstrate our ability to capture beat-level contractile and temporal dynamics in beating patterns. In both male and female groups, we see a large flattening across 1-week and 5-week fractional shortening distributions, indicating a large increase in contractility variance (Supplementary Fig. 3 a, b). Furthermore, a general decrease in contractility with aging is depicted on a beat-level, as expected from cohort-level data. The reverse trend is true in heart period distribution, with flattening across both male and female aging groups but an increase, on average, in beat length (Supplementary Fig. 3c, d). Furthermore, we employ beat-level dynamics to detect brachy- and tachycardia arrythmias. Supplementary Fig. 4 quantifies prevalence and significant shifts in exhibited tachy- and brachy- cardiac arrythmia across our aging model. We find a significant decrease in average SI length during tachycardiac events for female specimen with aging (Supplementary Fig. 4). Additionally, we observe a significant increase in average DI length during brachycardiac events for both male and female specimen through aging. We note an exceptionally small 𝑛𝑛-value at 𝑛𝑛 = 3 for brachycardiac events in young female hearts. We visualize cohort-level and beat-level data for SI and DI distributions (Supplementary Fig. 4c). Beat-level data suggests a widening distribution in both SI and DI for aging male and female specimen. Prediction of aging in Drosophila data g g p We investigate the use of machine learning approaches for age classification in Drosophila datasets (Figs. 5, 6). We apply logistic regression for age classification (Fig. 5). We note high accuracy in experimental classification, with an accuracy of 77.1% and AUROC of 0.85 (Fig. 5a, b). We observe the highest inaccuracies to be found in old hearts falsely classified as young. The presented results suggest that cardiac statistics are accurate predictors of aging phenotypes. We demonstrate the use of SHAP values for assessing drivers of aging function, enabling interpretable machine learning (Fig. 5c). Descriptors are sorted in predictive importance, from top to bottom. Thus, we note that SHAP-methods determine fractional shortening (FS) as a main predictor of fly age, followed by diastolic diameter (DD). Using a deep convolutional neural network, we train a video classifier for assessing fly age from cardiac video without knowledge of cardiac parameters. Results of this technique are presented in Fig. 6. The constructed model architecture along with data preparation are discussed in the Methods section. Using a k-fold cross-validation procedure, we perform experimental validation on 𝑛𝑛= 497 cardiac samples, with group breakdowns of 𝑛𝑛= 118 for 1wm, 𝑛𝑛= 156 for 1wf, 𝑛𝑛= 121 for 5wm, and 𝑛𝑛= 98 for 5wf. We observe an average AUROC score of 0.90 (Fig. 6b) and 6 an average accuracy of 83.3%, demonstrating excellent performance, further depicted in the confusion matrix (Fig. 6a). Qualitatively, we note excellent separation of age likelihood as determined by our model, with a high separation between young and old likelihoods (Fig. 6c). The presented results suggest a neural architecture can recover fly age from cardiac video based on spatial data from select frames. We note that the highest number of inaccuracies found in young hearts falsely predicted as old (22% of samples). an average accuracy of 83.3%, demonstrating excellent performance, further depicted in the confusion matrix (Fig. 6a). Qualitatively, we note excellent separation of age likelihood as determined by our model, with a high separation between young and old likelihoods (Fig. 6c). The presented results suggest a neural architecture can recover fly age from cardiac video based on spatial data from select frames. We note that the highest number of inaccuracies found in young hearts falsely predicted as old (22% of samples). Discussion We present methods for the machine and deep-learning analysis of Drosophila optical cardiac videos. We find that deep segmentation models can accurately recover important contractile and temporal dynamics in Drosophila heart beating patterns. Our approach allows us to bypass the time-consuming human involvement required in existing canonical software such as SOHA13. Moreover, our automated machine-learning analysis will be helpful to erase human errors in marking heart edges under contraction and relaxation conditions. This is one of the vital steps for automated analysis as three of the main cardiac parameters, including diastolic and systolic diameters as well as fractional shortening (cardiac performance) depends on accurately marking cardiac edges. Such cardiac edge marking is automated in Dong et al.14 for OCM and Klassen et al.16 for fluorescent microscopy, but yet to be automated in an optical microscopy approach. Dong et al.14 provides utilities for heart beat calculation. We provide a full suite of cardiac statistics that may be automatically calculated using our deep model. The presented code can readily provide calculated cardiac statistics including diastolic and systolic diameters/intervals, fractional shortening, ejection fraction, heart period/rate as well as quantify heartbeat arrhythmicity. Our model may be applied on readily available consumer hardware. In our study, we employ the Tesla P100, but suggest the model can be used with lower-end, consumer graphics models. Our method may potentially aid researchers with a higher fidelity, reproducible, and more automatic approach to Drosophila cardiac modeling beyond the capabilities of human technicians. We use a 2D deep segmentation model to generate heart-wall segmentations on a frame- by-frame basis. We find that the employed segmentation model allows us to recover accurate heart-wall segmentations, thus, recover accurate beating patterns and cardiac statistics on a frame-by-frame resolution. Using our deep learning method, we note fly aging expresses significant reduction in cardiac function (contractility) and an increase in cardiac dysrhythmia. Similarly, our model detects significant changes in aged heart rate and heart period, as well as underlying parameters including diastolic and systolic intervals. Furthermore, such annotations open opportunities in precise time-resolved study of Drosophila cardiac morphologies in optical micrography assays (Fig. 2). Such analysis is not possible using canonical analysis software. For example, deep learning assisted modeling of congenital muscular dystrophies in Drosophila cardiac morphologies may reveal unique physiological information30,31. Prediction of aging in Drosophila data Overall, we determine that deep learning classification models can model aging directly on raw cardiac video with a high accuracy, suggesting such models may possess capabilities for capturing morphological or rhythmic features in image pairs. Furthermore, we determine that both cardiac parameters and videos can be applied for highly accurate aging prediction in Drosophila models. Discussion To our knowledge, this is the first platform for deep learning assisted segmentation applied to standard high-resolution, high-speed optical microscopy of Drosophila hearts and is the first to quantify all relevant parameters, including directly quantifying ejection fraction. Cited works such as Ouyang et. al26 report a limited amount of cardiac parameters and provide ejection fraction as 7 a model-derived value on human echocardiography data. Ouyang et. al studied failing hearts as a contrasting model to demonstrate differences in beat-level ejection fraction but did not include age dependence. Similarly, Lee et. al15 report segmentation on Drosophila OCM video, with fewer provided cardiac parameters including heart-rate, ESD, EDD, and FS, with some discussion on aging phenotypes. We further enable understanding of contractile dynamics via our beat-level capabilities (Figs. 3, 4, Supplementary Fig. 3). Through per-frame analysis, we quantify contractility through measurement of morphological parameters quantifying stroke performance (Figs. 3, 4) and latency (Fig. 4). We note significant reduction in spatial beating modes (FS, SV, CO) with aging, but little to no dependence on aging for temporal beating character (AI, latency) (Fig. 4). However, we do note significant shifts with aging in beat lengths, indicated in modeling of HP and derivative parameters. Beat-level investigation elucidates per-beat information regarding cardiac arrythmia’s including tachycardia and brachycardiac arrythmias (Supplementary Fig. 3). We observe a significant, large increase in DI length during brachycardiac events in 5wk male flies. We demonstrate capabilities of predicting fly age using experimentally calculated cardiac statistics with excellent agreement (Fig. 5). We also use a 2D video classification model to predict fly ages between 1-week and 5-week groups (Fig. 6). The ability to classify age via both raw video frames (Fig. 6) and cardiac statistics suggests that experimentally calculated cardiac statistics are physiologically salient and model age dependence. Additionally, the ability to predict age with only video frames suggests that deep learning models can capture morphological and rhythmic patterns in Drosophila cardiac video data. This has important implications for detecting phenotypes mimicking or delaying aging of Drosophila hearts. To our knowledge, we are the first work to determine that deep neural networks can capture such cardiac physiological features of the heart that induce aging in cardiac videos. In the future, such classification could be extended to classify healthy and compromised hearts in mutant studies and may include further quantification through methods such as GradCAM32. High-speed cardiac recording g p g Physiological cardiac parameters such as heart rate, heart period, diastolic and systolic diameters, diastolic and systolic intervals, cardiac rhythmicity, and cardiac performance (% fractional shortening) will be determined for each fly group to detect cardiac defects using established protocols4–7. To avoid any circadian variability in cardiac function, all assays will be performed between ZT4 and ZT8. Briefly, in these semi-intact heart preparations, nerve input is eliminated so that endogenous pacing can be observed. Direct immersion optics is used in conjunction with a digital high-speed camera (up to 150 frame/s, Hamamatsu EM-CCD) to record contraction movements using the image capture software HC Image (Hamamatsu Corp.). Discussion Current limitations include validation of parameters including calculation region of interest and suitable confidence thresholds. In the future, we hope to overcome this limitation. We report preliminary results in the Supplementary Fig. 3 and Supplementary Fig. 4 of using our trained segmentation approach without specifying a region of interest for morphological calculation (referred to as “No ROI”). Furthermore, we homogenously employ a pre-selected threshold, enabling a user to analyze samples with no human input or supervision. Presented results demonstrate excellent agreement in temporal statistics, including high accuracy in heart rate/period, diastolic and systolic intervals (with comparison to data collected via SOHA software), but strong disagreement in the arrythmia index. Spatial statistics, however, demonstrate strong disagreement in systolic diameter, driving further disagreement in fractional shortening. We propose further developments in dataset size, domain adaptability, and self-supervision may lead to stronger spatial performance in the case of completely hands-free usage. Future applications of the discussed techniques include enabling deep learning assisted studies of cardiac mutation models, other small animal models (e.g., commonly studied zebrafish and mice models), and parameter calculation in human heart models. Furthermore, quantification of measured uncertainty techniques may be applied to qualify certainty of heart analyses. Massive volumes of Drosophila cardiac data collected in the lab necessitate advanced methods for automated analysis of cardiac physiologies and morphologies on a beat-by-beat basis. In summary, we evaluate the use of deep segmentation models for high-fidelity analysis of cardiac physiologies in high-speed Drosophila cardiac optical recordings. We demonstrate that the presented deep segmentation models can be applied for accurately expressing known 8 Drosophila phenotypes in aging across male and female 1-week and 5-week groups. Furthermore, we demonstrate that developed deep video classification methods can be successfully applied to Drosophila fly age-classification using only video clips with exceptional accuracy. We hope these models can be applied in the future to expedite laboratory analysis and power next-generation Drosophila cardiological model analysis. Deep-learning development and training p g p g Model design was performed in Python using the PyTorch deep learning framework29. Our study employs a modified Attention-UNet architecture for semantic segmentation22. The model contains a symmetric encoder/decoder architecture containing 8, 16, 32, 64, and 128 filters, employing a rectangular convolutional kernel. Validation loss was optimized via Dice Loss33. Our model employs a random weight initialization and the Adam34 optimizer and a 16-image batch size. For experimental testing, the model epoch with the lowest validation loss was employed. During the training process, the neural network samples data via a data point’s corresponding diameter, as measured via its human-annotated mask. 75 frames per measured diameter are randomly sampled to be included in the training dataset – this is done to minimize class imbalance in our segmentation task. After frame sampling, 3D samples are constructed by appending frames at (𝑡𝑡−4, 𝑡𝑡, 𝑡𝑡+ 4) indices, allowing us to encode temporal information in 3D convolutions. After sampling and augmentation, the model used a total of 114750 images for training with 19350 images used for validation. The model was trained for a total of 30 epochs, yielding a total of approximately 30 compute hours. Models were trained on a Tesla P100 GPU on the UAB Cheaha supercomputer. Dataset curation A standard library of high-speed cardiac optical recordings is procured for training. We employ a total of 54 training videos, with an 85%/15% train/validation split. Training videos are captured in grayscale format with a total of 500 frames per video from a complete 6000 frames, indicating 2.5 seconds of video. Training videos include 40 videos with wildtype and 14 videos with genetically engineered flies. Researchers used the Computer Vision Annotation Tool (CVAT) software to produce high-quality masks on a per-frame basis. Annotations spanned heart walls that were clearly identifiable – annotations were clipped in the presence of pericardial occlusion, change of heart regions, and unclear tissue. Annotation masks were reviewed for accuracy before usage in deep-learning experiments. Testing videos are captured in identical formats. We procure 177 videos for experimental validation, with 𝑛𝑛 = 46 for 1-week males, 𝑛𝑛 = 43 for 1-week females, 𝑛𝑛 = 44 for 5-week males, and 𝑛𝑛 = 44 for 5-week females. Experimental validation 𝑛 We procure high-speed videos of 𝑛𝑛 = 46 for 1-week males, 𝑛𝑛 = 43 for 1-week females, 𝑛𝑛  =  44 for 5-week males, and 𝑛𝑛 = 44 for 5-week females to experimentally validate our model. For each heart, we calculate time-resolved beating patterns and cardiac statistics using our deep- learning approach. For each heart, an end-to-end analysis takes approximately 2 minutes. Next, each heart was identically examined in the canonical software for small-animal cardiac analysis, titled SOHA13. We employ the SOHA software for experimental validation of our model using blind data. Statistics comparing age-dependent phenotypes are calculated using generally available Python packages. For comparison of aging groups, we calculate t-Test significance values. Furthermore, for a quantitative view of our model performance, we calculate pairwise coefficient of determination information (𝑅𝑅2) score via the Scikit-Learn Python library35. A close agreement between deep-learning calculated data and SOHA data indicates high model performance. Calculation of cardiac parameters 𝑡𝑡 Calculation of cardiac parameters 𝑡𝑡 p Once a video has been selected for analysis, each frame 𝑡𝑡 is converted into a three-channel image with the 𝑡𝑡−4 frame and the 𝑡𝑡+ 4 frame. Images are then processed via neural network. 9 On a Tesla P100, we estimate this process to take approximately 103 seconds for a video with 5990 frames (58.16 FPS). From sigmoidal activations, the user determines an acceptable threshold and region-of-interest (ROI) through visual confirmation. Using provided values, average heart diameter for each frame is measured. Processing codes for the identification of diastolic intervals (DI) and systolic intervals (SI) is provided in the paper repository (GitHub). The diastolic diameter (DD) for each DI is taken to be the largest diameter attained during the DI. Similarly, the systolic diameter (SD) for each SI is taken to be the smallest diameter attained over its duration. We derive additional cardiac parameters from the collected DD, SD, DI, and SI statistics. Equations for fractional shortening (FS), ejection fraction (EF), heart period (HP), heart rate (HR), and arrhythmia index (AI) can be found in literature6. Stroke volume and cardiac output calculations follow those in Klassen et al.16, along with peak contraction velocity and peak contraction to relaxation latencies16. Velocity information is procured via numeric differentiation of frame-level time-resolved diameter data. Extraction of beat-level data enables capturing of SI and DI arrythmias, referred to as tachycardiac and brachycardiac arrythmias. After identifying all SI and DI at the beat-level, tachycardiac data is selectively filtered from all SI events with a length over 0.5 sec. Brachycardiac events are extracted from DI data with a length over 1.0 sec. These filtering parameters are extracted analysis performed by Occur et al.4. 𝑛𝑛𝑛 is then flattened and combined with the duration data before being passed into three dense layers and a final sigmoid output layer. Our dataset consisted of 𝑛𝑛 = 118 for 1-week males, 𝑛𝑛 = 156 for 1-week females, 𝑛𝑛 = 121 for 5-week males, and 𝑛𝑛 = 98 for 5-week females. Again, we utilized k-fold cross-validation to evaluate model performance with 𝑘𝑘= 5 folds. Classification of Drosophila age Calculated cardiac statistics are exported from experimental studies. A dataset is labelled with cardiac statistics and corresponding fly age (1-week, 5-week). We produce a predictive model combining calculated cardiac statistics and fly age via logistic classification. To fit this logistic model, we employed the Scikit-Learn Python library. Our logistic model was fit on experimentally predicted DD, SD, FS, DI, SI, HP, and AI parameters. A k-fold cross-validation with 𝑘𝑘= 5 folds was used to evaluate model performance. Using such a configuration achieved a high testing accuracy with our model, as quantified in the Results section. To elucidate the connection between cardiac physiological parameters and age-dependence, we employ the SHapley Additive exPlanations (SHAP)36 technique. Doing so enables model transparency and insights into main determinants of aging phenotypes. We also investigate the use of deep learning models for the classification of Drosophila age from cardiac recordings. For this task, the sum of the squared difference of each pixel between a frame 𝑡𝑡0 and frame t is saved as timeseries data and are normalized between 0 and 1. This data is binarized with a threshold of 0.5. The resulting timeseries is composed of alternating regions of consecutive ones and consecutive zeros. The frame corresponding to the center of each region was saved until 96 frames had been collected. This along with the duration between each frame was taken as the input for the model. The model passes the 96-frame clip through 3 convolution blocks each consisting of two 2D convolutional layers followed by a max pooling layer. The result 10 is then flattened and combined with the duration data before being passed into three dense layers and a final sigmoid output layer. Our dataset consisted of 𝑛𝑛 = 118 for 1-week males, 𝑛𝑛 = 156 for 1-week females, 𝑛𝑛 = 121 for 5-week males, and 𝑛𝑛 = 98 for 5-week females. Again, we utilized k-fold cross-validation to evaluate model performance with 𝑘𝑘= 5 folds. is then flattened and combined with the duration data before being passed into three dense layers and a final sigmoid output layer. Our dataset consisted of 𝑛𝑛 = 118 for 1-week males, 𝑛𝑛 = 156 for 1-week females, 𝑛𝑛 = 121 for 5-week males, and 𝑛𝑛 = 98 for 5-week females. Again, we utilized k-fold cross-validation to evaluate model performance with 𝑘𝑘= 5 folds. 11 11 Data availability Data used within this paper and connected findings of this study are available from the corresponding author upon request. Author Contributions Under G.C.M supervision, A.P. and Y.M. developed and trained the presented deep learning algorithms, developed methods for cardiac parameter calculation, and performed statistical analysis. A.P. and Y.M. created all the figures and drafted the manuscript. G.C.M provided studied Drosophila cardiac recordings, including cardiac aging datasets, writing and editing the manuscript. Acknowledgements This work was partially supported by NIH-NIA 1R01AG065992, UAB Heersink School of Medicine AMC21 grant, and Pathology Department start-up funds to G.C.M. The authors thankful to Melkani lab members for their help. 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For the segmentation task, users upload videos for analysis, perform segmentation using a trained neural network, select validated parameters, and extract beat-level parameters. The middle pipelines enable age- prediction of hearts via calculated cardiac statistics, further enabling transparent modeling of physiological parameters. Lastly, the bottom-most pipeline details an age-prediction task using a neural network, thus providing an alternate method for age-prediction. Figure 2: Representative output of neural network. (a) Select frames extracted from cardiac video, with overlaid mask from neural network denoting heart walls. (b, c) Using annotated frames, an annotated mechanical mode (M-Mode) image is generated. (d, e) Detected heart masks are used to calculate average stationary diameter on a per frame resolution. (b, d, f) represent generated M-Mode, time-series beating pattern, and calculated cardiac parameters for a representative 1w male heart. (c, e, g) represent generated M-Mode, time-series beating pattern, and calculated cardiac parameters for a representative 5w male heart. Green (start of DI) and red (end of DI) lines are annotated in (b, c) and as gray periods in (d, e). Times of max contraction (red) and max relaxation (green) is annotated by x indicators in (d, e). Figure 3: Deep learning recovers aging phenotypes in Drosophila cardiac model. (a) Diastolic diameter, in micron, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (b) Systolic diameter, in micron, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (c) Radial contractility (fractional shortening), in percentage, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (d) Heart rate, in Hertz, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (e) Beating dysrhythmia (arrythmia index) calculated by our model (left), SOHA (right), and agreement between two datasets (right). All error bars report ± SEM. Age-dependent statistics compared with paired t-Test statistics. Statistics are calculated via the use of a restricted ROI, selected by a trained user. Figure 3: Deep learning recovers aging phenotypes in Drosophila cardiac model. (a) Diastolic diameter, in micron, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (b) Systolic diameter, in micron, calculated by our model (left), SOHA (right), and agreement between two datasets (right). Figure Legends (c) Radial contractility (fractional shortening), in percentage, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (d) Heart rate, in Hertz, calculated by our model (left), SOHA (right), Figure 4: Contractile dynamics detected by deep learning. (a) Stroke volume for male and female aging groups is depicted, in picolitres. A significant reduction in stroke volume is exhibited with aging in both genders. (b) Cardiac output visualized via integration of per-beat stroke volumes and normalized by beat-times, measured in pL s-1. Aging depicts a strong reduction in cardiac output. (c) Time to peak contraction (negative) heart-wall velocity visualized for aging. (d) Time between peak contraction (negative) and relaxation (positive) velocities visualized for aging. Both (c) and (d) reflect no significant change with aging. All error bars report ± SEM. Age- dependent statistics compared with paired t-Test statistics. Figure 5: Machine learning classification of aging data. Logistic classification models enable accurate prediction of fly age from model-calculated cardiac statistics. (a) Heatmap of test-dataset predictions, indicating high agreement. (b) Average ROC-curve of all folds from presented model, reporting a mean AUROC of 0.85. (c) Physiological feature relevancies for predicting fly age, as 15 determined by SHAP study. SHAP study suggests large dependence on fractional shortening (FS) in aging. Figure 6: Deep learning classification of aging videos (n = 497). Deep learning models enable accurate prediction of fly age from only video data. (a) Heatmap of test-dataset predictions, indicating high agreement. (b) Average ROCcurve of all folds from presented model, reporting a mean test accuracy of 83.3% and mean AUROC of 0.900. (c) Model class log-likelihood distribution for investigated test predictions. determined by SHAP study. SHAP study suggests large dependence on fractional shortening (FS) in aging. Figure 6: Deep learning classification of aging videos (n = 497). Deep learning models enable accurate prediction of fly age from only video data. (a) Heatmap of test-dataset predictions, indicating high agreement. (b) Average ROCcurve of all folds from presented model, reporting a mean test accuracy of 83.3% and mean AUROC of 0.900. (c) Model class log-likelihood distribution for investigated test predictions. determined by SHAP study. SHAP study suggests large dependence on fractional shortening (FS) in aging. determined by SHAP study. SHAP study suggests large dependence on fractional shortening (FS) in aging. Figure 6: Deep learning classification of aging videos (n = 497). Figure Legends Deep learning models enable accurate prediction of fly age from only video data (a) Heatmap of test-dataset predictions Figure 6: Deep learning classification of aging videos (n = 497). Deep learning models enable accurate prediction of fly age from only video data. (a) Heatmap of test-dataset predictions, indicating high agreement. (b) Average ROCcurve of all folds from presented model, reporting a mean test accuracy of 83.3% and mean AUROC of 0.900. (c) Model class log-likelihood distribution for investigated test predictions. Figure 6: Deep learning classification of aging videos (n = 497). Deep learning models enable accurate prediction of fly age from only video data. (a) Heatmap of test-dataset predictions, indicating high agreement. (b) Average ROCcurve of all folds from presented model, reporting a mean test accuracy of 83.3% and mean AUROC of 0.900. (c) Model class log-likelihood distribution for investigated test predictions. y ( ) class log-likelihood distribution for investigated test predictions. 16 Figures Figures Figure 1 Proposed deep learning pipeline. One pipeline enables frame-level segmentation of heart walls, while two alternate pipelines are used for age classi¦cation. For the segmentation task, users upload videos for analysis, perform segmentation using a trained neural network, select validated parameters, and extract beat-level parameters. The middle pipelines enable age-prediction of hearts via calculated cardiac statistics, further enabling transparent modeling of physiological parameters. Lastly, the bottom- most pipeline details an age-prediction task using a neural network, thus providing an alternate method for age-prediction. most pipeline details an age-prediction task using a neural network, thus providing an alternate method for age-prediction. Figure 2 Representative output of neural network. (a) Select frames extracted from cardiac video, with overlaid mask from neural network denoting heart walls. (b, c) Using annotated frames, an annotated mechanical mode (M-Mode) image is generated (d e) Detected heart masks are used to calculate average Figure 2 Representative output of neural network. (a) Select frames extracted from cardiac video, with overlaid mask from neural network denoting heart walls. (b, c) Using annotated frames, an annotated mechanical mode (M-Mode) image is generated. (d, e) Detected heart masks are used to calculate average stationary diameter on a per frame resolution. (b, d, f) represent generated M-Mode, time-series beating Representative output of neural network. (a) Select frames extracted from cardiac video, with overlaid mask from neural network denoting heart walls. (b, c) Using annotated frames, an annotated mechanical mode (M-Mode) image is generated. (d, e) Detected heart masks are used to calculate average stationary diameter on a per frame resolution. (b, d, f) represent generated M-Mode, time-series beating Representative output of neural network. (a) Select frames extracted from cardiac video, with overlaid mask from neural network denoting heart walls. (b, c) Using annotated frames, an annotated mechanical mode (M-Mode) image is generated. (d, e) Detected heart masks are used to calculate average stationary diameter on a per frame resolution. (b, d, f) represent generated M-Mode, time-series beating pattern, and calculated cardiac parameters for a representative 1w male heart. (c, e, g) represent generated M-Mode, time-series beating pattern, and calculated cardiac parameters for a representative 5w male heart. Green (start of DI) and red (end of DI) lines are annotated in (b, c) and as gray periods in (d, e). Times of max contraction (red) and max relaxation (green) is annotated by x indicators in (d, e). (d, e). Times of max contraction (red) and max relaxation (green) is annotated by x indicators in ( Figure 3 Deep learning recovers aging phenotypes in Drosophila cardiac model. (a) Diastolic diameter, in micron, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (b) Systolic diameter, in micron, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (c) Radial contractility Deep learning recovers aging phenotypes in Drosophila cardiac model. (a) Diastolic diameter, in micron, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (b) Systolic diameter, in micron, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (c) Radial contractility Deep learning recovers aging phenotypes in Drosophila cardiac model. (a) Diastolic diameter, in micron, calculated by our model (left), SOHA (right), and agreement between two datasets (right). Figure 2 (b) Systolic diameter, in micron, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (c) Radial contractility (fractional shortening), in percentage, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (d) Heart rate, in Hertz, calculated by our model (left), SOHA (right), and agreement between two datasets (right). (e) Beating dysrhythmia (arrythmia index) calculated by our model (left), SOHA (right), and agreement between two datasets (right). All error bars report ± SEM. Age- dependent statistics compared with paired t-Test statistics. Statistics are calculated via the use of a restricted ROI, selected by a trained user. Figure 4 Contractile dynamics detected by deep learning. (a) Stroke volume for male and female aging groups is depicted, in picolitres. A signi¦cant reduction in stroke volume is exhibited with aging in both genders. (b) Cardiac output visualized via integration of per-beat stroke volumes and normalized by beat-times, measured in pL s-1. Aging depicts a strong reduction in cardiac output. (c) Time to peak contraction (negative) heart-wall velocity visualized for aging. (d) Time between peak contraction (negative) and relaxation (positive) velocities visualized for aging. Both (c) and (d) re§ect no signi¦cant change with aging. All error bars report ± SEM. Age-dependent statistics compared with paired t-Test statistics. Figure 5 Machine learning classi¦cation of aging data. Logistic classi¦cation models enable accurate prediction of §y age from model-calculated cardiac statistics. (a) Heatmap of test-dataset predictions, indicating high agreement. (b) Average ROC-curve of all folds from presented model, reporting a mean AUROC of 0.85. (c) Physiological feature relevancies for predicting §y age, as determined by SHAP study. SHAP study suggests large dependence on fractional shortening (FS) in aging. Machine learning classi¦cation of aging data. Logistic classi¦cation models enable accurate prediction of §y age from model-calculated cardiac statistics. (a) Heatmap of test-dataset predictions, indicating high agreement. (b) Average ROC-curve of all folds from presented model, reporting a mean AUROC of 0.85. (c) Physiological feature relevancies for predicting §y age, as determined by SHAP study. SHAP study suggests large dependence on fractional shortening (FS) in aging. study suggests large dependence on fractional shortening (FS) in aging. Figure 6 Deep learning classi¦cation of aging videos (n = 497). Deep learning models enable accurate prediction of §y age from only video data. (a) Heatmap of test-dataset predictions, indicating high agreement. (b) Figure 6 Figure 6 Deep learning classi¦cation of aging videos (n = 497). Deep learning models enable accurate prediction of §y age from only video data. (a) Heatmap of test-dataset predictions, indicating high agreement. (b) Average ROCcurve of all folds from presented model, reporting a mean test accuracy of 83.3% and mean AUROC of 0.900. (c) Model class log-likelihood distribution for investigated test predictions. class log-likelihood distribution for investigated test predictions. Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SupplementaryInformation.pdf
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Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation
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RESEARCH ARTICLE Chih-Chung Lin1, I-Ta Lee2, Chun-Hao Hsu2, Chih-Kai Hsu2, Pei-Ling Chi2, Li-Der Hsiao1, Chuen-Mao Yang2* 1 Department of Anesthetics, Chang Gung Memorial Hospital at Lin-Kou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan, 2 Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan * chuenmao@mail.cgu.edu.tw * chuenmao@mail.cgu.edu.tw OPEN ACCESS Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflam- mation via adhesion molecules induction, and then causes lung injury. However, the mech- anisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these re- sponses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and trans- fection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was me- diated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/ PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/ p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrat- ed that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation. Citation: Lin C-C, Lee I-T, Hsu C-H, Hsu C-K, Chi P- L, Hsiao L-D, et al. (2015) Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation. PLoS ONE 10(3): e0118473. doi:10.1371/journal.pone.0118473 Academic Editor: Rajesh Mohanraj, Faculty of Medicine & Health Sciences, UNITED ARAB EMIRATES Academic Editor: Rajesh Mohanraj, Faculty of Medicine & Health Sciences, UNITED ARAB EMIRATES Received: June 4, 2014 Accepted: January 19, 2015 Published: March 3, 2015 Copyright: © 2015 Lin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Lin 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. * chuenmao@mail.cgu.edu.tw Sphingosine-1-Phosphate Mediates ICAM-1- Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation Chih-Chung Lin1, I-Ta Lee2, Chun-Hao Hsu2, Chih-Kai Hsu2, Pei-Ling Chi2, Li-Der Hsiao1, Chuen-Mao Yang2* Introduction role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Lung inflammation is a pivotal event in the pathogenesis of chronic obstructive pulmonary dis- ease and asthma. These inflammatory responses are mediated by complex interactions between both circulating polymorphonuclear cells (PMNs) and the vascular endothelium. Several stud- ies indicate that expression of adhesion molecules on the cell surface of endothelial cells plays a critical role in the inflammatory responses [1]. Raised levels of adhesion molecules might con- tribute to the recruitment of PMNs to the regions of inflammatory tissue. These adhesion mol- ecules are classified into two major families: the Ig superfamily (e.g., ICAM-1 and VCAM-1) and the selectins (e.g., P-selectin and E-selectin) [2]. ICAM-1 is an inducible cell surface glyco- protein on several cell types, which mediates the tight adhesiveness of PMNs and thus facili- tates PMNs migration across the vascular endothelium barrier and then interacts with lung epithelium [3]. Competing Interests: The authors have declared that no competing interests exist. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite that plays important roles in allergic responses, including asthma and anaphylaxis [4]. S1P regulates numerous cel- lular responses, including motility and cytoskeletal rearrangements, formation of adherens junctions, proliferation, survival, angiogenesis, and the trafficking of immune cells [5]. These myriad effects are partly elicited by binding of S1P to a family of five G protein-coupled recep- tors (S1PRs), termed S1PR1–5. Moreover, S1P has been shown to induce lung injury and in- flammation [6]. In addition, S1P has been also shown to induce ICAM-1 or VCAM-1 i i i ll [ ] H h h i f S P l d ICAM Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite that plays important roles in allergic responses, including asthma and anaphylaxis [4]. S1P regulates numerous cel- lular responses, including motility and cytoskeletal rearrangements, formation of adherens junctions, proliferation, survival, angiogenesis, and the trafficking of immune cells [5]. These myriad effects are partly elicited by binding of S1P to a family of five G protein-coupled recep- tors (S1PRs), termed S1PR1–5. Moreover, S1P has been shown to induce lung injury and in- flammation [6]. In addition, S1P has been also shown to induce ICAM-1 or VCAM-1 expression in various cell types [7,8]. S1P Mediates ICAM-1-Dependent Monocyte Adhesion Data Availability Statement: All relevant data are within the paper. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by NSC102- 2321-B-182-011, NSC101-2320-B-182-039-MY3, and NSC101-2314-B-182-182A-112 from National Science Council, Taiwan; EMRPD1D0231 and EMRPD1D0241 from Ministry of Education, Taiwan; and CMRPD1C0102, CMRPD1B0382, Funding: This work was supported by NSC102- 2321-B-182-011, NSC101-2320-B-182-039-MY3, and NSC101-2314-B-182-182A-112 from National Science Council, Taiwan; EMRPD1D0231 and EMRPD1D0241 from Ministry of Education, Taiwan; and CMRPD1C0102, CMRPD1B0382, CMRPD1C0561, CMRPD1B0331, CMRPG3C1301, and CMRPG3B1092 from Chang Gung Medical Research Foundation, Taiwan. The funders had no CMRPD1C0561, CMRPD1B0331, CMRPG3C1301, and CMRPG3B1092 from Chang Gung Medical Research Foundation, Taiwan. The funders had no 1 / 21 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 Cell culture Human pulmonary alveolar epithelial cells (HPAEpiCs) were purchased from the ScienCell Re- search Laboratories (San Diego, CA) and grown as previously described [19]. Experiments were performed with cells from passages 4 to 8. Treatment of HPAEpiCs with DMSO or the pharmacological inhibitors alone had no significant effect on cell viability determined by a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay (data not shown). Western blot Growth-arrested cells were incubated with S1P at 37°C for the indicated time intervals. The cells were washed, scraped, collected, and centrifuged at 45000 × g at 4°C for 1 h to yield the whole cell extract, as previously described [19]. Samples were denatured, subjected to SDS- PAGE using a 10% running gel, and transferred to nitrocellulose membrane. Membranes were incubated with an anti-ICAM-1 antibody for 24 h, and then membranes were incubated with an anti-rabbit horseradish peroxidase antibody for 1 h. The immunoreactive bands were de- tected by ECL reagents. Materials Anti-ICAM-1, anti-GAPDH, anti-S1PR1, anti-S1PR2, anti-S1PR3, anti-c-Src, anti-EGFR, anti-PDGFR, anti-JNK1, anti-p42, anti-p38, anti-c-Jun, and anti-c-Fos antibodies and ICAM-1 neutralizing antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-c- Src, anti-phospho-EGFR, anti-phospho-PDGFR, anti-phospho-JNK1/2, anti-phospho-p42/ p44 MAPK, anti-phospho-p38 MAPK, anti-phospho-Akt, and anti-phospho-c-Jun antibodies were from Cell Signaling (Danver, MA). W123, JTE-013 and CAY10444 were from Cayman (Ann Arbor, MI). PP1, U0126, SP600125, SB202190, AG1478, AG1296, Genistein, Tanshinone IIA, and LY294002 were from Biomol (Plymouth Meetings, PA). BCECF/AM was from Molec- ular Probes (Eugene, OR). SDS-PAGE reagents were from MDBio Inc (Taipei, Taiwan). All other reagents were from Sigma (St. Louis, MO). Transient transfection with siRNAs Human siRNAs of scrambled, S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, and c-Fos were from Sigma (St. Louis, MO). Transient transfection of siRNAs was carried out using Metafectene transfection reagent (Biontex, Germany). siRNA (100 nM) was formulated with Metafectene transfection reagent according to the manufacturer’s instruction. S1P Mediates ICAM-1-Dependent Monocyte Adhesion the increased expression of ICAM-1 and monocyte adhesion on S1P-challenged HPAEpiCs are mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-depen- dent AP-1 activation. These results provide new insights into the mechanisms of S1P action on HPAEpiCs to regulate the expression of ICAM-1 and thus exaggerate the inflammation responses. Introduction However, the mechanisms of S1P-regulated ICAM-1 ex- pression in human pulmonary alveolar epithelial cells (HPAEpiCs) are not completely under- stood. Thus, to clarify the mechanisms of ICAM-1 induction by S1P in lung epithelium was recognized as a new therapeutic approach in the management of respiratory diseases. c-Src, a common modular participating in the crosstalk between the cytoplasmic protein ty- rosine kinases and receptors, has been shown to mediate ICAM-1 expression in various cell types [9,10]. On the other hand, previous studies indicated that c-Src regulates platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) transactivation [11], which further promotes inflammatory responses. Mitogen-activated protein kinases (MAPKs) are important components of signaling modules activated by neurotransmitters, cy- tokines, and growth factors, as well as chemical and mechanical stressors. MAPKs are also im- plicated in S1P-induced inflammatory responses [12,13]. Recent studies suggested that numerous components of the PI3K/Akt pathway play a crucial role in the expression and acti- vation of inflammatory mediators, inflammatory cell recruitment, immune cell function, air- way remodeling, and corticosteroid insensitivity in chronic inflammatory respiratory diseases [2]. Indeed, previous studies indicated that PI3K/Akt regulates the expression of adhesion mol- ecules in various cell types [10,14]. S1P has been shown to enhance Akt activation [15,16]. Al- though these studies have demonstrated that ICAM-1 induction was regulated via various signaling components, whether these signalings also participated in ICAM-1 expression and monocyte adhesion on HPAEpiCs challenged with S1P remains unknown. The ICAM-1 promoter has been shown to contain several binding sequences for various transcription factors, including AP-1 [17]. AP-1 is a heterogeneous collection of dimeric tran- scription factors comprising Jun, Fos, and ATF subunits. Among AP-1 subunits, c-Jun is the most important transcriptional activator in inflammatory status [2]. AP-1 activity is regulated by multiple mechanisms, including phosphorylation by various MAPKs [18]. Thus, in this study, we also investigated the role of AP-1 in ICAM-1 expression in HPAEpiCs challenged with S1P. In addressing these questions, experiments were undertaken to investigate the effects of S1P on expression of ICAM-1 and monocyte adhesion on HPAEpiCs. These findings suggest that PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 2 / 21 RT-PCR analysis RT-PCR analysis Total RNA was isolated using TRIzol according to the protocol of the manufacturer. The cDNA obtained from 0.5 μg total RNA was used as a template for PCR amplification as previ- ously described [11]. The primers used were as follows: ICAM-1: 50-CAAGGGGAGGTCACCCGCGAGGTG-30 (sense) 50-TGCAGTGCCCATTATGACTG-30 (anti-sense) β-actin: 50-CTAGAAGCATTTGCGGTGGACGATGGAGGG-30 (sense) 50-TGACGGGGTCACCCACACTGTGCCCATCTA-30 (anti-sense) S1PR1: 50-CCACAACGGGAGCAATAACT-30 (sense) 50-GTAAATGATGGGGTTGGTGC-30 (anti-sense) S1PR2: 50-GCAGCTTGTACTCGGAGTACCTGAAC-30 (sense) 50-CGATGGCCAACAGGATGATGGAGAAG-30 (anti-sense) S1PR3: 50-CGTCTGTGAATGCCAAGTGATGGCAACTG-30 (sense) 50-CGAGTTGTTGTGGTTGGCCACCTTACG-30 (anti-sense) S1PR4: 50-ATCACGCTGAGTGACCTGCTCA-30 (sense) 50-TGCGGAAGGAGTAGATGA-30 (anti-sense) S1PR5: 50-CTACTGTCGGGGCCGCTCAC-30 (sense) 50-CGGTTGGTGAACGTGTAGATGA-30 (anti-sense) Total RNA was isolated using TRIzol according to the protocol of the manufacturer. The cDNA obtained from 0.5 μg total RNA was used as a template for PCR amplification as previ- ously described [11]. The primers used were as follows: ICAM-1: 50-CAAGGGGAGGTCACCCGCGAGGTG-30 (sense) 50-TGCAGTGCCCATTATGACTG-30 (anti-sense) Real-time PCR Total RNA was extracted using TRIzol reagent. mRNA was reverse-transcribed into cDNA and analyzed by real-time PCR using SYBR Green PCR reagents (Applied Biosystems, Branch- burg, NJ) with primers specific for ICAM-1, c-Fos, c-Jun, and GAPDH. The levels of ICAM-1, c-Fos, and c-Jun expression were determined by normalizing to that of GAPDH expression. 3 / 21 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 S1P Mediates ICAM-1-Dependent Monocyte Adhesion S1P Mediates ICAM-1-Dependent Monocyte Adhesion power field well using a fluorescence microscope (Zeiss, Axiovert 200M). Experiments were performed in triplicate and repeated at least three times. S1P induces ICAM-1-dependent monocyte adhesion To investigate the effect of S1P on ICAM-1 expression, HPAEpiCs were incubated with various concentrations of S1P for the indicated time intervals. As shown in Fig. 1A, S1P induced ICAM-1 expression in a time- and concentration-dependent manner. In addition, S1P also en- hanced ICAM-1 mRNA expression and promoter activity in a time-dependent manner in HPAEpiCs (Fig. 1B and C). Finally, we demonstrated that adhesion of THP-1 to HPAEpiCs challenged with S1P was enhanced, which was inhibited by an ICAM-1 neutralizing antibody but not by anti-IgG Ab (Fig. 1D). Taken together, we suggest that S1P induces monocyte adhe- sion via an ICAM-1-dependent pathway in HPAEpiCs. Co-immunoprecipitation assay Cell lysates containing 1 mg of protein were incubated with 2 μg of an anti-c-Src antibody at 4°C for 24 h, and then 10 μl of 50% protein A-agarose beads was added and mixed at 4°C for 24 h. The immunoprecipitates were collected and washed thrice with a lysis buffer without Tri- ton X-100. 5X Laemmli buffer was added and subjected to electrophoresis on SDS-PAGE, and then blotted using an anti-EGFR, anti-PDGFR, or anti-c-Src antibody. Luciferase activity assay The human ICAM-1 (pIC-339) firefly luciferase was kindly provided by Dr. P. T. van der Saag (Hubrecht Laboratory, Utrecht, The Netherlands). All plasmids were prepared by using QIA- GEN plasmid DNA preparation kits. ICAM-1-luc activity was determined as previously de- scribed using a luciferase assay system (Promega, Madison, WI) [3]. Analysis of data. All the data were expressed as the mean or mean±S.E.M. The data were analyzed using a GraphPad Prism Program (GraphPad, San Diego, CA) by one-way analysis of variance (ANOVA) followed with Tukey’s post-hoc test. A P<0.05 value was considered significant. PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 Adhesion assay HPAEpiCs were grown to confluence in 6-well plates, incubated with S1P for 16 h, and then adhesion assays were performed. Briefly, THP-1 cells (human acute monocytic leukemia cell line) were labeled with a fluorescent dye, 10 μM BCECF/AM, at 37°C for 1 h in RPMI-1640 medium (Gibco BRL, Grand Island, NY) and subsequently washed by centrifugation. Conflu- ent HPAEpiCs in 6-well plates were incubated with THP-1 cells (2 × 106 cells/ml) at 37°C for 1 h. Non-adherent THP-1 cells were removed and plates were gently washed twice with PBS. The numbers of adherent THP-1 cells were determined by counting four fields per 200X high- PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 4 / 21 S1P Mediates ICAM-1-Dependent Monocyte Adhesion Fig 1. S1P induces ICAM-1-dependent monocyte adhesion. (A) HPAEpiCs were incubated with various concentrations of S1P for the indicated time intervals. ICAM-1 protein expression was detected by Western blot. (B) Cells were incubated with 10 M S1P for the indicated time intervals. The ICAM-1 mRNA expression was determined by RT-PCR. (C) Cells were incubated with 10 M S1P for the indicated time intervals. The ICAM-1 mRNA expression and promoter activity were analyzed by real-time PCR and promoter assay, respectively. (D) Cells were pretreated with an ICAM-1 neutralizing antibody or IgG antibody, and then incubated with S1P for 16 h. Then THP-1 cells adherence was measured. Data are expressed as mean±S.E.M. of three independent experiments. *P<0.05; #P<0.01, as compared with the cells exposed to vehicle alone (A-C) or S1P alone (D). Fig 1. S1P induces ICAM-1-dependent monocyte adhesion. (A) HPAEpiCs were incubated with various concentrations of S1P for the indicated time intervals. ICAM-1 protein expression was detected by Western blot. (B) Cells were incubated with 10 M S1P for the indicated time intervals. The ICAM-1 mRNA expression was determined by RT-PCR. (C) Cells were incubated with 10 M S1P for the indicated time intervals. The ICAM-1 mRNA expression and promoter activity were analyzed by real-time PCR and promoter assay, respectively. (D) Cells were pretreated with an ICAM-1 neutralizing antibody or IgG antibody, and then incubated with S1P for 16 h. Then THP-1 cells adherence was measured. Data are expressed as mean±S.E.M. of three independent experiments. *P<0.05; #P<0.01, as compared with the cells exposed to vehicle alone (A-C) or S1P alone (D). doi:10.1371/journal.pone.0118473.g001 S1PR3 play critical roles in S1P-induced ICAM-1 expression in HPAEpiCs and monocyte adhesion. S1PR3 play critical roles in S1P-induced ICAM-1 expression in HPAEpiCs and monocyte adhesion. S1PR1 and S1PR3 play key roles in S1P-induced ICAM-1 expression S1PR1, S1PR2, and S1PR3 are ubiquitously expressed, whereas the levels of S1PR4 and S1PR5 expression are predominantly existed in immune cells, CNS, and some organs [5]. Which types of S1P receptors expressed on HPAEpiCs are still unclear. Therefore, we identified the expres- sion of S1P receptors on HPAEpiCs. As shown in Fig. 2A, S1PR1, S1PR2, and S1PR3 are ex- pressed on HPAEpiCs, determined by RT-PCR. On the other hand, we observed that pretreatment with the selective inhibitor of S1PR1 (W123) or S1PR3 (CAY10444) markedly re- duced S1P-induced ICAM-1 protein levels (Fig. 2B). Indeed, pretreatment with the inhibitor of S1PR2 (JTE-013) had no effect on S1P-induced ICAM-1 expression (data not shown). In addi- tion, pretreatment with W123 or CAY10444 markedly inhibited S1P-induced ICAM-1 mRNA levels and promoter activity (Fig. 2C). These two inhibitors also attenuated the monocyte adhe- sion to HPAEpiCs challenged with S1P (Fig. 2D). To further confirm the roles of S1PR1 and S1PR3 in S1P-induced ICAM-1 expression, siRNA of S1PR1, S1PR2, or S1PR3 was used. As shown in Fig. 2E, transfection with S1PR1 and S1PR3 but not S1PR2 siRNA significantly re- duced S1P-induced ICAM-1 expression in HPAEpiCs. Thus, we demonstrate that S1PR1 and S1PR1, S1PR2, and S1PR3 are ubiquitously expressed, whereas the levels of S1PR4 and S1PR5 expression are predominantly existed in immune cells, CNS, and some organs [5]. Which types of S1P receptors expressed on HPAEpiCs are still unclear. Therefore, we identified the expres- sion of S1P receptors on HPAEpiCs. As shown in Fig. 2A, S1PR1, S1PR2, and S1PR3 are ex- pressed on HPAEpiCs determined by RT-PCR On the other hand we observed that pretreatment with the selective inhibitor of S1PR1 (W123) or S1PR3 (CAY10444) markedly re- duced S1P-induced ICAM-1 protein levels (Fig. 2B). Indeed, pretreatment with the inhibitor of S1PR2 (JTE-013) had no effect on S1P-induced ICAM-1 expression (data not shown). In addi- tion, pretreatment with W123 or CAY10444 markedly inhibited S1P-induced ICAM-1 mRNA levels and promoter activity (Fig. 2C). These two inhibitors also attenuated the monocyte adhe- sion to HPAEpiCs challenged with S1P (Fig. 2D). To further confirm the roles of S1PR1 and S1PR3 in S1P-induced ICAM-1 expression, siRNA of S1PR1, S1PR2, or S1PR3 was used. As shown in Fig. 2E, transfection with S1PR1 and S1PR3 but not S1PR2 siRNA significantly re- duced S1P-induced ICAM-1 expression in HPAEpiCs. Thus, we demonstrate that S1PR1 and PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 5 / 21 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 c-Src plays a critical role in S1P-induced ICAM-1 expression c-Src, a common modulator participating in the crosstalk between the cytoplasmic protein ty- rosine kinases and receptors, has been shown to mediate ICAM-1 expression in various cell types [9,10]. Thus, we investigated whether c-Src was involved in S1P-induced ICAM-1 expres- sion in HPAEpiCs. As shown in Fig. 3A and B, pretreatment with the inhibitor of c-Src (PP1) markedly reduced S1P-induced ICAM-1 protein and mRNA expression and promoter activity. We confirmed the role of c-Src in S1P-induced ICAM-1 expression in HPAEpiCs by using c-Src siRNA. As shown in Fig. 3C, transfection with c-Src siRNA markedly reduced the c-Src protein expression, and then inhibited S1P-induced ICAM-1 expression. In addition, c-Src in- hibition by PP1 also attenuated monocyte adhesion to HPAEpiCs challenged with S1P (Fig. 3D). Finally, we observed that S1P could enhance c-Src phosphorylation in a time- 6 / 21 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 *P<0.05; #P<0.01, as compared with the cells exposed to S1P alone (B, C, and D) or transfected with siRNA of scrambled+S1P (E). doi:10.1371/journal.pone.0118473.g002 Fig 3. S1P induces ICAM-1 expression via c-Src. (A) HPAEpiCs were pretreated with PP1 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with PP1 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were transfected with siRNA of scrambled or c-Src, and then incubated with S1P (10 μM) for 16 h. The levels of c-Src and ICAM-1 proteins were determined by Western blot. (D) Cells were pretreated with PP1 (10 M) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (E) Cells were transfected with siRNA o scrambled or c-Src, and then incubated with S1P for the indicated time intervals. The levels of phospho-c-Src protein were determined by Western blot. Data are expressed as mean (C, E) or mean±S.E.M (A, B, D) of three independent experiments. *P<0.05; #P<0.01, as compared with the cells exposed to S1P alone (A, B, D) or transfected with siRNA of scrambled+S1P (C, E). doi:10.1371/journal.pone.0118473.g003 Fig 3. S1P induces ICAM-1 expression via c-Src. (A) HPAEpiCs were pretreated with PP1 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with PP1 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were transfected with siRNA of scrambled or c-Src, and then incubated with S1P (10 μM) for 16 h. The levels of c-Src and ICAM-1 proteins were determined by Western blot. (D) Cells were pretreated with PP1 (10 M) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (E) Cells were transfected with siRNA of scrambled or c-Src, and then incubated with S1P for the indicated time intervals. The levels of phospho-c-Src protein were determined by Western blot. Data are expressed as mean (C, E) or mean±S.E.M (A, B, D) of three independent experiments. scrambled, S1PR1, S1PR2, or S1PR3, and then incubated with S1P (10 μM) for 16 h. The levels of S1PR1, S1PR2, S1PR3, and ICAM-1 proteins were determined by Western blot. Data are expressed as mean (E) or mean±S.E.M. (B, C, and D) of three independent experiments. *P<0.05; #P<0.01, as compared with the cells exposed to S1P alone (B, C, and D) or transfected with siRNA of scrambled+S1P (E). S1P Mediates ICAM-1-Dependent Monocyte Adhesion S1P Mediates ICAM-1-Dependent Monocyte Adhesion Fig 2. S1P up-regulates ICAM-1 expression via S1PR1 and S1PR3 in HPAEpiCs. (A) The mRNA expression of various S1P receptors on HP determined by RT-PCR. (B) Cells were pretreated with W123 or CAY10444 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein ex was determined by Western blot. (C) Cells were pretreated with W123 (10 M) or CAY10444 (10 M) for 1 h, and then incubated with S1P for 4 h. T mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (D) Cells were pretreated with W1 CAY10444 (10 M) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (E) Cells were transfected with siRN Fig 2. S1P up-regulates ICAM-1 expression via S1PR1 and S1PR3 in HPAEpiCs. (A) The mRNA expression of various S1P receptors on HPAEpiCs was determined by RT-PCR. (B) Cells were pretreated with W123 or CAY10444 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (C) Cells were pretreated with W123 (10 M) or CAY10444 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (D) Cells were pretreated with W123 (10 M) or CAY10444 (10 M) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (E) Cells were transfected with siRNA of PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 7 / 21 S1P Mediates ICAM-1-Dependent Monocyte Adhesion S1P Mediates ICAM-1-Dependent Monocyte Adhesion scrambled, S1PR1, S1PR2, or S1PR3, and then incubated with S1P (10 μM) for 16 h. The levels of S1PR1, S1PR2, S1PR3, and ICAM-1 proteins were determined by Western blot. Data are expressed as mean (E) or mean±S.E.M. (B, C, and D) of three independent experiments. *P<0.05; #P<0.01, as compared with the cells exposed to S1P alone (B, C, and D) or transfected with siRNA of scrambled+S1P (E). scrambled, S1PR1, S1PR2, or S1PR3, and then incubated with S1P (10 μM) for 16 h. The levels of S1PR1, S1PR2, S1PR3, and ICAM-1 proteins were determined by Western blot. Data are expressed as mean (E) or mean±S.E.M. (B, C, and D) of three independent experiments. S1P induces ICAM-1 expression via EGFR and PDGFR activation Previous studies indicated that c-Src regulates platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) transactivation [11], which further promotes the expression of inflammatory genes. Thus, we investigated whether EGFR or PDGFR was involved in S1P-induced ICAM-1 expression in HPAEpiCs. As shown in Fig. 4A and B, pretreatment with the inhibitor of EGFR (AG1478) or PDGFR (AG1296) markedly re- duced S1P-induced ICAM-1 protein expression, mRNA levels, and promoter activity. We con- firmed the role of EGFR or PDGFR in S1P-induced ICAM-1 expression in HPAEpiCs by using siRNA of EGFR or PDGFR. As shown in Fig. 4C, transfection with EGFR or PDGFR siRNA markedly reduced the respective protein expression, and then inhibited S1P-induced ICAM-1 expression. We observed that S1P could enhance EGFR or PDGFR phosphorylation in a time- dependent manner in these cells, which was inhibited by AG1478 or AG1296, respectively. We further investigated the relationship of c-Src, EGFR, and PDGFR in S1P-stimulated HPAE- piCs. As shown in Fig. 4E, we found that pretreatment with PP1 markedly reduced S1P-stimu- lated EGFR and PDGFR phosphorylation, suggesting that c-Src plays as an upstream molecule in regulating S1P-stimulated EGFR and PDGFR phosphorylation. Finally, we investigated the physical association of EGFR, PDGFR, and c-Src in S1P-stimulated HPAEpiCs by immunopre- cipitation using an anti-c-Src, anti-EGFR, or anti-PDGFR antibody and Western blot. As shown in Fig. 4F, we observed that S1P time-dependently induced the formation of a c-Src/ EGFR/PDGFR complex in these cells. Taken together, we suggest that S1P-induced ICAM-1 expression is mediated through a c-Src/EGFR or a c-Src/PDGFR pathway in HPAEpiCs. *P<0.05; #P<0.01, as compared with the cells exposed to S1P alone (A, B, D) or transfected with siRNA of scrambled+S1P (C, E). g 3. S1P induces ICAM-1 expression via c-Src. (A) HPA doi:10.1371/journal.pone.0118473.g003 doi:10.1371/journal.pone.0118473.g003 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 8 / 21 S1P Mediates ICAM-1-Dependent Monocyte Adhesion dependent manner in these cells, which was inhibited by c-Src siRNA (Fig. 3E). Taken together, we suggest that S1P induces ICAM-1 expression via a c-Src-dependent pathway in HPAEpiCs. dependent manner in these cells, which was inhibited by c-Src siRNA (Fig. 3E). Taken together, we suggest that S1P induces ICAM-1 expression via a c-Src-dependent pathway in HPAEpiCs. S1P induces ICAM-1 expression via MAPKs MAPK cascades are highly conserved signaling modules downstream of receptors/sensors that relay extracellular stimuli into intracellular responses in eukaryotes. MAPKs also have been shown to regulate S1P-induced inflammatory responses [12,13]. In this study, we found that pretreatment with the inhibitor of p38 MAPK (SB202190), MEK1/2 (U0126), or JNK1/2 (SP600125) significantly reduced S1P-induced ICAM-1 protein and mRNA expression and promoter activity (Fig. 5A and B). In addition, all these three inhibitors could attenuate mono- cyte adhesion to HPAEpiCs challenged with S1P (Fig. 5C). We further used siRNA of p38, p42, or JNK1 to confirm the roles of MAPKs in S1P-induced ICAM-1 expression in HPAEpiCs. As shown in Fig. 5D, transfection with siRNA of p38, p42, or JNK1 down-regulated the expression of respective proteins and markedly reduced S1P-induced ICAM-1 expression in HPAEpiCs. To investigate whether S1P-induced ICAM-1 expression was mediated through MAPKs activa- tion, the phosphorylation of p42/p44 MAPK, JNK1/2, or p38 MAPK was observed in S1P- stimulated HPAEpiCs. As shown in Fig. 5E, S1P markedly stimulated the phosphorylation of these three MAPKs in a time-dependent manner, which was reduced by their respective inhibi- tors. Thus, we demonstrated that S1P-induced ICAM-1 expression was mediated through a MAPKs signaling pathway in HPAEpiCs. S1P Mediates ICAM-1-Dependent Monocyte Adhesion Fig 4. S1P induces ICAM-1 expression via EGFR and PDGFR. (A) HPAEpiCs were pretreated with AG1296 or AG1478 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with AG1296 (10 M) or AG1478 (10 μM) for 1 h, an then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively (C) Cells were transfected with siRNA of scrambled, EGFR, or PDGFR, and then incubated with S1P (10 μM) for 16 h. The levels of EGFR, PDGFR, and ICAM-1 proteins were determined by Western blot. (D, E) Cells were pretreated without or with AG1478, AG1296, or PP1 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-EGFR or phospho-PDGFR were determined by Western blot. (F) Cells were treated with S1P fo the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-c-Src, anti-EGFR, or anti-PDGFR antibody. The Fig 4. S1P induces ICAM-1 expression via EGFR and PDGFR. (A) HPAEpiCs were pretreated with AG1296 or AG1478 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with AG1296 (10 M) or AG1478 (10 μM) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were transfected with siRNA of scrambled, EGFR, or PDGFR, and then incubated with S1P (10 μM) for 16 h. The levels of EGFR, PDGFR, and ICAM-1 proteins were determined by Western blot. (D, E) Cells were pretreated without or with AG1478, AG1296, or PP1 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-EGFR or phospho-PDGFR were determined by Western blot. (F) Cells were treated with S1P for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-c-Src, anti-EGFR, or anti-PDGFR antibody. The immunoprecipitates were analyzed by Western blot using an anti-PDGFR, anti-EGFR, or anti-c-Src antibody. Data are expressed as mean (C, D, E) or mean ±S.E.M (A, B) of three independent experiments. S1P induces c-Src/EGFR, PDGFR-dependent p42/p44 MAPK and p38 MAPK activation Here, we investigated the relationship of c-Src, EGFR, PDGFR, and MAPKs in S1P-stimulated HPAEpiCs. As shown in Fig. 6A and B, S1P markedly induced p42/p44 MAPK and p38 MAPK phosphorylation in a time-dependent manner, which was inhibited by PP1, AG1478, 9 / 21 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 doi:10.1371/journal.pone.0118473.g004 S1P Mediates ICAM-1-Dependent Monocyte Adhesion Fig 5. S1P induces ICAM-1 expression via MAPKs. (A) HPAEpiCs were pretreated with U0126, SB202190, S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with S (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were assay, respectively. (C) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled, p38, p42, or JNK1 Fig 5. S1P induces ICAM-1 expression via MAPKs. (A) HPAEpiCs were pretreated with U0126, SB202190, or SP600125 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled, p38, p42, or JNK1, and then incubated with S1P (10 μM) for 16 Fig 5. S1P induces ICAM-1 expression via MAPKs. (A) HPAEpiCs were pretreated with U0126, SB202190, or SP600125 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled, p38, p42, or JNK1, and then incubated with S1P (10 μM) for 16 Fig 5. S1P induces ICAM-1 expression via MAPKs. (A) HPAEpiCs were pretreated with U0126, SB202190, or SP600125 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. *P<0.05; #P<0.01, as compared with the cells exposed to S1P alone (A, B, D, E) or transfected with siRNA of scrambled+S1P (C). doi:10.1371/journal.pone.0118473.g004 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 10 / 21 S1P Mediates ICAM-1-Dependent Monocyte Adhesion h. The levels of p38, p42, JNK1, and ICAM-1 proteins were determined by Western blot. (E) Cells were pretreated without or with U0126 (1 μM), SB202190 (10 M), or SP600125 (10 M) for 1 h, and then incubated with S1P (10 M) for the indicated time intervals. The levels of phospho-p42/p44 MAPK, phospho-p38 MAPK, and phospho-JNK1/2 were determined by Western blot. Data are expressed as mean (A, D) or mean±S.E.M (B, C) of three independent experiments. *P<0.05; #P<0.01, as compared with the cells exposed to S1P alone (A, B, C) or transfected with siRNA of scrambled+S1P (D). doi:10.1371/journal.pone.0118473.g005 or AG1296 in HPAEpiCs. However, we found that pretreatment with PP1 was attenuated S1P- induced JNK1/2 activation, but not AG1296 and AG1478 in these cells (Fig. 6C). To further confirm the roles of c-Src-dependent EGFR/PDGFR in S1P-induced MAPKs activation, siR- NAs of c-Src, EGFR, and PDGFR were used. As shown in Fig. 6D, transfection with siRNA of c-Src was significantly reduced S1P-induced MAPKs activation. However, S1P-induced phos- phorylation of p42/p44 MAPK and p38 MAPK but not JNK was significantly reduced by trans- fection with siRNA of EGFR, or PDGFR. Thus, we demonstrated that S1P stimulated p38 MAPK and p42/p44 MAPK phosphorylation via a c-Src/EGFR and PDGFR- dependent pathway. S1P induces ICAM-1 expression via PI3K/Akt in HPAEpiCs Previous studies indicated that PI3K/Akt regulates the expression of adhesion molecules in various cell types [10,14]. S1P has been shown to enhance Akt activation [15,16]. Thus, we in- vestigated whether PI3K/Akt were involved in S1P-induced ICAM-1 expression in HPAEpiCs. In this study, we found that pretreatment with the inhibitor of PI3K (LY294002) significantly reduced S1P-enhanced ICAM-1 protein and mRNA expression and promoter activity (Fig. 7A and B). In addition, monocyte adhesion to HPAEpiCs challenged with S1P was also reduced by AG1478, AG1296, or LY294002 (Fig. 7C). We found that S1P markedly stimulated Akt phos- phorylation in a time-dependent manner, which was inhibited by LY294002 (Fig. 7D). We fur- ther investigated the relationship of c-Src, EGFR, PDGFR, and Akt in S1P-stimulated HPAEpiCs. As shown in Fig. 7E, S1P-stimulated Akt phosphorylation was reduced by Genis- tein (an inhibitor of tyrosine protein kinases), PP1, AG1478, or AG1296 in these cells. On the other hand, we also observed the relationship between MAPKs and Akt in S1P-stimulated HPAEpiCs. As shown in Fig. 7F, we found that S1P-stimulated Akt phosphorylation was inhib- ited by SB202190 or U0126, but not SP600125. Indeed, we found that pretreatment with LY294002 had no effects on S1P-stimulated p42/p44 MAPK, JNK1/2, and p38 MAPK phos- phorylation (data not shown). Thus, we suggested that S1P-stimulated Akt phosphorylation is mediated through p42/p44 MAPK and p38 MAPK in HPAEpiCs. (B) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled, p38, p42, or JNK1, and then incubated with S1P (10 μM) for 16 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 11 / 21 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 S1P induces ICAM-1 expression via AP-1 in HPAEpiCs The ICAM-1 promoter has been shown to contain several binding sequences for various tran- scription factors, including AP-1 [17]. To investigate whether AP-1 was involved in S1P-in- duced ICAM-1 expression, the inhibitor of AP-1 (Tanshinone IIA) was used. As shown in Fig. 8A and B, pretreatment with Tanshinone IIA markedly reduced S1P-induced ICAM-1 protein and mRNA expression and promoter activity. In addition, Tanshinone IIA also attenu- ated monocyte adhesion to HPAEpiCs challenged with S1P (Fig. 8C). AP-1 is a heterogeneous collection of dimeric transcription factors comprising Jun, Fos, and ATF subunits. Among AP- 1 subunits, c-Jun and c-Fos are the most important transcriptional activators in inflammatory status. In this study, we found that S1P markedly induced c-Fos, but not c-Jun mRNA expres- sion in these cells (Fig. 8D). To further confirm the roles of c-Jun and c-Fos in S1P-induced ICAM-1 expression in HPAEpiCs, as shown in Fig. 8E, transfection with c-Jun or c-Fos siRNA significantly reduced c-Jun or c-Fos protein expression, and then inhibited S1P-induced ICAM-1 expression in HPAEpiCs. Finally, we observed that S1P time-dependently stimulated c-Jun phosphorylation, which was reduced by PP1, AG1296, AG1478, U0126, SP600125, PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 12 / 21 S1P Mediates ICAM-1-Dependent Monocyte Adhesion Fig 7. S1P induces ICAM-1 expression via PI3K/Akt. (A) HPAEpiCs were pretreated with LY294002 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with LY294002 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with AG1478 (10 M), AG1296 (10 μM), or LY294002 (10 μM) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D-F) Cells were pretreated with LY294002, Genistein, PP1, AG1478, AG1296, SB202190, SP600125, or U0126 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-Akt were determined by Western blot. Data are expressed as mean±S.E.M. of three independent experiments. #P<0.01, as compared with the cells exposed to S1P alone. doi:10.1371/journal.pone.0118473.g007 S1P Mediates ICAM-1-Dependent Monocyte Adhesion Fig 7 S1P induces ICAM-1 expression via PI3K/Akt (A) HPAEpiCs were pretreated with LY294002 for 1 h and then incubated with S1P for 16 h The Fig 7. S1P induces ICAM-1 expression via PI3K/Akt. (A) HPAEpiCs were pretreated with LY294002 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with LY294002 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with AG1478 (10 M), AG1296 (10 μM), or LY294002 (10 μM) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D-F) Cells were pretreated with LY294002, Genistein, PP1, AG1478, AG1296, SB202190, SP600125, or U0126 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-Akt were determined by Western blot. Data are expressed as mean±S.E.M. of three independent experiments. #P<0.01, as compared with the cells exposed to S1P alone. Fig 7. S1P induces ICAM-1 expression via PI3K/Akt. (A) HPAEpiCs were pretreated with LY294002 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. S1P Mediates ICAM-1-Dependent Monocyte Adhesion Fig 6. S1P induces c-Src-dependent p42/p44 MAPK and p38 MAPK activation. HPAEpiCs were pretreated without or with PP1, AG1478, or AG1296 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of (A) phospho-p42/p44 MAPK, (B) phospho-p38 MAPK, or (C) phospho-JNK1/2 were determined by Western blot. Data are expressed as representatives of three independent experiments. (D) Cells were transfected with siRNA of bl d S EGFR PDGFR d h i b d i h S1P (10 M) f h i di d i i l Th l l f h h 38 MAPK h h 42/ Fig 6. S1P induces c-Src-dependent p42/p44 MAPK and p38 MAPK activation. HPAEpiCs were pretreated without or with PP1, AG1478, or AG1296 for 1 h d h i b d i h S1P f h i di d i i l Th l l f (A) h h 42/ 44 MAPK (B) h h 38 MAPK (C) h h JNK1/2 Fig 6. S1P induces c-Src-dependent p42/p44 MAPK and p38 MAPK activation. HPAEpiCs were pretreated without or with PP1, AG1478, or AG1296 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of (A) phospho-p42/p44 MAPK, (B) phospho-p38 MAPK, or (C) phospho-JNK1/2 were determined by Western blot. Data are expressed as representatives of three independent experiments. (D) Cells were transfected with siRNA of scrambled, c-Src, EGFR, or PDGFR, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-p38 MAPK, phospho-p42/ p44 MAPK, phospho-JNK1/2, and GAPDH proteins were determined by Western blot. Fig 6. S1P induces c-Src-dependent p42/p44 MAPK and p38 MAPK activation. HPAEpiCs were pretreated without or with PP1, AG1478, or AG1296 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of (A) phospho-p42/p44 MAPK, (B) phospho-p38 MAPK, or (C) phospho-JNK1/2 were determined by Western blot. Data are expressed as representatives of three independent experiments. (D) Cells were transfected with siRNA of scrambled, c-Src, EGFR, or PDGFR, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-p38 MAPK, phospho-p42/ p44 MAPK, phospho-JNK1/2, and GAPDH proteins were determined by Western blot. doi:10.1371/journal.pone.0118473.g006 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 13 / 21 (B) Cells were pretreated with LY294002 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with AG1478 (10 M), AG1296 (10 μM), or LY294002 (10 μM) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D-F) Cells were pretreated with LY294002, Genistein, PP1, AG1478, AG1296, SB202190, SP600125, or U0126 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-Akt were determined by Western blot. Data are expressed as mean±S.E.M. of three independent experiments. #P<0.01, as compared with the cells exposed to S1P alone. doi:10.1371/journal.pone.0118473.g007 doi:10.1371/journal.pone.0118473.g007 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 14 / 21 S1P Mediates ICAM-1-Dependent Monocyte Adhesion or c-Fos, and then incubated with S1P (10 μM) for 16 h. The levels of c-Fos, c-Jun, and ICAM-1 proteins were determined by Western blot. (F) Cells were pretreated without or with PP1, AG1296, AG1478, U0126, SP600125, SB202190, or U0126 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-c-Jun were determined by Western blot. Data are expressed as mean (A, E) or mean±S.E.M. of three independent experiments. #P<0.01, as compared with the cells exposed to S1P alone (A-C), vehicle alone (D), or transfected with siRNA of scrambled+S1P (E). doi:10.1371/journal.pone.0118473.g008 SB202190, or LY294002 (Fig. 8F). Taken together, we suggest that S1P induces ICAM-1 expres- sion via an AP-1-dependent signaling in HPAEpiCs. SB202190, or LY294002 (Fig. 8F). Taken together, we suggest that S1P induces ICAM-1 expres- sion via an AP-1-dependent signaling in HPAEpiCs. S1P-evoked c-Src/EGFR and PDGFR/p38 MAPK and p42/p44 MAPK/ Akt- or JNK1/2-dependent AP-1 activation are mediated via S1PR1/3 S1P-induced ICAM-1 expression was mediated through c-Src/EGFR/PDGFR/p38 MAPK, p42/p44 MAPK/Akt- or JNK1/2-dependent AP-1 activation. However, whether these signaling pathways regulated by distinct S1PRs are still unclear. As shown in Fig. 9, S1P-stimulated phos- phorylation of c-Src, EGFR, PDGFR, Akt, MAPKs, and AP-1 was reduced by transfection with siRNA of S1PR1 or S1PR3, suggesting that S1P-induced ICAM-1 expression is mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 ac- tivation in HPAEpiCs. S1P Mediates ICAM-1-Dependent Monocyte Adhesion Fig 8. S1P induces ICAM-1 expression via AP-1. (A) HPAEpiCs were pretreated with Tanshinone IIA for 1 h, and then incubated with S1 ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with Tanshinone IIA (100 nM) for 1 h, and then incu 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells w Tanshinone IIA (100 nM) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were treated indicated time intervals. The mRNA levels of c-Jun and c-Fos were determined by real-time PCR. (E) Cells were transfected with siRNA of PLOS ONE | DOI 10 1371/j l 0118473 M h 3 2015 -1 expression via AP-1. (A) HPAEpiCs were pretreated with Tanshinone IIA for 1 h, and then incubated with S1P for 16 h. The was determined by Western blot. (B) Cells were pretreated with Tanshinone IIA (100 nM) for 1 h, and then incubated with S1P for pression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with or 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were treated with S1P for the e mRNA levels of c-Jun and c-Fos were determined by real-time PCR (E) Cells were transfected with siRNA of scrambled c-Jun Fig 8. S1P induces ICAM-1 expression via AP-1. (A) HPAEpiCs were pretreated with Tanshinone IIA for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with Tanshinone IIA (100 nM) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with Tanshinone IIA (100 nM) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were treated with S1P for the indicated time intervals. The mRNA levels of c-Jun and c-Fos were determined by real-time PCR. (E) Cells were transfected with siRNA of scrambled, c-Jun, PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 15 / 21 PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 Discussion S1P stimulates c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt- or JNK1/2-dependent AP-1 activation are mediated via S1PR1/3. (A) Cells were transfected with siRNA of scrambled, S1PR1, or S1PR3, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-c-Src, phospho-EGFR, phospho-PDGFR, phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, phospho-c-Jun, and GAPDH proteins were determined by Western blot. The figure represents one of three individual experiments (n = 3). doi:10.1371/journal.pone.0118473.g009 Fig 9. S1P stimulates c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt- or JNK1/2-dependent AP-1 activation are mediated via S1PR1/3. (A) Cells were transfected with siRNA of scrambled, S1PR1, or S1PR3, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-c-Src, phospho-EGFR, phospho-PDGFR, phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, phospho-c-Jun, and GAPDH proteins were determined by Western blot. The figure represents one of three individual experiments (n = 3). doi:10.1371/journal.pone.0118473.g009 Fig 9. S1P stimulates c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt- or JNK1/2-dependent AP-1 activation are mediated via S1PR1/3. (A) Cells were transfected with siRNA of scrambled, S1PR1, or S1PR3, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-c-Src, phospho-EGFR, phospho-PDGFR, phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, phospho-c-Jun, and GAPDH proteins were determined by Western blot. The figure represents one of three individual experiments (n = 3). doi:10 1371/journal pone 0118473 g009 Fig 9. S1P stimulates c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt- or JNK1/2-dependent AP-1 activation are mediated via S1PR1/3. (A) Cells were transfected with siRNA of scrambled, S1PR1, or S1PR3, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-c-Src, phospho-EGFR, phospho-PDGFR, phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, phospho-c-Jun, and GAPDH proteins were determined by Western blot. The figure represents one of three individual experiments (n = 3). doi:10.1371/journal.pone.0118473.g009 doi:10.1371/journal.pone.0118473.g009 monocyte adhesion was attenuated by a c-Src inhibitor PP1. Several studies have reported that c-Src is an essential component for cytokine-stimulated PDGFR or EGFR transactivation via the phosphorylation of cytoplasmic domains of EGFR or PDGFR [11,18]. The PDGF family of growth factors consists of five different disulphide-linked dimers built up of four different poly- peptide chains encoded by four different genes. These isoforms, PDGF-AA, PDGF-AB, PDGF- BB, PDGF-CC, and PDGF-DD, act via two receptor tyrosine kinases, PDGF receptors α and β [18]. Discussion Asthma and COPD are respiratory disorders characterized by various degrees of inflammation and tissue remodeling. S1P is a bioactive sphingolipid metabolite that plays important roles in allergic responses, including asthma and anaphylaxis [4]. Moreover, S1P has been shown to play a key role in inflammation via adhesion molecules, such as ICAM-1 induction, and then causes lung injury. However, the molecular mechanisms by which S1P induces ICAM-1-de- pendent monocyte adhesion are not fully understood in HPAEpiCs. The present study clearly demonstrated that S1P-induced ICAM-1 expression was regulated via S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation. Genetic silencing through transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos and pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) attenuated S1P-induced ICAM-1 expression and monocyte adhesion. Therefore, activation of S1P receptors by S1P causes inflammatory responses through ICAM-1 up-regulation. Several lines of evidence have reported that S1P-induced diverse biological effects are medi- ated through S1PRs [20]. Moreover, S1PR1, S1PR2, and S1PR3 have been shown to be express- ed on various cell types [21,22]. Indeed, we also found that S1PR1, S1PR2, and S1PR3 are expressed on HPAEpiCs. Zhang et al. indicated that S1PR2-mediated NF-κB activation con- tributes to TNF-α-induced VCAM-1 and ICAM-1 expression in endothelial cells [23]. Interest- ingly, in our study, we demonstrated that the inhibition of S1PR1 or S1PR3, but not S1PR2, significantly attenuated S1P-induced ICAM-1 expression in HPAEpiCs. Thus, we suggest that S1PR1 and S1PR3 mainly play key roles in S1P-induced ICAM-1 expression in these cells. The non-receptor tyrosine kinases of the Src family (SFK) play important roles in signal transduc- tion induced by a large variety of extracellular stimuli [18]. SFKs are signaling enzymes that have long been recognized to regulate critical cellular processes, such as proliferation, survival, migration, and metastasis [1]. Moreover, Chang et al. showed that IFN-γ induces ICAM-1 ex- pression via PKC α/c-Src activation in human NCI-H292 epithelial cells [24]. In addition, we previously indicated that TNF-α induces ICAM-1 expression via a c-Src signaling in human airway smooth muscle cells [9]. Here, we observed that S1P-induced ICAM-1 expression and PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 16 / 21 S1P Mediates ICAM-1-Dependent Monocyte Adhesion monocyte adhesion was attenuated by a c Src inhibitor PP1 Several studies have reported that Fig 9. PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 Discussion The classic PDGFs, PDGF-A and PDGF-B, undergo intracellular activation during trans- port in the exocytic pathway for subsequent secretion, while the novel PDGFs, PDGF-C and PDGF-D, are secreted as latent factors that require activation by extracellular proteases [18]. EGFR exists on the cell surface and is activated by binding of its specific ligands, including EGF and transforming growth factor- α (TGF- α). Moreover, activation of EGFR and PDGFR has been shown to induce respiratory system inflammation [1,2]. Here, we established that S1P monocyte adhesion was attenuated by a c-Src inhibitor PP1. Several studies have reported that c-Src is an essential component for cytokine-stimulated PDGFR or EGFR transactivation via the phosphorylation of cytoplasmic domains of EGFR or PDGFR [11,18]. The PDGF family of growth factors consists of five different disulphide-linked dimers built up of four different poly- peptide chains encoded by four different genes. These isoforms, PDGF-AA, PDGF-AB, PDGF- BB, PDGF-CC, and PDGF-DD, act via two receptor tyrosine kinases, PDGF receptors α and β [18]. The classic PDGFs, PDGF-A and PDGF-B, undergo intracellular activation during trans- port in the exocytic pathway for subsequent secretion, while the novel PDGFs, PDGF-C and PDGF-D, are secreted as latent factors that require activation by extracellular proteases [18]. EGFR exists on the cell surface and is activated by binding of its specific ligands, including EGF and transforming growth factor- α (TGF- α). Moreover, activation of EGFR and PDGFR has been shown to induce respiratory system inflammation [1,2]. Here, we established that S1P PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 17 / 21 S1P Mediates ICAM-1-Dependent Monocyte Adhesion Fig 10. Proposed model to illustrate the signaling pathways involved in ICAM-1 expression and monocyte adhesion in HPAEpiCs challenged with S1P. S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR and PDGFR/p38 MAPK and p42/p44 MAPK/Akt- or S1PR1/3/JNK1/2-dependent AP-1 activation. doi:10 1371/journal pone 0118473 g010 Fig 10. Proposed model to illustrate the signaling pathways involved in ICAM-1 expression and monocyte adhesion in HPAEpiCs challenged with S1P. S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR and PDGFR/p38 MAPK and p42/p44 MAPK/Akt- or S1PR1/3/JNK1/2-dependent AP-1 activation. doi:10.1371/journal.pone.0118473.g010 doi:10.1371/journal.pone.0118473.g010 induced ICAM-1-dependent monocyte adhesion via c-Src/EGFR and c-Src/PDGFR activation in HPAEpiCs. In addition, we also demonstrated that S1P could induce the formation of a c-Src/EGFR/PDGFR complex in these cells. PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 S1P Mediates ICAM-1-Dependent Monocyte Adhesion involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells [18]. In this study, we observed that the inhibition of c-Src markedly reduced S1P-induced p42/p44 MAPK, p38 MAPK, and JNK1/2, activation in HPAEpiCs. On the other hand, we also showed that S1P induced p42/p44 MAPK and p38 MAPK, but not JNK1/2, phosphorylation via c-Src/EGFR- and PDGFR-dependent cascade. The PI3Ks are a conserved family of signal transduction enzymes that are involved in cellu- lar activation, inflammatory responses, chemotaxis, and apoptosis. PI3K/Akt have been shown to be a downstream component of EGFR or PDGFR activated by different stimuli in various cell types [11,18]. This is confirmed by our observation that S1P-induced Akt phosphorylation was reduced through the inhibition of c-Src, EGFR, or PDGFR. On the other hand, we found that pretreatment with LY294002 inhibited S1P-induced ICAM-1 expression, consistent with the results obtained with that ICAM-1 expression was mediated via a PI3K/Akt cascade in IL- 1β-challenged A549 cells [10]. PI3K/Akt has been shown to regulate MAPKs activation in re- sponse to various stimuli, such as Japanese encephalitis virus and TNF-α [18,26]. In this study, we found that S1P-induced MAPKs activation was not regulated via PI3K/Akt (data not shown). Interestingly, we observed that S1P-induced Akt phosphorylation was reduced by SB202190 or U0126, but not SP600125, suggesting that S1P induced p38 MAPK- or p42/p44 MAPK-dependent Akt activation in HPAEpiCs. Indeed, Takahashi et al. showed that VEGF may stimulate PI3K/Akt through activation of the PKC and p42/p44 MAPK pathway in hepat- ic stellate cells [27]. Shi et al. also indicated that inhibition of p38 MAPK decreases Akt phos- phorylation in proteasome inhibitors-stimulated breast carcinoma cells [28]. Thus, we suggest that in HPAEpiCs, S1P-stimulated p38 MAPK and p42/p44 MAPK phosphorylation plays key roles in mediating Akt activation leading to ICAM-1 expression. It has been well established that inflammatory responses following exposure to extracellular stimuli are highly dependent on activation of AP-1, which plays an important role in the ex- pression of several target genes. The ICAM-1 promoter has been shown to contain several binding sequences for various transcription factors, including AP-1 [17]. These studies suggest that AP-1 plays a critical role in the regulation of ICAM-1 expression in the inflammatory re- sponses. Moreover, we found that AP-1 inhibition could reduce S1P-induced ICAM-1 expres- sion. In S1P-stimulated HPAEpiCs, c-Fos mRNA expression was up-regulated. In addition, S1P also stimulated c-Jun phosphorylation in these cells. However, S1P had no effects on c-Jun mRNA levels. Previous studies indicated that AP-1 is regulated by various signaling compo- nents, such as c-Src and MAPKs [17,18,26], consistent with our results indicating that in HPAEpiCs, S1P stimulated c-Jun phosphorylation via a c-Src/EGFR and PDGFR/p42/p44 MAPK and p38 MAPK/PI3K/Akt- or JNK1/2-dependent pathway. In summary, as depicted in Fig. 10, our results showed that in HPAEpiCs, S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR and PDGFR/p38 MAPK and p42/p44 MAPK/Akt- or S1PR1/3/c-Src/JNK1/2-dependent AP-1 acti- vation. These results provide new insights into the mechanisms of S1P-induced the expression of ICAM-1 and monocyte adhesion and thus exaggerate the inflammatory responses. Increased understanding of signaling mechanisms underlying ICAM-1 gene regulation will create oppor- tunities for the development of anti-inflammation therapeutic strategies. Discussion Although the detailed protein-protein interactions among c-Src, EGFR, and PDGFR are not known, our results are the first time to show a novel role of c-Src/EGFR/PDGFR complex formation in S1P-induced ICAM-1 expression in HPAE- piCs. In the future, we will further determine which domains of c-Src, EGFR, and PDGFR are involved in protein-protein interactions caused by S1P. The MAPKs regulate diverse cellular programs by relaying extracellular signals to intracel- lular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the con- ventional MAPKs, including p42/p44 MAPK, JNK1/2, and p38 MAPK [2]. MAPKs also have been shown to regulate S1P-induced inflammatory responses [12,13]. In addition, MAPKs also have been shown to regulate ICAM-1 induction and monocyte adhesion in response to various stimuli [3,17]. Indeed, in HPAEpiCs, we found that all these three MAPKs were involved in ICAM-1 expression and monocyte adhesion induced by S1P. Thus, we suggest that MAPKs play key roles in S1P-induced inflammatory responses. Lin et al. indicated that thrombin- induced NF-κB activation is mediated by a c-Src-dependent p42/p44 MAPK pathway in lung epithelial cells [25]. Our group also showed that c-Src-dependent MAPKs/AP-1 activation is PLOS ONE | DOI:10.1371/journal.pone.0118473 March 3, 2015 18 / 21 References 1. Lee IT, Yang CM (2012) Role of NADPH oxidase/ROS in pro-inflammatory mediators-induced airway and pulmonary diseases. Biochem Pharmacol 84: 581–590. doi: 10.1016/j.bcp.2012.05.005 PMID: 22587816 2. Lee IT, Yang CM (2013) Inflammatory signalings involved in airway and pulmonary diseases. Mediators Inflamm 2013: 791231. doi: 10.1155/2013/791231 PMID: 23690670 3. 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Aromatic C-H addition of ketones to imines enabled by manganese catalysis
Nature communications
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ARTICLE 1 Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Molecular Recognition and Function, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. 2 University of Chinese Academy of Sciences, Beijing 100049, China. Correspondence and requests for materials should be addressed to C.W. (email: wangcy@iccas.ac.cn) Results Optimization of Reaction conditions. As shown in Fig. 1b, we intended to develop a manganese-catalyzed aromatic C–H addi- tion of ketones to imines. At the outset, in order to simplify the reaction outcome we chose t-butyl phenyl ketone 1a and imine 2a as model substrates to screen the reaction parameters (see Sup- plementary Table 1 for more details). The optimal reaction con- ditions were obtained by using MnBr(CO)5 as a catalyst, Me2Zn/ ZnBr2 as promoters in the solvent of 1,2-dichloroethane (DCE) at 60 °C. We then evaluated the reaction chemoselectivity by using acetophenone 1v bearing α-C–H bonds as a substrate which was commonly used in the Mannich reaction (Fig. 2). Interestingly, when the reaction was carried out in the absence of MnBr(CO)5, the Mannich reaction took place overwhelmingly followed by elimination of an amine of the β-amino carbonyl intermediate to afford chalcone 6 in 46% gas chromatography–mass spectrometry (GC-MS) yield. In a sharp contrast, under the manganese catalysis product 4a resulting from aromatic C–H addition/cyclization/ elimination was obtained in 66% isolated yield. Remarkably, this represents a reversal of the usual reactivity between ortho C (sp2)–H bonds and α-C(sp3)–H bonds of ketones with imines achieved by using a transition metal catalyst. g Recently, the directed C–H transformations of ketones4,5 have attracted immense attentions due to the prevalence of the car- bonyl group in natural products, pharmaceuticals, and organic synthesis. Since the pioneering work of Ru-catalyzed aromatic C–H alkylation by Murai and others6–10, the ketone-directed C–H alkenylation11–13, arylation14–16, and amination17–19, among others20–25 have been elegantly demonstrated. Note that in most of these protocols the undesirable reactions on α-C–H bonds of ketones are not notorious by choosing suitable reaction partners. Moreover, these reactions have heavily relied on late transition metals (Ru, Rh, Pd, and Ir) so far. Developments of earth-abundant base metal catalyzed site-selective aromatic C–H transformations of ketones with more challenging imine elec- trophiles have not been reported yet. Despite of their huge syn- thetic interests, considerable challenges still remain in these processes, such as the formidably competitive Mannich and/or Aldol-type reactions of α-C–H bonds of ketones, the relatively inert reactivity of aromatic C–H bonds with a weakly coordi- nating ketone group26, and the lower catalytic reactivity of base metals compared with the precious ones. Aromatic C-H addition of ketones to imines enabled by manganese catalysis DOI: 10.1038/s41467-017-01262-4 OPEN Bingwei Zhou1,2, Yuanyuan Hu1,2, Ting Liu1,2 & Congyang Wang 1,2 Selectivity control of varied C–H bonds in a complex molecule is a long-standing goal and still a great challenge in C–H activation field. Most often, such selectivity is achieved by the innate reactivity of different C–H bonds. In this context, the classic Mannich reaction of acet- ophenone derivatives and imines is ascribed to the more reactive C(sp3)–H bonds α to the carbonyl, with the much less reactive aromatic C(sp2)–H bonds remaining intact. Herein we report an aromatic C(sp2)–H addition of ketones to imines enabled by manganese catalysis, which totally reverses the innate reactivity of C–H bonds α to the carbonyl and those on the aromatic ring. Diverse products of ortho-C–H aminoalkylated ketones, cyclized exo-olefinic isoindolines, and three-component methylated isoindolines can be successfully accessed under mild reaction conditions, which significantly expands the synthetic utilities of ketones as simple bulk chemicals. 1 Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Molecular Recognition and Function, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. 2 University of Chinese Academy of Sciences, Beijing 100049, China. Correspondence and requests for materials should be addressed to C.W. (email: wangcy@iccas.ac.cn) NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01262-4 K K etones such as acetophenone are considered among the most easily accessible and practically useful building blocks in both laboratories and chemical industries. They undergo various transformations on the α-C–H bonds with a wide range of electrophiles, which now constitute an important chapter in many textbooks of organic chemistry. Among them, the Mannich reaction, enabling an addition of the α-C–H bond to an iminium ion or imine, has been known for a long time and represents one of the most classic reactions of ketones (Fig. 1a)1–3. It proceeds easily under either acidic or basic reaction conditions to afford the β-amino carbonyl and/or other derivatives. Of note, the C (sp2)–H bonds ortho to the carbonyl of ketones remain intact during this process, which shows that the reactivity of α-C–H bonds holds an absolute superiority over that of the ortho-C–H bonds on the benzene ring. 4 5 addition of ketones to imines under mild reaction conditions, while the conventional Mannich reaction is completely sup- pressed. Moreover, cyclized exo-olefinic isoindoline and three- component methylated isoindoline derivatives can be selectively obtained. Aromatic C-H addition of ketones to imines enabled by manganese catalysis DOI: 10.1038/s41467-017-01262-4 OPEN Thus, such diverse reactivity provides a straightforward and efficient way to access varied functionalized isoindolines from simple ketones and imines. Results To address these issues, we resort to manganese-promoted C–H activation27–43, in which the stoichiometric cyclomanganation of ketones was shown by Kaesz and Nicholson as early as in 197544,45. However, the manganese-catalyzed aromatic C–H transformations of ketones remain elusive. Investigations on substrate scopes. With the optimized condi- tions in hand, the scope of ketones was first explored (Fig. 3). Aromatic ketones bearing a wide range of electronically varied functional groups on the benzene ring delivered the corre- sponding aromatic C–H addition products successfully (3a–j). Ketones containing two sterically biased C–H bonds reacted with imine 2a at the less hindered positions exclusively giving products 3k and 3l respectively. Heteroaromatic ketone 2,2-dimethyl-1- (thiophen-2-yl)propan-1-one 1m was also a viable substrate affording the expected product 3m in synthetically useful yield. Here, we describe, as our continuous interest in manganese catalysis31–36, a manganese-catalyzed site-selective aromatic C–H O N R2 R3 Acid or base H H N R2 R3 N R2 R3 Me ÷ diverse reactivities and valuable products ÷ reversing the innate reactivity of C H bonds ÷ first Mn-catalyzed C H activation of ketones + H O R2 H or others R1 R1 O N R2 R3 Me2Zn/ZnBr2 R3 = sulfonyl H H + H R1 O R1 H NHR3 R2 R1 R1 H Mn α α o o α O NHR3 R2 H R1 o α o –R3NH2 β-amino carbonyl 3 4 5 1 2 b a e and reversed reactivity of C–H bonds in ketones with imines. a The classic Mannich reaction of innately reactive α-C(sp3)–H bonds. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01262-4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01262-4 O H R = S S 2a N Ph R 4a Ph O Ph Variations 6 Yields – 66%b 1%a No MnBr(CO)5 ND 46%a MnBr(CO)5 (10 mol%) Me2Zn (1.5 equiv) ZnBr2 (1.0 equiv) DCM/PhMe, 60 °C, 10 h o N H Ph R + + H α α o O O 1v Fig. 2 Evaluating the C–H bond selectivity of ketones by manganese-based catalytic system. aGC-MS yield. bIsolated yield. DCM dichloromethane, ND not detected O H R = S S 2a N Ph R 4a Ph O Ph Variations 6 Yields – 66%b 1%a No MnBr(CO)5 ND 46%a MnBr(CO)5 (10 mol%) Me2Zn (1.5 equiv) ZnBr2 (1.0 equiv) DCM/PhMe, 60 °C, 10 h o N H Ph R + + H α α o O O 1v Fig. 2 Evaluating the C–H bond selectivity of ketones by manganese-based catalytic system. aGC-MS yield. bIsolated yield. DCM dichloromethane, ND not detected O H 2a o N H Ph R + H α 1v Ph 1v 6 Fig. 2 Evaluating the C–H bond selectivity of ketones by manganese-based catalytic system. aGC-MS yield. bIsolated yield. DCM dichloromethane, ND not detected O tBu NHR3 Ph R1 3a: R1 = H, 80% 3b: R1 = Me, 63% 3c: R1 = Ph, 74% 3d: R1 = OMe, 73% 3e: R1 = SMe, 61% 3f: R1 = NMe2, 42% 3g R1 = F, 60% 3h: R1 = Cl, 64% 3i: R1 = Br, 55% 3j: R1 = I, 60% O tBu NHR3 Ph S tBu O NHR3 Ph 3m: 42% 3l: 59% O NHR3 Ph 3p: 53%c N N N Ph NHR3 3s: 58% N N NHR3 Ph 3t: 61% N N NHR3 Ph 3u: 52% O tBu NHR3 Ph Me 3k: 63% O R2 H N R3 Ph R3 = S O O S 1 2a MnBr(CO)5 (10mol%) Me2Zn, ZnBr2 DCE/PhMe, r.t.–60 °C, 10 h H R1 O R2 NHR3 Ph R1 3 + O NHR3 Ph O NHR3 Ph O NHR3 Ph O NHR3 Ph 3r: 82% Me 3q: 65% 3o: 58%b 3n: 71%a H H H H H H H H H H H Fig. 3 Scope of ketones for the mono-C–H addition reaction. Reaction conditions: 1 (1.5 mmol), 2a (0.5 mmol), MnBr(CO)5 (0.05 mmol), Me2Zn (0.75 mmol, 1.2 M in toluene), ZnBr2 (0.5 mmol), DCE (0.4 M), 60 °C, 10 h. Results b Mn- tho-C(sp2)–H addition by reversing the reactivity of C–H bonds (this work) O N R2 R3 Acid or base H H + H O R2 H or others R1 R1 α o O NHR3 R2 H R1 o α –R3NH2 β-amino carbonyl a O N R2 R3 Acid or base H H + H R1 α o O NHR3 R2 H R1 o α –R β-amino carbonyl a a or base O NHR3 R2 H R1 o α β-amino carbonyl O R2 H or others R1 –R3NH2 or others N R2 R3 N R2 R3 Me O R1 H NHR3 R2 R1 R1 H α o 3 4 5 N R2 R3 N R2 R3 Me ÷ diverse reactivities and valuable products ÷ reversing the innate reactivity of C H bonds ÷ first Mn-catalyzed C H activation of ketones O N R2 R3 Me2Zn/ZnBr2 R3 = sulfonyl H H + H R1 O R1 H NHR3 R2 R1 R1 H Mn α o α o 3 4 5 1 2 b N R2 R3 N R3 Me ÷ reversing the innate reactivity of C H bonds O N R2 R3 Me2Zn/ZnBr2 R3 = sulfonyl H H + H R1 O R1 H NHR3 R2 R1 R1 H Mn α o α o 3 4 1 2 b O N R2 R3 Me2Zn/ZnBr2 R3 = sulfonyl H H + H R1 Mn α o 1 2 b b 4 2 3 ÷ diverse reactivities and valuable products ÷ reversing the innate reactivity of C H bonds ÷ first Mn-catalyzed C H activation of ketones 5 Fig. 1 Innate and reversed reactivity of C–H bonds in ketones with imines. a The classic Mannich reaction of innately reactive α-C(sp3)–H bonds. b Mn- catalyzed ortho-C(sp2)–H addition by reversing the reactivity of C–H bonds (this work) NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01262-4 Next, the scope of imines was surveyed with ketone 1a as the model substrate (Fig. 4). Both electron-donating and electron- withdrawing groups were well tolerated in the reaction with the former ones giving relatively higher yields of the aromatic C–H addition products (3v-A). Ortho- and meta-substituents on the benzene ring of imines showed comparable effect on the reaction yields (3v vs. 3B, 3C). Naphthyl imines with extended conjugation delivered the expected products smoothly (3D, 3E). It seemed that the steric hindrance had limited influence on the reaction outcome (3B, 3D). Heteroaromatic imine and p- tolylsulfonyl imine were also amenable to this protocol (3F, 3G). Unfortunately, aliphatic imines failed to afford the corresponding products under the reaction conditions. isomer of 3H was in 96% ee, which reflected the ee value of ketone 1C. Furthermore, the structural configuration of the major diastereo-isomer was confirmed by single-crystal X-ray diffrac- tion analysis. y Interestingly, the exo-olefinic isoindoline products 4 could be selectively obtained from the reactions of imines and aryl alkyl ketones bearing α-C–H bonds by slightly tuning the reaction conditions (Fig. 6). Specifically, acetophenone 1v was treated with imine 2a at 60 °C for 2 h under the otherwise same conditions giving exo-olefinic isoindoline 4a in 66% isolated yield. The structure of 4a was unambiguously confirmed by single-crystal X- ray diffraction analysis. Introducing a methyl group into the para, meta, or ortho position of acetophenone resulted in the formation of the expected products successfully (4b–d). Remarkably, the reaction of propiophenone with imine 2a provided exclusively isoindoline 4e with an E-configuration of the exo-cyclic C = C bond, which was again unambiguously confirmed by single- When a chiral ketone, 1C of 96% ee, was used as a substrate and treated with imine 2a under the similar reaction conditions, the corresponding ortho-aminoalkylated product 3H was isolated in 66% yield with a dr value of 9.4:1 (Fig. 5). ARTICLE The major diastereo- O tBu H N R3 O tBu NHR3 1a 2 3 H R4 R4 O tBu NHR3 3v: R4 = Me, 64% 3w: R4 = OMe, 76% 3x: R4 = F, 56% 3y: R4 = Cl, 45% 3z: R4 = Br, 55% 3A: R4 = CF3, 42% R4 O tBu NHR3 Me 3B: 63% O tBu NHR3 3C: 77% Me O tBu NHR3 3D: 68% O tBu NHR3 3E: 74% O tBu NHR3 3F: 68% O O tBu NHTs 3G: 70% R3 = S O O S MnBr(CO)5 (10 mol%) Me2Zn, ZnBr2 DCE/PhMe, 60 oC, 10 h + Fig. 4 Scope of imines for the mono-C–H addition reaction. Reaction conditions: 1a (1.5 mmol), 2 (0.5 mmol), MnBr(CO)5 (0.05 mmol), Me2Zn (0.75 mmol, 1.2 M in toluene), ZnBr2 (0.5 mmol), DCE (0.4 M), 60 °C, 10 h. DCE 1,2-dichloroethane O tBu NHR3 3 R4 O tBu H N R3 O tBu NHR3 1a 2 3 H R4 R4 R3 = S O O S MnBr(CO)5 (10 mol%) Me2Zn, ZnBr2 DCE/PhMe, 60 oC, 10 h + O tBu H N R3 1a 2 H R4 R3 = S O O S MnBr(CO)5 (10 mol%) Me2Zn, ZnBr2 DCE/PhMe, 60 oC, 10 h + R3 = S O O S MnBr(CO)5 (10 mol%) Me2Zn, ZnBr2 DCE/PhMe, 60 oC, 10 h O tBu H N R3 1a 2 H R4 + O tBu H 1a NHR3 2 3 1a O tBu NHR3 R4 O tBu NHR3 3D: 68% O tBu NHR3 3C: 77% Me O tBu NHR3 Me 3B: 63% NHR3 NHR3 NHR3 Me 3B: 63% 3C: 77% O tBu NHR3 3E: 74% O tBu NHR3 3F: 68% O O tBu NHTs 3G: 70% NHR3 NHTs NHR3 3G: 70% 3D: 68% 3E: 74% Fig. 4 Scope of imines for the mono-C–H addition reaction. Reaction conditions: 1a (1.5 mmol), 2 (0.5 mmol), MnBr(CO)5 (0.05 mmol), Me2Zn (0.75 mmol, 1.2 M in toluene), ZnBr2 (0.5 mmol), DCE (0.4 M), 60 °C, 10 h. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01262-4 a1n (2.0 mmol), DCM (0.1 M), r.t., 16 h. b1o (2.0 mmol), DCM (0.1 M), r.t., 1 h. c1p (2.0 mmol), DCM (0.1 M), 40 °C, 1 h. DCE 1,2-dichloroethane, DCM dichloromethane O R2 H N R3 Ph R3 = S O O S 1 2a MnBr(CO)5 (10mol%) Me2Zn, ZnBr2 DCE/PhMe, r.t.–60 °C, 10 h H R1 O R2 NHR3 Ph R1 3 + O R2 H N R3 Ph 1 2a H R1 + % HR3 Ph O NHR3 % H 2a % O NHR3 H O tBu NHR3 Ph R1 H NHR3 Ph O tBu NHR3 Ph Me 3k: 63% H O tBu NHR3 Ph 3l: 59% H S tBu O NHR3 Ph 3m: 42% NHR3 3l: 59% 3k: 63% 3m: 42% 3l: O NHR3 Ph 3o: 58%b H O NHR3 Ph 3n: 71%a H O NHR3 Ph 3q: 65% H H H O NHR3 Ph 3p: 53%c H Ph 3o: 58%b 3p: 53%c 3n: 71%a O NHR3 Ph 3r: 82% Me 3n: 71%a H H N N NHR3 Ph 3t: 61% N N N Ph NHR3 3s: 58% N N NHR3 Ph 3u: 52% NHR3 NHR3 NHR3 NHR3 3t: 61% 3u: 52% 3s: 58% Fig. 3 Scope of ketones for the mono-C–H addition reaction. Reaction conditions: 1 (1.5 mmol), 2a (0.5 mmol), MnBr(CO)5 (0.05 mmol), Me2Zn (0.75 mmol, 1.2 M in toluene), ZnBr2 (0.5 mmol), DCE (0.4 M), 60 °C, 10 h. a1n (2.0 mmol), DCM (0.1 M), r.t., 16 h. b1o (2.0 mmol), DCM (0.1 M), r.t., 1 h. c1p (2.0 mmol), DCM (0.1 M), 40 °C, 1 h. DCE 1,2-dichloroethane, DCM dichloromethane Replacing the t-butyl group of 1a by other alkyl groups bearing α- C–H bonds, the reaction worked as well even at room tempera- ture or 40 °C leading to the expected products smoothly (3n–p). Of note, no Mannich-type products were detected in these reactions and the carbonyl-remaining products provide a handle for further synthetic elaborations. Importantly, benzophenone 1q and phenyl(o-tolyl)methanone 1r were also suitable substrates giving the mono-C–H addition products in good yields (3q, 3r). In addition, arenes and heteroarenes bearing nitrogen-containing directing groups could also undergo the corresponding C–H aminoalkylation reaction with the current reaction conditions (3s–u).46–51 Of note, Ackermann has elegantly disclosed the related C–H aminoalkylation of indoles with imines in the absence of zinc additives at higher tempreture.40 NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications 3 ARTICLE DCE 1,2-dichloroethane + Ph N H MnBr(CO)5 (10 mol%) ZnBr2, Me2Zn O Ph Me DCM/PhMe 40 oC, 1 h O Ph Me C27 C26 C28 S2 C4 C5 C6 C7 C8 C10 C13 C18 C17 C16 C15 C14 C12 C9 O3 O1 O2 S1 N1 C3 C25 C11 C2 C1 C20 C21 C22 C23 C24 C19 NHR3 Ph R3 = S O O S R3 3Ha 66% yieldb dr: 9.4:1c 96% ee 2a 1C 96% ee Fig. 5 A diastereoselective mono-C–H addition reaction using chiral ketone 1 C. aThe major diastereo-isomer was shown. bCombined isolated yield. cDetermined by 1H NMR analysis of the crude product DCM dichloromethane Fig. 5 A diastereoselective mono-C–H addition reaction using chiral ketone 1 C. aThe major diastereo-isomer was shown. bCombined isolated yield. cDetermined by 1H NMR analysis of the crude product. DCM dichloromethane Fig. 5 A diastereoselective mono-C–H addition reaction using chiral ketone 1 C. aThe major diastereo-isomer was shown. bCombined isolated yield. cDetermined by 1H NMR analysis of the crude product. DCM dichloromethane NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01262-4 O R2 H 1 R1 N Ph R3 4a: 66% N Ph R3 Me 4c: 53%a N Ph R3 Me 4d: 43% N Ph R3 4f: 61%b N Ph R3 4e: 42% N Ph R3 4g: 47%b R1 N R3 N R3 OMe 4h: 53% N R3 Me 4l: 62% N Ts 4m: 65% R4 4 Me R3 = S O O S MnBr(CO)5 (10 mol%) Me2Zn, ZnBr2 DCM/PhMe, 60 oC, 2 h N R3 2 H R4 + R N Ph R3 4b: 62% Me N R3 Br 4k: 42% 4j: 67% N R3 F N R3 Me 4i: 43% C18 C17 C16 C15 C2 C6 C19 C5 O2 S2 C1 C2 C3 C4 S1 O1 N1 C8 C9 C10 C11 C12 C13 C10 C9 S2 C1 O1 C17 C18 C19 C20 C4 C3 C2 C5 C7 N1 S1 O2 C8 C11 C12 C6 C16 C13 C14 C15 C14 Fig. 6 Substrate scope for the [3 + 2] annulations giving exo-olefinic isoindolines 4. Reaction conditions: 1 (2.0 mmol), 2 (0.5 mmol), MnBr(CO)5 (0.05 mmol), Me2Zn (0.75 mmol, 1.2 M in toluene), ZnBr2 (0.5 mmol), DCM (0.1 M), 60 °C, 2 h. aCombined yield of two regioisomers (3.9/1), major isomer 4c was shown. bMe2Zn (2.0 equiv.), 100 °C, 10 h. ARTICLE DCM dichloromethane 2 1 3 N Ph R3 Me 4d: 43% N Ph R3 4b: 62% Me N Ph R3 4a: 66% N Ph R3 4b: 62% Me C18 C17 C16 C15 C2 C6 C19 C5 O2 S2 C1 C2 C3 C4 S1 O1 N1 C8 C9 C10 C11 C12 C13 C14 N Ph R3 Me 4c: 53%a Ph 4d: 43% 4h: 53% N R3 Me Me 4h: 53% Me 4f: 61%b N R3 Br 4k: 42% 4h: 53% M 4e: 42% 4f: 61%b 4g: 47%b 4e: 42% N R3 OMe 4j: 67% N R3 F 4i: 43% 4e: 42% N R3 OMe 4i: 43% N R3 Me 4l: 62% N Ts 4m: 65% N R3 4m: 65% 4j: 67% 4l: 62% 4k: 42% 4i: 43% Fig. 6 Substrate scope for the [3 + 2] annulations giving exo-olefinic isoindolines 4. Reaction conditions: 1 (2.0 mmol), 2 (0.5 mmol), MnBr(CO)5 (0.05 mmol), Me2Zn (0.75 mmol, 1.2 M in toluene), ZnBr2 (0.5 mmol), DCM (0.1 M), 60 °C, 2 h. aCombined yield of two regioisomers (3.9/1), major isomer 4c was shown. bMe2Zn (2.0 equiv.), 100 °C, 10 h. DCM dichloromethane N R4 R3 Me R2 5 O R2 H N R3 R4 R3 = S O O S 1 2 MnBr(CO)5 (10 mol%) Me2Zn (2.0 equiv.) ZnBr2 (1.0 equiv.) DCM/PhMe, 100 oC, 10 h N Ph R3 Me 5a: 63% (3.1 : 1) 5b: 53% (9 : 1) N Ph R3 Me N Ph R3 Me 5d: 65% (7.6 : 1) H 5e: 41% (16 : 1) N Ph Ts Me + N Ph R3 Me 5c: 44% (3.3 : 1) 5g: 46% (9.8 : 1) N (p-F)C6H4 R3 Me N (p-OMe)C6H4 R3 Me 5f: 57% (5.8 : 1) C11 C12 C10 C3 C4 C2 C7 C5 C6 C15 C16 C17 C18 C13 C8 C14 O2 S1 N1 C1 C9 O1 C20 C19 C21 C22 S2 Fig. 7 Substrate scope for the three-component reaction giving isoindolines 5. Reaction conditions: 1 (2.0 mmol), 2 (0.5 mmol), MnBr(CO)5 (0.05 mmol), Me2Zn (2.0 mmol, 1.2 M in toluene), ZnBr2 (0.5 mmol), DCM (0.1 M), 100 °C, 10 h. The ratio of diastereoisomers (dr) was shown in parentheses. ARTICLE The steric compulsion between the methyl group and (2-thienyl)sulfonyl group might account for the observed configuration of the double bond. 1-Tetralone and 1-benzosuberone were also proved to be suitable substrates affording the corresponding tricyclic products successfully under the slightly modified reaction conditions (4f, 4g). A series of imines bearing electronically varied functional groups were applicable to this reaction leading to the expected exo-olefinic isoindoline products smoothly (4h–l). p-Tolylsulfonyl imine was again susceptible to the reaction conditions giving the corre- sponding product in comparable yield (4m). butyl phenyl ketone 1a could not afford the corresponding three- component product presumably due to the increased steric hindrance of the congested tetra-substituted carbon center. Mechanistic studies. To clarify the possible reaction pathways, a range of mechanistic experiments were conducted. First, the stoichiometric reaction of ketone 1a with MnBr(CO)5 was examined and no product was detected (Fig. 8a). While no reaction occurred with the assistance of ZnBr2, the addition of Me2Zn to the reaction resulted in the formation of five-membered manganacycle Mn-I in 28% isolated yield. Also, enolizable acet- ophenone 1v could delivered the corresponding manganacycle Mn-I′ in comparable yield, whose structure was confirmed by single-crystal X-ray diffraction analysis. MnMe(CO)5, generated in situ from the transmetalation of MnBr(CO)5 with Me2Zn, might play a critical role in the step of C–H bond cleavage35. Second, treatment of Mn-I with imine 2a afforded the C–H addition product 3a in 27% 1H NMR yield (Fig. 8b). The reaction yields could be further improved by adding either Me2Zn or ZnBr2. Finally, the reactions of ketone 1a and imine 2a using During our further investigations on the reaction parameters, we surprisingly found that a three-component reaction of ketone, imine, and dimethylzinc could be achieved simply by utilizing two equivalents of dimethylzinc at an elevated temperature under the otherwise same conditions (Fig. 7). Thus, a range of isoindolines bearing a tetra-substituted carbon center could be easily accessed from simple ketones and imines with moderate to good diastereoselectivity (5a–g). The structures of the major cis- diastereoisomers 5c and 5d were both confirmed by single-crystal X-ray diffraction analysis. It should be noted that the use of t- R O H MnBr(CO)5 Additive DCE/PhMe 60 oC, 10 h O R Mn(CO)4 Additive isolated yield Me2Zn (1.5 equiv.) 28% Mn-I′ (R = Me): 24% – n.r. ZnBr2 (1.0 equiv.) n.r. ARTICLE DCM dichloromethane N R4 R3 Me R2 5 O R2 H N R3 R4 R3 = S O O S 1 2 MnBr(CO)5 (10 mol%) Me2Zn (2.0 equiv.) ZnBr2 (1.0 equiv.) DCM/PhMe, 100 oC, 10 h H + 5 1 5g: 46% (9.8 : 1) N (p-F)C6H4 R3 Me C11 C12 C10 C3 C4 C2 C7 C5 C6 C15 C16 C17 C18 C13 C8 C14 O2 S1 N1 C1 C9 O1 C20 C19 C21 C22 S2 5b: 53% (9 : 1) N Ph R3 Me N Ph R3 Me 5a: 63% (3.1 : 1) N Ph R3 Me 5c: 44% (3.3 : 1) Ph Ph Ph 5b: 53% (9 : 1) Ph Ph 5c: 44% (3.3 : 1) 5c: 44% (3.3 : 1) 5a: 63% (3.1 : 1) 5b: 53% (9 : 1) N (p-OMe)C6H4 R3 Me 5f: 57% (5.8 : 1) N Ph R3 Me 5d: 65% (7.6 : 1) 5e: 41% (16 : 1) N Ph Ts Me 5e: 41% (16 : 1) 5d: 65% (7.6 : 1) Fig. 7 Substrate scope for the three-component reaction giving isoindolines 5. Reaction conditions: 1 (2.0 mmol), 2 (0.5 mmol), MnBr(CO)5 (0.05 mmol), Me2Zn (2.0 mmol, 1.2 M in toluene), ZnBr2 (0.5 mmol), DCM (0.1 M), 100 °C, 10 h. The ratio of diastereoisomers (dr) was shown in parentheses. DCM dichloromethane Fig. 7 Substrate scope for the three-component reaction giving isoindolines 5. Reaction conditions: 1 (2.0 mmol), 2 (0.5 mmol), MnBr(CO)5 (0.05 mmol), Me2Zn (2.0 mmol, 1.2 M in toluene), ZnBr2 (0.5 mmol), DCM (0.1 M), 100 °C, 10 h. The ratio of diastereoisomers (dr) was shown in parentheses. DCM dichloromethane NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunication 5 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01262-4 crystal X-ray diffraction analysis. The steric compulsion between the methyl group and (2-thienyl)sulfonyl group might account for the observed configuration of the double bond. 1-Tetralone and 1-benzosuberone were also proved to be suitable substrates affording the corresponding tricyclic products successfully under the slightly modified reaction conditions (4f, 4g). A series of imines bearing electronically varied functional groups were applicable to this reaction leading to the expected exo-olefinic isoindoline products smoothly (4h–l). p-Tolylsulfonyl imine was again susceptible to the reaction conditions giving the corre- sponding product in comparable yield (4m). crystal X-ray diffraction analysis. NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications ARTICLE 1a, R = tBu 1v, R = Me Mn-I (R = tBu) a + Mn-I 2a Additive DCE/PhMe 60 oC, 10 h 3a Additive 1H NMR yield Me2Zn (1.5 equiv.) 27% – 68% ZnBr2 (1.0 equiv.) 54% b O tBu Mn(CO)4 + N H Ph R3 O tBu NHR3 Ph R3 = S O O S cat. [Mn] MnMe(CO)5 74% Mn-I 80% c tBu O H 1a 2a + N H Ph R3 3a O tBu NHR3 Ph cat. [Mn] (10 mol%) Me2Zn, ZnBr2 DCE/PhMe 60 oC, 10 h 1H NMR yield O5 O4 O3 O2 C9 O1 C7 C8 C11 C10 C2 C12 C1 C3 C6 C5 C4 echanistic experiments. a Isolation of key intermediates Mn-I and Mn-I′. b Stoichiometric reactions of Mn-I and imine 2a. c Reactions using Mn-I Me(CO)5 as a catalyst. DCE 1,2-dichloroethane NATURE COMMUNICATIONS| 8 1169 | DOI 10 1038/ 4146 01 01262 4| / i i R O H MnBr(CO)5 Additive DCE/PhMe 60 oC, 10 h O R Mn(CO)4 Additive isolated yield Me2Zn (1.5 equiv.) 28% Mn-I′ (R = Me): 24% – n.r. ZnBr2 (1.0 equiv.) n.r. 1a, R = tBu 1v, R = Me Mn-I (R = tBu) a + O5 O4 O3 O2 C9 O1 C7 C8 C11 C10 C2 C12 C1 C3 C6 C5 C4 a R H MnBr(CO)5 1a, R = tBu 1v, R = Me + O5 O4 O3 O2 C9 O1 C7 C8 C11 C10 C2 C12 C1 C3 C6 C5 C4 H DCE/PhMe 60 oC, 10 h Mn(CO)4 Additive isolated yield Me2Zn (1.5 equiv.) 28% Mn-I′ (R = Me): 24% – n.r. ZnBr2 (1.0 equiv.) n.r. 1a, R = tBu 1v, R = Me Mn-I (R = tBu) Mn-I 2a Additive DCE/PhMe 60 oC, 10 h 3a Additive 1H NMR yield Me2Zn (1.5 equiv.) 27% – 68% ZnBr2 (1.0 equiv.) 54% b O tBu Mn(CO)4 + N H Ph R3 O tBu NHR3 Ph R3 = S O O S c O R3 tBu cat. ARTICLE [Mn] (10 mol%) Me2Zn ZnBr2 O5 O4 O3 O2 C9 O1 C7 C8 C11 C10 C2 C12 C1 C3 C6 C5 C4 , O5 O4 O3 O2 C9 O1 C7 C8 C11 C10 C2 C12 C1 C3 C6 C5 C4 Mn-I′ (R = Me): 2 Mn-I 2a Additive DCE/PhMe 60 oC, 10 h 3a Additive 1H NMR yield b O tBu Mn(CO)4 + N H Ph R3 O tBu NHR3 Ph R3 = S O O S O3 O2 C10 b Me2Zn (1.5 equiv.) 27% – 68% ZnBr2 (1.0 equiv.) 54% cat. [Mn] MnMe(CO)5 74% Mn-I 80% c tBu O H 1a 2a + N H Ph R3 3a O tBu NHR3 Ph cat. [Mn] (10 mol%) Me2Zn, ZnBr2 DCE/PhMe 60 oC, 10 h 1H NMR yield h i ti i t I l ti f k i t di t M I d M I′ b St i hi t i ti f M I d i i 2 R t c 1a Fig. 8 Mechanistic experiments. a Isolation of key intermediates Mn-I and Mn-I′. b Stoichiometric reactions of Mn-I and imine 2a. c Reactions using Mn-I and MnMe(CO)5 as a catalyst. DCE 1,2-dichloroethane NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications 6 6 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01262-4 ARTICLE deuterium was observed at the ortho positions of 1a-d5, which suggested an irreversible C–H bond cleavage step in the reaction. Next, two parallel reactions of 1a and 1a-d5 with imine 2a respectively were conducted (Fig. 9b). As a result, a kinetic isotope effect (KIE) value of 3.2 implied the C–H bond cleavage might be involved in the turnover-limiting step or in a prior step with a lower activation barrier52. 3 43 3 manganacycle Mn-I or MnMe(CO)5 as a catalyst were examined and the corresponding product 3a was formed in 74 and 80% yield, respectively (Fig. 8c). These results suggested that both the manganacycle Mn-I and MnMe(CO)5 might be the key inter- mediates in the reaction. Furthermore, deuterium-labeling experiments were carried out in order to probe the nature of the C–H bond cleavage. First, tert- butyl(pentadeuteriophenyl)methone 1a-d5 was prepared and then subjected to the reaction conditions (Fig. 9a). No loss of Based on the above results and literature clues35,43,53, a plausible reaction mechanism was depicted in Fig. 10. ARTICLE The tBu O D5 No deuterium loss kH kD KIE = kH/kD = 3.2 1a-d5: > 99% D DCE/PhMe, 60 oC, 10 h 93% a b tBu O H 1a 2a + N H Ph R3 3a O tBu NHR3 Ph MnBr(CO)5 (10 mol%) Me2Zn, ZnBr2 DCE/PhMe, 60 oC MnBr(CO)5 (10 mol%) Me2Zn, ZnBr2 tBu O D5 1a-d5: > 99% D 2a DCE/PhMe, 60 oC 3a-d4 + N H Ph R3 O tBu NHR3 Ph MnBr(CO)5 (10 mol%) Me2Zn, ZnBr2 tBu O D3 D D (>99%) (>99%) D D3 H H Fig. 9 Deuterium-labeling experiments. a Probing the reversibility of the C–H bond cleavage. b Probing the kinetic isotope effect. DCE 1,2-dichloroethane tBu O D5 1a-d5: > 99% D a a a tBu O D3 D D (>99%) (>99%) (>99%) DCE/PhMe, 60 oC, 10 h 93% MnBr(CO)5 (10 mol%) Me2Zn, ZnBr2 No deuterium loss kH b tBu O H 1a 2a + N H Ph R3 3a O tBu NHR3 Ph MnBr(CO)5 (10 mol%) Me2Zn, ZnBr2 DCE/PhMe, 60 oC H H b NHR3 3a 1a 2a 3a 3a-d4 O tBu NHR3 Ph D D3 NHR3 1a-d5: > 99% D 2a Fig. 9 Deuterium-labeling experiments. a Probing the reversibility of the C–H bond cleavage. b Probing the kinetic iso ents. a Probing the reversibility of the C–H bond cleavage. b Probing the kinetic isotope effect. DCE 1,2-dichloroethane ZnBr2 R2 O Mn CO CO CO N R4 Mn-I Mn-III Me2Zn OC R2 O ZnBr NR3 R4 1 3 H+ 2 MnBr(CO)5 + Me2Zn MnMe(CO)5 1 R2 O Mn CO CO CO Me R4 NR3 BrZn OC O Mn CO CO CO Me OC R2 R2 O Mn CO CO CO CO Mn-II Mn-IV Me Zn-I N R3 R4 ZnBr2 H H H Me H NR3 BrZnO R4 NR3 R4 R NR3 Me R4 –Zn(OH)Br Me2Zn 4 5 R R Zn-II R3 –CO Fig. 10 A proposed reaction mechanism. Key steps include formation of Mn-I followed by addition to imine yielding Mn-II, transmetalation of Mn-II with Me2Zn giving Mn-III, then producing Mn-IV and Zn-I by a ligand exchange with 1, and C–H activation of Mn-IV regenerating Mn-I. NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications Methods G l 13. Li, G. et al. Pd(II)-catalyzed C–H functionalizations directed by distal weakly coordinating functional groups. J. Am. Chem. Soc. 137, 4391–4397 (2015). General procedure for the formation of products 3. To a 25 ml flame-dried Schlenk tube was added ZnBr2 (0.5 mmol, 112.5 mg, stored in glove box), MnBr (CO)5 (0.05 mmol, 10.0 mol%, 13.8 mg), DCE (1.25 mL), 2,2-dimethyl-1-phenyl- propan-1-one 1a (1.5 mmol, 243.0 mg), (E)-N-benzylidenethiophene-2-sulfona- mide 2a (0.5 mmol, 125.5 mg), and Me2Zn (0.75 mmol, 1.2 M in toluene, 0.625 mL) sequentially under nitrogen. The tube was sealed and stirred at 60 °C for 10 h. After completion, the reaction mixture was diluted with ethyl acetate (5.0 mL) and fil- tered through a short pad silica gel washing with ethyl acetate (20 mL). The filtrate was concentrated and purified by silica gel column chromatography to provide the product 3a in 80% yield. 14. Kakiuchi, F., Kan, S., Igi, K., Chatani, N. & Murai, S. A ruthenium-catalyzed reaction of aromatic ketones with arylboronates: a new method for the arylation of aromatic compounds via C–H bond cleavage. J. Am. Chem. Soc. 125, 1698–1699 (2003). 15. Kakiuchi, F., Matsuura, Y., Kan, S. & Chatani, N. A RuH2(CO)(PPh3)3- catalyzed regioselective arylation of aromatic ketones with arylboronates via carbon−hydrogen bond cleavage. J. Am. Chem. Soc. 127, 5936–5945 (2005). 16. Gandeepan, P., Parthasarathy, K. & Cheng, C.-H. Synthesis of phenanthrone derivatives from sec-alkyl aryl ketones and aryl halides via a palladium- catalyzed dual C–H bond activation and enolate cyclization. J. Am. Chem. Soc. 132, 8569–8571 (2010). General procedure for the formation of products 4. To a Schlenk tube was added ZnBr2 (0.5 mmol, 112.5 mg), MnBr(CO)5 (0.05 mmol, 10.0 mol%, 13.8 mg), DCM (5.0 mL), acetophenone 1v (2.0 mmol, 240.0 mg), (E)-N-benzylidene thiophene-2-sulfonamide 2a (0.5 mmol, 125.5 mg), and Me2Zn (0.75 mmol, 1.2 M in toluene, 0.625 mL) sequentially under nitrogen. The tube was sealed and stirred at 60 °C for 2 h. After completion, the reaction mixture was diluted with ethyl acetate (10 mL) and filtered through a short pad silica gel washing with ethyl acetate (20 mL). The filtrate was concentrated and purified by silica gel column chromatography to provide 4a in 66% yield. 17. Xiao, B., Gong, T.-J., Xu, J., Liu, Z.-J. & Liu, L. Palladium-catalyzed intermolecular directed C–H amidation of aromatic ketones. J. Am. Chem. Soc. 133, 1466–1474 (2011). 18. Shin, K., Baek, Y. & Chang, S. ARTICLE Intramolecular cyclization of Zn-I yields Zn-II followed by either an elimination giving 4 or an intermolecular nucleophilic substitution with Me2Zn forming 5 NR3 BrZnO R4 NR3 R4 R NR3 Me R4 –Zn(OH)Br Me2Zn 4 5 R R Zn-II MnBr(CO)5 + Me2Zn Mn-I Mn-IV BrZnO Mn-III 5 Mn-II Fig. 10 A proposed reaction mechanism. Key steps include formation of Mn-I followed by addition to imine yielding Mn-II, transmetalation of Mn-II with Me2Zn giving Mn-III, then producing Mn-IV and Zn-I by a ligand exchange with 1, and C–H activation of Mn-IV regenerating Mn-I. Intramolecular cyclization of Zn-I yields Zn-II followed by either an elimination giving 4 or an intermolecular nucleophilic substitution with Me2Zn forming 5 Fig. 10 A proposed reaction mechanism. Key steps include formation of Mn-I followed by addition to imine yielding Mn-II, transmetalation of Mn-II with Me2Zn giving Mn-III, then producing Mn-IV and Zn-I by a ligand exchange with 1, and C–H activation of Mn-IV regenerating Mn-I. Intramolecular cyclization of Zn-I yields Zn-II followed by either an elimination giving 4 or an intermolecular nucleophilic substitution with Me2Zn forming 5 NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications 7 ARTICLE Discussion 7. Grellier, M. et al. Synthesis, neutron structure, and reactivity of the bis (dihydrogen) complex RuH2(η2-H2)2(PCyp3)2 stabilized by two tricyclopentylphosphines. J. Am. Chem. Soc. 127, 17592–17593 (2005). In conclusion, aromatic C–H addition of ketones to imines was developed via manganese catalysis, which enabled to reverse the reactivity of labile C(sp3)–H bonds α to the carbonyl and inert C (sp2)–H bonds on the benzene ring of ketones. Thus, the classic Mannich reaction was completely depressed and a series of valuable products, namely the ortho-C–H aminoalkylated ketones, cyclized exo-olefinic isoindolines, and three-component methylated isoindolines, can be selectively achieved. Meanwhile, this protocol also represents a manganese-catalyzed aromatic C–H bond transformation of ketones since the parent stoichio- metric cyclomanganation reaction was reported in 197544,45. Further explorations on the manganese-catalyzed C–H activation reactions of ketones are underway in our laboratory. y p p z, R. et al. C–C bond formation via C–H bond activation using a 8. Martinez, R. et al. C–C bond formation via C–H bond activation using an in situ-generated ruthenium catalyst. J. Am. Chem. Soc. 131, 7887–7895 (2009 9. Santhoshkumar, R., Mannathan, S. & Cheng, C.-H. Cobalt-catalyzed hydroarylative cyclization of 1,6-enynes with aromatic ketones and esters via C–H activation. Org. Lett. 16, 4208–4211 (2014). 10. Shang, R., Ilies, L. & Nakamura, E. Iron-catalyzed ortho C–H methylation of aromatics bearing a simple carbonyl group with methylaluminum and tridentate phosphine ligand. J. Am. Chem. Soc. 138, 10132–10135 (2016). 11. Patureau, F. W., Basset, T. & Glorius, F. 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Transition metal- catalyzed ketone-directed or mediated C–H functionalization. Chem. Soc. Rev. 44, 7764–7786 (2015). 5. Zheng, Q.-Z. & Jiao, N. Transition-metal-catalyzed ketone-directed ortho-C–H functionalization reactions. Tetrahedron Lett. 55, 1121–1126 (2014). 6. Murai, S. et al. Efficient catalytic addition of aromatic carbon-hydrogen bonds to olefins. Nature 366, 529–531 (1993). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01262-4 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01262-4 reaction starts with the formation of MnMe(CO)5 from MnBr (CO)5 and Me2Zn. It further reacts with ketone 1 to give five- membered manganacycle Mn-I followed by addition to imine 2 yielding seven-membered manganacycle Mn-II. Transmetalation of Mn-II with Me2Zn affords intermediate Mn-III, which undergoes a ligand exchange with substrate 1 to produce species Mn-IV and Zn-I. An intramolecular C–H activation occurs in Mn-IV regenerating Mn-I and releasing methane35. Hydrolysis of Zn-I gives product 3. Meanwhile, Zn-I may also undergo an intramolecular cyclization to yield intermediate Zn-II, which is followed by either an elimination of zinc salt giving exo-olefinic isoindoline 4 or an intermolecular nucleophilic substitution with Me2Zn forming isoindoline 5 under well-controlled reaction conditions. Received: 11 March 2017 Accepted: 31 August 2017 Additional information ( ) 39. Liu, W., Richter, S. C., Zhang, Y. & Ackermann, L. Manganese(I)-catalyzed substitutive C−H allylation. Angew. Chem. Int. Ed. 55, 7747–7750 (2016). Supplementary Information accompanies this paper at doi:10.1038/s41467-017-01262-4. Competing interests: The authors declare no competing financial interests. Competing interests: The authors declare no competing financial interests. ( ) 40. Liang, Y.-F., Massignan, L., Liu, W. & Ackermann, L. Catalyst-guided C = Het hydroarylations by manganese-catalyzed additive-free C−H activation. Chem. Eur. J. 22, 14856–14859 (2016). 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 43. Yahaya, N. P. et al. Manganese(I)-catalyzed C−H activation: the key role of a 7- membered manganacycle in H-transfer and reductive elimination. 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ARTICLE Manganese-catalyzed dehydrogenative [4 + 2] annulation of N-H imines and alkynes by C–H/N–H activation. Angew. Chem. Int. Ed. 53, 4950–4593 (2014). g 53. Liu, W., Richter, S. C., Mei, R., Feldt, M. & Ackermann, L. Synergistic heterobimetallic manifold for expedient manganese(I)-catalyzed C−H cyanation. Chem. Eur. J. 22, 17958–17961 (2016). 34. He, R. et al. Mn-catalyzed three-component reactions of imines/nitriles, Grignard reagents, and tetrahydrofuran: an expedient access to 1,5-amino/keto alcohols. J. Am. Chem. Soc. 136, 6558–6561 (2014). Acknowledgements Financial support from the National Natural Science Foundation of China (21322203, 21272238, and 21521002) are gratefully acknowledged. We thank Mr. W. Bi (ICCAS) for the synthesis of ketone 1C and Prof. Z. Xi (PKU) and Dr. L. Liu (PKU) for the help in HPLC analysis. We also thank the Alexander von Humboldt Foundation for the Equipment Subsidy (GC-MS). Financial support from the National Natural Science Foundation of China (21322203, 21272238, and 21521002) are gratefully acknowledged. We thank Mr. W. Bi (ICCAS) for the synthesis of ketone 1C and Prof. Z. Xi (PKU) and Dr. L. Liu (PKU) for the help in HPLC analysis. We also thank the Alexander von Humboldt Foundation for the Equipment Subsidy (GC-MS). 35. Zhou, B., Hu, Y. & Wang, C. Manganese-catalyzed direct nucleophilic C (sp2)–H addition to aldehydes and nitriles. Angew. Chem. Int. Ed. 54, 13659–13663 (2015). 36. Yang, X., Jin, X. & Wang, C. Manganese-catalyzed ortho-C–H alkenylation of aromatic N–H imidates with alkynes: versatile access to mono-alkenylated aromatic nitriles. Adv. Synth. Catal. 358, 2436–2442 (2016). Author contributions 37. Liu, W., Zell, D., John, M. & Ackermann, L. Manganese-catalyzed synthesis of cis-β-amino acid esters through organometallic C–H activation of ketimines. Angew. Chem. Int. Ed. 54, 4092–4096 (2015). B.Z. and C.W. conceived and designed the project. B.Z. and Y.H. performed the experiments. T.L. prepared some of starting materials. All authors participated in data analyses and discussions. B.Z. and C.W. co-wrote the manuscript. g 38. Liu, W., Bang, J., Zhang, Y. & Ackermann, L. Manganese(I)-catalyzed C−H aminocarbonylation of heteroarenes. Angew. Chem. Int. Ed. 54, 14137–14140 (2015). Methods G l Direct C–H amination of arenes with alkyl azides under rhodium catalysis. Angew. Chem. Int. Ed. 52, 8031–8036 (2013). 19. Kim, J. & Chang, S. Iridium-catalyzed direct C–H amidation with weakly coordinating carbonyl directing groups under mild conditions. Angew. Chem. Int. Ed. 53, 2203–2207 (2014). General procedure for the formation of products 5. To a Schlenk tube was added ZnBr2 (0.5 mmol, 112.5 mg), MnBr(CO)5 (0.05 mmol, 10.0 mol%, 13.8 mg), DCM (5.0 mL), propiophenone 1z (2.0 mmol, 276.0 mg), (E)-N-benzylidenethio- phene-2 -sulfonamide 2a (0.5 mmol, 125.5 mg), and Me2Zn (1.0 mmol, 1.2 M in toluene, 0.83 mL) sequentially under nitrogen. The tube was sealed and stirred at 100 °C for 10 h. After completion, the reaction mixture was diluted with ethyl acetate (10 mL) and filtered through a short pad silica gel washing with ethyl acetate (20 mL). The filtrate was concentrated and purified by silica gel column chromatography to provide 5a in 63% yield (dr = 3.1:1). 20. Schroeder, N., Wencel-Delord, J. & Glorius, F. High-yielding, versatile, and practical [Rh(III)Cp*]-catalyzed ortho bromination and iodination of arenes. J. Am. Chem. Soc. 134, 8298–8301 (2012). 21. Shan, G., Yang, X., Ma, L. & Rao, Y. Pd-catalyzed C–H oxygenation with TFA/ TFAA: expedient access to oxygen-containing heterocycles and late-stage drug modification. Angew. Chem. Int. Ed. 51, 13070–13074 (2012). 22. Mo, F., Trzepkowski, L. & Dong, G. Synthesis of ortho-acylphenols via Pd- catalyzed ketone-directed hydroxylation of arenes. Angew. Chem. Int. Ed. 52, 13075–13079 (2012). 23. Thirunavukkarasu, V. S. & Ackermann, L. Ruthenium-catalyzed C–H bond oxygenations with weakly coordinating ketones. Org. Lett. 14, 6206–6209 (2012). Data availability. All data supporting the findings of this study are available within the article and its Supplementary Information file or from the authors on rea- sonable request. 24. Itoh, H., Kikuchi, T., Ishiyama, T. & Miyaura, N. Iridium-catalyzed ortho-C–H borylation of aryl ketones with bis(pinacolato)diboron. Chem. Lett. 40, 1007–1008 (2011). Supplementary crystallographic information files, which include structure factors, have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as deposition numbers CCDC 1563929, 3H; CCDC: 1532722, 4a; CCDC: 1532723, 4e; CCDC: 1532725, 5c; CCDC: 1532724, 5d; CCDC 1563930, Mn-I′. These data files can be obtained free of charge from http://www.ccdc.cam.ac.uk/ data_request/cif. 25. Padala, K. & Jeganmohan, M. Ruthenium-catalyzed ortho-alkenylation of aromatic ketones with alkenes by C–H bond activation. Org. Lett. 13, 6144–6147 (2011). NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications 8 Additional information Synthesis of o-deuterio- and o- halogeno-acetophenones via oxidation of η2-(2-acetylphenyl) tetracarbonylmanganese derivatives and the determination of a primary kinetic isotope effect in ortho-metallation of acetophenones. J. Chem. Soc. Chem. Commun. 1986, 12–13 (1986). 46. Tsai, A. S., Tauchert, M. E., Bergman, R. G. & Ellman, J. A. Rhodium(III)- catalyzed arylation of boc-imines via C−H bond functionalization. J. Am. Chem. Soc. 133, 1248–1250 (2011). © The Author(s) 2017 © The Author(s) 2017 NATURE COMMUNICATIONS| 8: 1169 | DOI: 10.1038/s41467-017-01262-4| www.nature.com/naturecommunications 9 9
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Comparison of Imported<i>Plasmodium ovale curtisi</i>and<i>P. ovale wallikeri</i>Infections among Patients in Spain, 2005–2011
Emerging infectious diseases
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Comparison of Imported Plasmodium ovale curtisi and P. ovale wallikeri Infections among Patients in Spain, 2005–2011 Gerardo Rojo-Marcos, José Miguel Rubio-Muñoz, Germán Ramírez-Olivencia, Silvia García-Bujalance, Rosa Elcuaz-Romano, Marta Díaz-Menéndez, María Calderón, Isabel García-Bermejo, José Manuel Ruiz-Giardín, Francisco Jesús Merino-Fernández, Diego Torrús-Tendero, Alberto Delgado-Iribarren, Mónica Ribell-Bachs, Juan Arévalo-Serrano, and Juan Cuadros-González Sequencing data from Plasmodium ovale genotypes co-circulating in multiple countries support the hypothesis that P. ovale curtisi and P. ovale wallikeri are 2 separate species. We conducted a multicenter, retrospective, com- parative study in Spain of 21 patients who had imported P. ovale curtisi infections and 14 who had imported P. ovale wallikeri infections confirmed by PCR and gene sequenc- ing during June 2005–December 2011. The only significant finding was more severe thrombocytopenia among patients with P. ovale wallikeri infection than among those with P. ovale curtisi infection (p = 0.031). However, we also found nonsignificant trends showing that patients with P. ovale wallikeri infection had shorter time from arrival in Spain to onset of symptoms, lower level of albumin, higher median maximum core temperature, and more markers of hemo- lysis than did those with P. ovale curtisi infection. Larger, prospective studies are needed to confirm these findings. Diagnosis of P. ovale malaria can be difficult because of low parasitemia levels, mixed infections with other Plas- modium species, and false negatives from malaria rapid di- agnostic tests (RDTs) (1). However, recent epidemiologic studies conducted by using PCR techniques have found P. ovale infections in most of sub-Saharan Africa, South- east Asia, and the Indian subcontinent (2–5), including prevalence as high as 15% according to results of cross- sectional studies conducted in rural Nigeria (6) and Papua New Guinea (7). In addition, severe complications such as spleen rupture, severe anemia, or acute respiratory distress syndrome (ADRS) (8) may occur in patients with P. ovale malaria. Thus, the global burden of P. ovale infection might have been underestimated. On the basis of differences in its gene sequences, P. ovale was considered to be dimorphic or to comprise 2 sub- species (2,3,9,10). This difference has hampered molecular diagnosis in some cases because of lack of DNA ampli- fication by PCR with gene-specific primers for the small subunit ribosomal RNA (ssrRNA) (10). These subspecies had been named classic and variant P. ovale, but a compre- hensive study recently described differences between these subspecies in at least 6 genes (4). These findings demon- strate that P. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Microbiological Diagnosis The initial diagnosis of imported Plasmodium spp. in- fection was made by thick and/or thin smears and/or by using the second-generation RDT NOW Malaria Test Kit (Binax Inc., Scarborough, ME, USA) for histidine-rich pro- tein 2 antigen of P. falciparum and aldolase enzyme com- mon to all Plasmodium spp. Blood smears were stained by a standard technique with Giemsa solution for 30 min and were reviewed by an expert microbiologist. Parasite count was measured by determining the proportion of parasitized erythrocytes or the number of trophozoites per microliter. P. ovale Subtype Characterization and Confirmation Partial sequencing of the ssrRNA gene was used to differentiate P. ovale curtisi from P. ovale wallikeri. ssr- RNA amplification was performed by using a nested PCR specific for Plasmodium. The first reaction included UNR (5′-GACGGT ATCTGATCGTCTTC-3′) and PLF (5′- AGTGTGTATCCAATCGAGTTTC-3′) primers, which correspond to the first reaction of the seminested multiplex malaria PCR. The second reaction incorporated the prod- ucts of the first reaction, along with NewPLFsh (5′-CTAT- CAGCTTTTGATGTTAG-3′) and NewRevsh (5′-CCTTA- ACTTTCGTTCTTG-3′) primers. Infection with different malaria species yielded products of 710–740 bp. Growth in international travel and migration has in- creased the incidence of imported malaria in industrialized countries. P. ovale infection may represent up to 8% of im- ported malaria cases, according to some published series of patients primarily from West Africa (16,17), where the proportion of sub-Saharan immigrants is high and PCR has been systematically performed. Yet, it is difficult to gather a substantial number of cases with clinico-epidemiologic cor- relation and molecular data. To identify clinical or analytical differences between P. ovale wallikeri and P. ovale curtisi infections and expand data on these infections, we conducted a multicenter, retrospective, comparative study of imported P. ovale infections diagnosed in Spain during 2005–2011. The PCR mixture in both reactions consisted of 75 mmol/L Tris-HCl (pH 9.0), 2 mmol/L MgCl2, 50 mmol/L KCl, 20 mmol/L (NH4)2SO4, 200 μmol/L dNTP, 0.075 μmol/L of the corresponding PCR primers, 1.25 units Taq DNA polymerase (Biotools B&M Labs, S.A., Madrid, Spain), and the template DNA in a reaction volume of 50 μL. The amount of template was 5 μL of DNA extracted by using a QIAamp DNA Blood Mini Kit (QIAGEN). For the second reaction mixture, 2 μL of the PCR product of the first reaction was used as template. For both reactions, a GeneAmp PCR System 2700 thermal cycler (Applied Bio- systems, Foster City, CA, USA) was used, beginning with 7 min at 94°C, followed by 40 cycles of 20 s at 94°C, 20 s at 62°C, and 30 s at 72°C for the first round; or 35 cycles of 20 s at 94°C, 20 s at 53°C, and 20 s at 72°C for the second round. The final cycle was followed by an extension time of 10 min at 72°C.ii Sample Selection During June 2005–December 2011, blood samples from all patients with positive PCR results for imported infection with P. ovale were sent from public hospitals in Spain to the reference Malaria & Emerging Parasitic Dis- eases Laboratory of the National Centre of Microbiology in Madrid. The samples were shipped to the laboratory 1) to confirm the diagnosis of malaria and the species or 2) to study fever, anemia, or suspected malaria in patients with negative results on thick and/or thin smears and RDTs who were considered at high risk for malaria (i.e., immigrants and travelers to malaria-endemic areas). The amplified products were purified by using Illustra DNA and Gel Band Purification Kit (GE Healthcare, Buck- inghamshire, UK) and sequenced by using the Big Dye Terminator v3.1 Cycle Sequencing Kit and ABI PRISM 3700 DNA Analyzer (Applied Biosystems). All amplified products were sequenced in both directions twice. To con- firm P. ovale subtyping, a nested PCR amplification plus sequencing targeting cytochrome (Cyt) b was performed (3) in 3 samples of each group, by using a unique second amplification (nested) reaction with primers Cyt b 2F and Cyt b 2R. Comparison of Imported Plasmodium ovale curtisi and P. ovale wallikeri Infections among Patients in Spain, 2005–2011 ovale actually consists of 2 subspecies that co- circulate in Africa and Asia and that are unable to recom- bine genetically; the differences seem to be explained by real biological factors, rather than ecologic or geographic factors (11). P. ovale curtisi and P. ovale wallikeri were the names proposed for these species (4). M alaria caused by Plasmodium ovale infection has been considered a low-prevalence disease with lim- ited geographic distribution, benign clinical course, and easy treatment; therefore, little attention has been paid to it. Author affiliations: Príncipe de Asturias University Hospital, Madrid, Spain (G. Rojo-Marcos, J. Arévalo-Serrano, J. Cuadros-González,); Instituto de Salud Carlos III, Madrid (J.M. Rubio-Muñoz); Carlos III Hospital, Madrid (G. Ramírez-Olivencia); La Paz University Hospi- tal, Madrid (S García-Bujalance); Doctor Negrín University Hospital, Las Palmas de Gran Canaria, Spain (R. Elcuaz-Romano); Ramón y Cajal Hospital, Madrid (M. Díaz-Menéndez); Gregorio Marañón University Hospital, Madrid (M. Calderón); Getafe University Hospi- tal, Madrid (I. García-Bermejo); University Hospital of Fuenlabrada, Madrid (J. M. Ruiz-Giardín); Severo Ochoa University Hospital, Madrid (F.J. Merino-Fernández); University General Hospital of Alicante, Alicante, Spain (D. Torrús-Tendero); University Hospital Fundación Alcorcón, Madrid (A. Delgado-Iribarren); and Hospital General de Granollers, Barcelona, Spain (M. Ribell-Bachs). M Scant information is available on differences in clini- cal and analytical features, relapse profile, or accuracy of RDT results between these proposed species. Relatively high parasitemia levels were found in some patients with P. ovale wallikeri infection in Thailand (12), Vietnam (13), and Flores Island (14). A study published from a disease-endem- ic area of Bangladesh reported on the clinical features and degree of parasitemia in 13 patients with P. ovale wallikeri DOI: http://dx.doi.org/10.3201/eid2003.130745 409 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 RESEARCH infection and 10 with P. ovale curtisi infection (5). These infections were diagnosed by PCR; only 4 of the 23 patients were symptomatic. Another recent study compared parasit- emia levels, RDT results, and patient country of origin for 31 patients from Côte d’Ivoire and the Comoros Islands with imported P. ovale wallikeri infection and 59 with P. ovale curtisi infection, but no clinical data were provided (15). Clearly, information on these infections is limited. Hilden, Germany), according to the manufacturer’s proto- col. P. ovale molecular diagnosis was confirmed by using seminested multiplex malaria PCR (18), which enables the discernment of the 4 most prevalent human malaria species by amplified fragments of DNA in 2 sequential PCRs. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Isolation of Parasite DNA and Molecular Diagnosis Confirmation Hospitals that submitted PCR-confirmed and P. ovale subtype–identified samples were invited to collaborate in the study. A database was designed and completed after the retrospective review of medical reports and laboratory DNA isolation from whole blood was performed by using the QIAamp DNA Blood Mini Kit (QIAGEN, Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 410 Imported Plasmodium ovale Infections, Spain samples from 28 patients and P. ovale wallikeri in 24 sam- ples from to 22 patients. registries. Patient data collected included sex, age, ethnicity, underlying diseases, time living in non–malaria-endemic countries, dates and purpose of travel, countries visited, ma- laria chemoprophylaxis, date of admission and diagnosis, symptoms and clinical signs, physical examinations, and complications of severe malaria according to World Health Organization criteria (19). The closest possible date of in- fection was defined as the day of departure from a malaria- endemic area. The time between date of arrival in Spain and onset of illness or diagnosis was calculated once asymp- tomatic patients were excluded. Patients were classified as early immigrant if they had stayed in a country without ma- laria for <1 year before diagnosis and as visiting friends and relatives if they had traveled to a malaria-endemic country after 1 year living in a non–malaria-endemic area. Recent Plasmodium infection was defined as probable or definite malaria infection in the 3 months before P. ovale infection was diagnosed. Twelve hospitals agreed to participate in the study and provided complete epidemiologic, microbiological, biochemical, clinical, and therapeutic information for 35 of the 50 patients for which P. ovale genetic characteriza- tion was available. Of these, 21 patients had P. ovale curtisi infection and 14 had P. ovale wallikeri infection. Table 1 shows the demographic and epidemiologic data for these 35 patients. Patient age and sex were virtually the same for patients with P. ovale curtisi and P. ovale wallikeri infec- tions and corresponded mostly to young persons from Af- rica who traveled to visit their friends and relatives for a long period or immigrants who had recently arrived. The lapses between time of arrival in Spain, onset of illness, and diagnosis were longer for patients with P. ovale curtisi infection than for those with P. ovale wallikeri in- fection, but not significantly. Most travelers did not take any malaria prophylaxis or did not adhere to the full regi- men. Isolation of Parasite DNA and Molecular Diagnosis Confirmation All but 2 infections were acquired in West Africa, and both Plasmodium species were found in patients from Ni- geria, Equatorial Guinea, Ghana, and Guinea-Conakry. A high rate of underlying disease was found among patients in both parasite groups; the P. ovale curtisi group included 3 early immigrants who were chronically infected with HIV, hepatitis B virus, and hepatitis C virus and carried filarial and intestinal parasites. Recorded laboratory results included microbiological data; complete blood count with leukocytes, hemoglobin, and platelet levels; levels ​of creatinine, albumin, trans- aminases, lactate dehydrogenase, and direct bilirubin in plasma; and glucose-6-phosphate dehydrogenase activity in erythrocytes. Abdominal ultrasonography and serologic studies to detect infection with HIV and hepatitis A, B, and C viruses were reviewed. Recorded treatments and compli- ance, clinical and microbiological evolution, and duration of hospital stay for those admitted were included. Microbiological and laboratory data for the patients are shown in Table 2. Statistically significant worse levels of thrombocytopenia were found among patients with P. ovale wallikeri infection compared with those who had P. ovale curtisi infection, but no other significant difference was found. One mixed infection with P. falciparum was found among each patient group. The RDT technique used showed a low sensitivity (<30%) for detecting P. ovale once mixed infections were excluded. Statistical Analysis Differences of proportions were evaluated by χ2 test or Fisher exact test, as appropriate for sample size. Means between groups were calculated by using the Student t-test for independent samples if the normal distribution could be assumed; we used the Levene test for homogeneity of variances. If normality was not valid, we used the Mann- Whitney U test for nonparametric variables. Clinical and therapeutic data for the patients are shown in Table 3. All 3 asymptomatic patients had P. ovale curtisi infection; 1 infection was detected as an incidental finding in a blood smear for sickle cell disease, and the other 2 were found during studies of anemia after negative results were found by thick film examination and RDT results. The remaining 7 patients with negative thick smear and positive PCR results reported at least fever. Symptomatic patients showed no difference in clinical signs and symptoms, but those with P. ovale wallikeri did have a higher mean num- ber of symptoms (3.5 per patient) than did those with P. ovale curtisi (2.7). The number of complications was simi- lar in both groups; 3 cases of severe anemia occurred, 2 of them related to sickle cell disease, and 1 case of ADRS oc- curred in a patient with P. ovale wallikeri infection. To test for normality, we used either the Shapiro- Wilks test for small samples or the Kolmogorov-Smirnov test with the Lilliefors correction for large samples. Val- ues were reported as means and SDs or, for nonparametric distributions, medians and interquartile ranges (IQRs). A 2-sided p value of <0.05 was considered to indicate statisti- cal significance. Statistical analyses were performed by us- ing SPSS version 15 (SPSS Inc., Chicago, IL, USA). Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Results During June 2005–December 2011, a total of 102 samples positive by PCR for P. ovale were analyzed at the reference laboratory; of these, we were able to amplify and genotype 55 samples. Poor quality of long-term stored DNA prevented amplification of the other samples. Genetic analyses of the cytb gene identified P. ovale curtisi in 31 Most patients were admitted to a hospital and received inpatient treatment. Roughly half the patients in each group 411 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 RESEARCH Table 1. Demographic and epidemiologic characteristics of patients with imported Plasmodium ovale curtisi or P. ovale wallikeri infections, Spain, 2005–2011* Characteristic P. ovale curtisi, n = 21 P. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Results ovale wallikeri, n = 14 p value Patient sex 0.332 M 10 (47.6) 9 (64.3) F 11 (52.4) 5 (35.7 ) Patient age, y, median (IQR) 36.50 (23.04–52.66) 38.33 (11.79–45.27) 0.377 Age <15 3 (14.3) 4 (28.6) 0.401 Ethnicity 0.721 Black 15 (71.4) 9 (64.3) White 6 (28.6) 5 (35.7) Type of patient 0.260 Early immigrant 6 (28.6) 4 (28.6) Traveler 14 (66.7) 10 (71.4) Reason for travel Visiting friends and relatives 9 (42.8) 7 (50.0) Tourism 1 (7.1) Work 3 (14.3) 2 (14.3) Cooperation 2 (9.5) Unknown 1 (4.8) Duration of travel, d, median (IQR) 75 (23.25–91.50) 23 (15.00–81.50) 0.279 Country of infection 0.486 Equatorial Guinea 12 (57.1) 7 (50.0) Nigeria 2 (9.5) 3 (21.4) Equatorial Guinea or Cameroon 1 (4.8) 0 Ghana 1 (4.8) 1 (7.1) Ethiopia 1 (4.8) 0 Guinea-Conakry 1 (4.8) 0 Liberia 1 (4.8) 0 Angola 1 (4.8) 0 Guinea-Bissau 1 (4.8) 0 Guinea-Conakry or Senegal 0 1 (7.1) Côte d’Ivoire 0 1 (7.1) Mozambique 0 1 (7.1) Chemoprophylaxis 0.627 No prophylaxis 17 (81.0) 13 (92.9) Mefloquine, incomplete 1 (4.8) 1 (7.1) Mefloquine 1 (4.8) 0 Doxycycline 1 (4.8) 0 Atovaquone/proguanil 1 (4.8) 0 Days from arrival to onset of symptoms, median (IQR) 94.5 (12.5–297.2) 9.5 (2.7–58.2) 0.077 Days from onset of symptoms to diagnosis, median (IQR) 8 (2.7–16.5) 3.5 (2.0–7.7) 0.206 Recent Plasmodium infection 3 (14.3) 3 (21.4) >0.999 Other infections Hepatitis B virus >0.999 Active 1/11 (9.1) 0/10 Cured or vaccinated 6/11 (54.5) 5/10 (50.0) Negative 4/11 (36.4) 5/10 (50.0) Hepatitis C virus 1/7 (14.3) 0/10 0.412 HIV 1/7 (14.3) 0/10 0.412 Filariasis† 3/6 (50.0) 0/4 0.200 Intestinal parasites‡ 3/6 (50.0) 1/4 (25.0) 0.571 Other underlying conditions 9 (42.8) 6 (42.8) >0.999 Diabetes mellitus 2 (9.5) 1 (7.1) Drepanocytosis 2 (9.5) 0 Hypertension 4 (19.0) 2 (14.3) Obesity 1 (4.8) 0 Acute pancreatitis 0 1 (7.1) Policystosis and nephrectomy 0 1 (7.1) Oligoarthritis 0 1 (7.1) Glucose-6-phosphate dehydrogenase deficiency 2/14 (14.3) 0/8 0.515 Pregnancy 1 (4.8) 0 >0.999 *Values are no. (%) patients or no. positive/total no. (%) patients unless otherwise indicated. IQR, interquartile range. †Mansonella perstans (n = 2), Loa loa (n = 1). ‡Trichiuris trichiura (n = 3), hookworms (n = 2), Ascaris lumbricoides (n = 2), Strongyloides stercoralis (n = 1), Entamoeba hystolitica (n = 1). ics of patients with imported Plasmodium ovale curtisi or P. ovale wallikeri nd epidemiologic characteristics of patients with imported Plasmodium ovale curtisi or P. ns (n = 2), Loa loa (n = 1). (n = 3), hookworms (n = 2), Ascaris lumbricoides (n = 2), Strongyloides stercoralis (n = 1), Entamoeba hystolitica (n = 1). Results ovale wallikeri 2011* 412 Imported Plasmodium ovale Infections, Spain Table 2. Microbiological characteristics of patients with imported Plasmodium ovale curtisi or P. ovale wallikeri infections, Spain, 2005– 2011* Characteristic P. ovale curtisi, n = 21 P. ovale wallikeri, n = 14 p value Positive thick smear, no. (%) patients 16 (76.2) 10 (71.4) >0.999 Positive by PCR only, no. (%) patients 5 (23.8) 4 (28.6) >0.999 Parasitemia, L 2,800 (773.25–5,484.25) 1,243.50 (337.75–6,200.00) 0.699 Mixed infection, no. (%) patients 1† (4.8) 1† (7.1) >0.999 Rapid diagnostic test result, no. positive/total no. patients (%) Common antigen positive 4/16 (25.0) 4/12 (33.3) 0.691 P. falciparum antigen positive 1/15 (6.7) 2/12 (16.6) 0.569 Leukocyte count,  109 cells/L 7.2 (4.9–8.7) 5.5 (4.2–8.2) 0.309 Hemoglobin, g/dL 11.6 (9.7–13.6) 10.9 (9.6–12.1) 0.364 Platelet count,  109 cells/L 126 (106.0–182.5) 91.5 (54.7–117.7) 0.031 Albumin, g/dL 3.7 (3.3–4.1) 3.4 (2.8–3.7) 0.063 Creatinine, mg/dL 0.88 (0.6–1.1) 0.97 (0.5–1.1) 0.730 Lactate dehydrogenase, IU/L 434.5 (358.7–807.7) 563 (462.5–731.7) 0.200 Aspartate aminotransferase, IU/L 24.5‡ (20.0–40.2) 31 (22–41) 0.624 Alanine aminotransferase, IU/L 25.5‡ (16.0–49.7) 23 (18.5–47.0) 0.785 Total bilirubin level, mg/dL 0.68‡ (0.6–1.2) 0.87 (0.6–1.4) 0.426 *Values are median (interquartile range) unless otherwise indicated. Boldface indicates significance. †P. falciparum was second infection for both patients. ‡One patient had active hepatitis B virus infection. among those with P. ovale wallikeri infection, a symptom that was absent among those with P. ovale curtisi infec- tion. However, because most cases were asymptomatic or had mild to moderate clinical features, finding significant differences within this narrow clinical spectrum may be especially difficult and might require a much larger study. received chloroquine alone. Almost all patients showed good tolerance to treatment and favorable clinical evolu- tion. More than a quarter (33.3% of P. ovale curtisi and 28.6% of P. ovale wallikeri) did not receive primaquine for radical cure, 2 because of glucose-6-phosphate dehydroge- nase deficiency. One patient was lost to follow-up and did not receive any treatment or monitoring. Because the time of infection is more likely to be accu- rately known, patients with imported malaria might make a better group for study of the epidemiologic and clinical characteristics of different Plasmodium species than those living in malaria-endemic countries. Moreover, signs or symptoms may be less affected by other tropical infections, including mixed Plasmodium infections, or by patient im- munity; imported malaria occurs among a larger number of nonimmune patients and patients who are visiting friends and relatives. Results Differentiation among primary infection or relapse continues to be practically impossible, but the lon- ger the time of latency, the more probable a relapse. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Discussion Our comparative study of the epidemiologic and clini- cal characteristics of patients with P. ovale curtisi and P. ovale wallikeri infection found only 1 statistically signifi- cant result, a higher rate of severe thrombocytopenia among patients with P. ovale wallikeri infection (p = 0.031). Nevertheless, we noted nonsignificant results, including a shorter time from arrival to onset of symptoms in travelers who acquired P. ovale wallikeri infection (p = 0.077). This finding fits with findings from a recently published larger series from the United Kingdom in which a significantly shorter latency was found for P. ovale wallikeri compared with P. ovale curtisi infection (20). We also found a trend toward a shorter stay in Africa and shorter interval be- tween onset of symptoms and diagnosis among patients with P. ovale wallikeri infection, which could reflect easier transmission, shorter latency, or higher relapse rates. This finding might also mean that slightly more severe illness, including higher median fever (39.7°C vs. 38.4°C) and a greater number of symptoms (3.5 vs. 2.7 per symptomatic patient), led more patients with P. ovale wallikeri infection than those with P. ovale curtisi infection to seek medical attention earlier. However, the higher percentage of travel- ers who took at least partial prophylaxis among the P. ovale curtisi group (19.2%) might also explain a longer time of onset. A high frequency of nausea and vomiting was found Although criteria for admission to each hospital in- volved in the study were different, P. ovale wallikeri pa- tients were admitted more frequently. The number of com- plications was similar in both groups; 3 cases of severe anemia were reported, 2 related to sickle cell disease, and 1 case of ADRS occurred in a patient infected with P. ovale wallikeri (8). Sickle cell trait was described as a risk factor for infection with P. ovale in Senegal (21), and a recent series showed 3 of 16 patients with P. ovale infection were homozygous for sickle cell disease (22), a clearly dispro- portionate number. Regarding symptomatic patients, the Bangladesh study (5) showed a trend toward a larger number of asymptom- atic P. ovale curtisi infections (90% vs. 75% for P. ovale wallikeri), which would be consistent with our results. In that study, all 13 mixed infections of P. ovale with other 413 erging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 RESEARCH Table 3. Discussion Clinical and therapeutic characteristics of patients with imported Plasmodium ovale curtisi or P. ovale wallikeri infections, Spain, 2005–2011* Characteristic P. ovale curtisi, n = 21 P. ovale wallikeri, n = 14 p value Asymptomatic 3 (14.3) 0 0.259 Fever 18 (85.7) 14 (100.0) 0.259 Tertian fever 1 (4.8) 3 (21.4) 0.279 Maximum temperature, ºC, median (IQR) 38.4 (37.5–40.0) 39.7 (38.9–40.5) 0.088 Chills 3 (14.3) 3 (21.4) 0.664 Sweating 0 1 (7.1) 0.400 Headache 6 (28.6) 4 (28.6) >0.999 Nauseas 0 3 (21.4) 0.056 Vomitus 0 3 (21.4) 0.056 Astenia 2 (9.5) 3 (21.4) 0.369 Epigastralgia 2 (9.5) 0 0.506 Arthralgia 5 (23.8) 3 (21.4) >0.999 Myalgia 6 (28.6) 4 (28.6) >0.999 Diarrhea 1 (4.8) 1 (7.1) >0.999 Chest pain 1 (4.8) 1 (7.1) >0.999 Cough 4 (19.0) 3 (21.4) >0.999 Dyspnea 0 1 (7.1) 0.400 Dizziness 2 (9.5) 0 >0.999 Splenomegaly 5 (23.8) 3 (21.4) >0.999 Complications or severe malaria 2 (9.5) 2 (14.3) >0.999 Hemolytic crisis 1 (4.8) 0 Severe anemia, hemoglobin <7 g/dL 1 (4.8) 1 (7.1) Acute respiratory distress syndrome 0 1 (7.1) Admission to hospital 13 (61.9) 13 (92.9) 0.056 Duration of hospitalization, d, median (IQR) 4 (3.0–7.5) 5 (3.5–7.5) 0.390 Treatment 0.563 Chloroquine 12 (57.1) 7 (50.0) Other treatment 8 (38.1) 7 (50.0) Quinine + doxycycline 3 (14.3) 4 (28.6) Atovaquone/proguanil 3 (14.3) 1 (7.1) Quinine + clindamycin + chloroquine/proguanil 1 (4.8) 0 Quinine + clindamycin + chloroquine 0 1 (7.1) Mefloquine 0 1 (7.1) Atovaquone/proguanil + chloroquine 1 (4.8) 0 No treatment 1 (4.8) 0 Primaquine 14 (66.7) 10 (71.4) >0.999 Compliance 19/21 (90.5) 13/13 (100.0)† 0.513 *Values are no. (%) patients or no. positive/total no. (%) patients unless otherwise indicated. IQR, interquartile range. †One patient was lost to follow-up. rapeutic characteristics of patients with imported Plasmodium ovale curtisi or P. ovale wallikeri infections, related to a greater severity of illness (25). Some studies also suggest an inverse correlation between the level of parasitemia and platelet count (26). Plasmodium species were asymptomatic; in our study, the 2 patients who had mixed infections with P. falciparum had at least fever. The number of P. ovale monoinfections in our study confirmed by PCR is high, unlike the number in malaria-endemic areas, where most infections are mixed (5,7,23) and a high rate of submicroscopic carriage occurs (24). Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Discussion In part, our finding may result from previous anti- malarial treatment or prophylaxis in a number of patients, which could have minimized the number of Plasmodium parasites in the blood. i We found that indirect parameters of hemolysis, such as hemoglobin, lactate dehydrogenase, and bilirubin levels, were less impaired among patients with P. ovale curtisi in- fection than those with P. ovale wallikeri infection, even including 2 patients with sickle cell disease, who had more severe anemia. Albumin values, diminished by other multi- ple types of infections, ​also tend to be lower in patients with P. ovale wallikeri infection. For transaminases, if we were to exclude a patient with chronic hepatitis B and hypertrans- aminasemia, we would also find higher values ​for patients with P. ovale wallikeri infection. These data collectively raise the hypothesis that P. ovale wallikeri is slightly more pathogenic, which warrants further investigation.i Among the laboratory results, the only significant difference was found in platelet count, with more severe thrombocytopenia seen among patients with P. ovale wal- likeri infection than among those with P. ovale curtisi infection. Thrombocytopenia is a common finding in pa- tients with malaria; a previous series found 10 (66.6%) of 15 patients with imported P. ovale infection had platelet counts of <140,000/mL (22). The mechanisms that pro- duce thrombocytopenia in malaria are not known but seem Parasitemia levels were not significantly different be- tween the 2 groups (p = 0.699). In 2 recent studies of P. ovale infection that included determination of parasitemia 414 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 Imported Plasmodium ovale Infections, Spain patient selection was not systematically planned but was done on the basis of decisions of physicians from many hospitals who sent samples to the reference laboratory and subsequently agreed to participate in this study. Fourth, the study’s retrospective design led to gaps in the informa- tion collected. Fifth, only strains of P. ovale from Africa were analyzed, and patients were from Africa and Europe; a study of infections and patients from Asia or Oceania might show different results. Last, more diversity in the geographic origin of the strains and a mix of nonimmune and semiimmune patients would lead to more heteroge- neous study groups. levels and genetic analysis (5,15), no differences were de- scribed. Case reports of high parasitemia levels in patients with P. References In industrialized countries, improved clinical and mi- crobiological control after treatment and radical cure with primaquine can be achieved for malaria. P. ovale seems to remain sensitivity to chloroquine and other antimalarial drugs. Patients in our study showed good clinical response that could be followed up without any relapse, including among those who had complications. The treatment dif- ferences reflect the different hospital managing protocols, patient age and pregnancy status, or difficulty in identifying P. ovale initially. 1. Mueller I, Zimmerman PA, Reeder JC. Plasmodium malariae and Plasmodium ovale—the “bashful” malaria parasites. Trends Parasitol. 2007;23:278–83. http://dx.doi.org/10.1016/j.pt.2007.04.009 1. Mueller I, Zimmerman PA, Reeder JC. Plasmodium malariae and Plasmodium ovale—the “bashful” malaria parasites. Trends Parasitol. 2007;23:278–83. http://dx.doi.org/10.1016/j.pt.2007.04.009 2. Tachibana M, Tsuboi T, Kaneko O, Khuntirat B, Torii M. Two types of Plasmodium ovale defined by SSU rRNA have distinct sequences for ookinete surface proteins. Mol Biochem Parasitol. 2002;122:223–6. http://dx.doi.org/10.1016/S0166-6851(02)00101-9 2. Tachibana M, Tsuboi T, Kaneko O, Khuntirat B, Torii M. Two types of Plasmodium ovale defined by SSU rRNA have distinct sequences for ookinete surface proteins. Mol Biochem Parasitol. 2002;122:223–6. http://dx.doi.org/10.1016/S0166-6851(02)00101-9 3. Win TT, Jalloh A, Tantular IS, Tsuboi T, Ferreira MU, Kimura M, et al. Molecular analysis of Plasmodium ovale variants. Emerg Infect Dis. 2004;10:1235–40. http://dx.doi.org/10.3201/eid1007.030411 3. Win TT, Jalloh A, Tantular IS, Tsuboi T, Ferreira MU, Kimura M, et al. Molecular analysis of Plasmodium ovale variants. Emerg Infect Dis. 2004;10:1235–40. http://dx.doi.org/10.3201/eid1007.030411 4. Sutherland CJ, Tanomsing N, Nolder D, Oguike M, Jennison C, Pukrittayakamee S, et al. Two non-recombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally. J Infect Dis. 2010;201:1544–50. http://dx.doi.org/10.1086/652240 4. Sutherland CJ, Tanomsing N, Nolder D, Oguike M, Jennison C, Pukrittayakamee S, et al. Two non-recombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally. J Infect Dis. 2010;201:1544–50. http://dx.doi.org/10.1086/652240 Our study has limitations. First, the small number of patients may lack sufficient statistical power to show dif- ferences between infections with different Plasmodium species. Second, the low performance of genetic amplifica- tion may have caused some sample selection bias (e.g., a higher number of samples that were stored short term or came from patients with higher parasitemia levels). Third, 5. Fuehrer H-P, Habler VE, Fally MA, Harl J, Starzengruber P, Swo- boda P, et al. Plasmodium ovale in Bangladesh: genetic diversity and the first known evidence of the sympatric distribution of Plas- modium ovale curtisi and Plasmodium ovale wallikeri in southern Asia. Discussion ovale wallikeri infection in Southeast Asia are iso- lated and noncomparative (12–14). Sooner and better access to health care in industrial- ized countries might provide a broader range of diagnostic tools for malaria, including RDT, thick and thin film ex- amination, or PCR. Current techniques of RDT still show a low sensitivity for detecting P. ovale (27,28). This prob- lem is usually explained by the genetic variability of the 2 subspecies and the low levels of parasitemia detected. After discarding mixed infections, in our study, the aldol- ase-based RDT used as a common antigen of Plasmodium obtained 20% sensitivity for detection of P. ovale curtisi and 27.27% for P. ovale wallikeri. Results are poorer than those recently published by Bauffe et al., who found a high- er false-negative rate of infection for P. ovale curtisi than for P. ovale wallikeri (60% vs. 43%, respectively) and no significant differences in parasitemia levels (15). In summary, after comparing epidemiologic, clinical, and analytic data for patients with P. ovale wallikeri and P. ovale curtisi infections, we found significantly more marked thrombocytopenia among patients with P. ovale wallikeri infection, but we found no other significant differ- ences. However, some trends toward slightly greater patho- genicity were observed for P. ovale wallikeri infection. The description of both genotypes occurring in sympatry without hybrid forms in an increasing number of countries supports the idea of ​2 well-defined species. Larger prospec- tive studies should be conducted to more fully explore this hypothesis. Although circulation of P. ovale was known in these countries, our study provides PCR confirmation for P. ovale curtisi infections from Guinea-Conakry, Ethiopia, and Li- beria and P. ovale wallikeri infections from Mozambique. Both species have been described as sympatric in time and space in Equatorial Guinea, Republic of Congo, Uganda, Bangladesh, and Angola (5,11,29), maintaining genetic differentiation, which supports the hypothesis of 2 distinct subspecies. Moreover, 2 cases of P. ovale curtisi and P. ovale wallikeri co-infection have been reported (5,29). Acknowledgments We thank Francisco Javier Vilar Izquierdo for his assistance in the translation and critical review of this manuscript. Dr Rojo-Marcos is a consultant in tropical and travel medicine with the Department of Internal Medicine, Príncipe de Asturias Uni- versity Hospital, Alcalá de Henares, Madrid, Spain. His research interests include malaria, leishmaniasis, schistosomiasis, imported tropical diseases, and social and health problems of immigration. We found a high rate of underlying chronic diseases among the patients in this study, especially homozygous sickle cell anemia and diabetes mellitus. Previous stud- ies have shown that carriage of the sickle cell trait confers increased susceptibility to P. ovale infection (20) and that diabetes and HIV infection confer increased susceptibility to P. falciparum infection (30). References Int J Parasitol. 2012;42:693–9. http://dx.doi.org/10.1016/j. ijpara.2012.04.015 415 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 RESEARCH 6. May J, Mockenhaupt FP, Ademowo OG, Falusi AG, Olumese PE, Bienzle U, et al. High rate of mixed and subpatent malarial infec- tions in southwest Nigeria. Am J Trop Med Hyg. 1999;61:339–43. 19. World Health Organization. Severe and complicated malaria. Trans R Soc Trop Med Hyg. 1990;84 Suppl 2:1–65. http://dx.doi. org/10.1016/0035-9203(90)90363-J 20. Nolder D, Oguike MC, Maxwell-Scott H, Niyazi HA, Smith V, Chiodini PL, et al. An observational study of malaria in British travellers: Plasmodium ovale wallikeri and Plasmodium ovale curtisi differ significantly in the duration of latency. BMJ Open. 2013;3:e002711. http://dx.doi.org/10.1136/bmjopen-2013-002711 7. Mehlotra RK, Lorry K, Kastens W, Miller SM, Alpers MP, Bockarie M, et al. Random distribution of mixed species malaria in- fections in Papua New Guinea. Am J Trop Med Hyg. 2000;62:225–31. 8. Rojo-Marcos G, Cuadros-González J, Mesa-Latorre JM, Culebras-López AM, de Pablo-Sánchez R. Acute respiratory distress syndrome in a case of Plasmodium ovale malaria. Am J Trop Med Hyg. 2008;79:391–3. 21. Faye FB, Spiegel A, Tall A, Sokhna C, Fontenille D, Rogier C, et al. Diagnostic criteria and risk factors for Plasmodium ovale malaria. J Infect Dis. 2002;186:690–5. http://dx.doi.org/10.1086/342395 9. Li J, Wirtz RA, McConkey GA, Sattabongkot J, Waters AP, Rogers MJ, et al. Plasmodium: genus-conserved primers for species identification and quantitation. Exp Parasitol. 1995;81: 182–90. http://dx.doi.org/10.1006/expr.1995.1107 22. Rojo-Marcos G, Cuadros-González J, Gete-García L, Gómez- Herruz P, López-Rubio M, Esteban-Gutierrez G. Plasmodium ovale infection: description of 16 cases and a review [in Spanish.]. Enferm Infecc Microbiol Clin. 2011;29:204–8. http://dx.doi.org/10.1016/ j.eimc.2010.09.004 10. Calderaro A, Piccolo G, Perandin F, Gorrini C, Peruzzi S, Zuelli C, et al. Genetic polymorphisms influence Plasmodium ovale PCR detection accuracy. J Clin Microbiol. 2007;45:1624–7. http://dx.doi. org/10.1128/JCM.02316-06 23. Kawamoto F, Liu Q, Ferreira MU, Tantular IS. How prevalent are Plasmodium ovale and P. malariae in East Asia? Parasitol Today. 1999;15:422–6. http://dx.doi.org/10.1016/S0169-4758(99) 01511-2 11. Oguike MC, Betson M, Burke M, Nolder D, Stothard JR, Kleinschmidt I, et al. Plasmodium ovale curtisi and Plasmodium ovale wallikeri circulate simultaneously in African communities. Int J Parasitol. 2011;41:677–83. http://dx.doi.org/10.1016/j.ijpara. 2011.01.004 24. Bruce MC, Macheso A, Kelly-Hope LA, Nkhoma S, McConnachie A, Molyneux ME. Effect of transmission setting and mixed species infections on clinical measures of malaria in Malawi. PLoS ONE. 2008;3:e2775. http://dx.doi.org/10.1371/journal.pone.0002775 12. Win TT, Lin K, Mizuno S, Zhou M, Liu Q, Ferreira MU, et al. References Wide distribution of Plasmodium ovale in Myanmar. Trop Med Int Health. 2002;7:231–9. http://dx.doi.org/10.1046/j.1365-3156.2002.00857.x 25. Lacerda MV, Mourão MP, Coelho HC, Santos JB. Thrombocytopenia in malaria: who cares? Mem Inst Oswaldo Cruz. 2011;106(Suppl 1):52–63. http://dx.doi.org/10.1590/S0074-02762011000900007 13. Kawamoto F, Miyake H, Kaneko O, Kimura M, Nguyen TD, Nguyen TD, et al. Sequence variation in the 18S rRNA gene, a target for PCR-based malaria diagnosis in Plasmodium ovale from southern Vietnam. J Clin Microbiol. 1996;34:2287–9. 26. Saravu K, Docherla M, Vasudev A, Shastry BA. Thrombocytopenia in vivax and falciparum malaria: an observational study of 131 patients in Karnataka, India. Ann Trop Med Parasitol. 2011;105:593– 8. http://dx.doi.org/10.1179/2047773211Y.0000000013 14. Win TT, Tantular IS, Pusarawati S, Kerong H, Lin K, Matsuoka H, et al. Detection of Plasmodium ovale by the ICT Malaria P.f/P.v. immunochromatographic test. Acta Trop. 2001;80:283–4. http://dx.doi.org/10.1016/S0001-706X(01)00155-3 27. Moody A. Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev. 2002;15:66–78. http://dx.doi.org/10.1128/ CMR.15.1.66-78.2002 15. Bauffe F, Desplans J, Fraisier C, Parzy D. Real-time PCR assay for discrimination of Plasmodium ovale curtisi and Plasmodium ovale wallikeri in the Ivory Coast and in the Comoros Islands. Malar J. 2012;11:307. http://dx.doi.org/10.1186/1475-2875-11-307 28. Bigaillon C, Fontan E, Cavallo JD, Hernandez E, Spiegel A. Ineffectiveness of the Binax NOW malaria test for diagnosis of Plasmodium ovale malaria. J Clin Microbiol. 2005;43:1011. http://dx.doi.org/10.1128/JCM.43.2.1011.2005 29. Fançony C, Gamboa D, Sebastião Y, Hallett R, Sutherland C, Sousa-Figueiredo JC, et al. Various pfcrt and pfmdr1 geno- types of Plasmodium falciparum cocirculate with P. malariae, P. ovale spp., and P. vivax in northern Angola. Antimicrob Agents Chemother. 2012;56:5271–7. http://dx.doi.org/10.1128/ AAC.00559-12 16. Rojo-Marcos G, Cuadros-González J, Gete-García L, Prieto-Ríos B, Arcos-Pereda P. Imported malaria in a general hospital in Madrid [in Spanish.]. Enferm Infecc Microbiol Clin. 2007;25:168–71. http://dx.doi.org/10.1157/13099367 17. Calderaro A, Gorrini C, Peruzzi S, Piccolo G, Dettori G, Chezzi C. An 8-year survey on the occurrence of imported malaria in a nonen- demic area by microscopy and molecular assays. Diagn Microbiol Infect Dis. 2008;61:434–9. http://dx.doi.org/10.1016/j.diagmicrobio. 2008.03.016 30. Danquah I, Bedu-Addo G, Mockenhaupt FP. Type 2 diabetes mellitus and increased risk for malaria infection. Emerg Infect Dis. 2010;16:1601–4. http://dx.doi.org/10.3201/eid1610.100399 18. Rubio JM, Post RJ, van Leeuwen WM, Henry MC, Lindergard G, Hommel M. Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semin- ested multiplex malaria PCR (SnM-PCR). Trans R Soc Trop Med Hyg. 2002;96 Suppl 1:S199–204. http://dx.doi.org/10.1016/S0035- 9203(02)90077-5 G, Hommel M. References Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semin- ested multiplex malaria PCR (SnM-PCR). Trans R Soc Trop Med Hyg. 2002;96 Suppl 1:S199–204. http://dx.doi.org/10.1016/S0035- 9203(02)90077-5 Address for correspondence: Gerardo Rojo-Marcos, Department of Internal Medicine, Príncipe de Asturias University Hospital, Ctra de Meco s/n, 28805 Alcalá de Henares, Madrid, Spain; email: grojo.hupa@salud.madrid.org Zombies—A Pop Culture Resource for Public Health Awareness Reginald Tucker reads an abridged version of the Emerging Infectious Diseases Another Dimension, Zombies— A Pop Culture Resource for Public Health Awareness. http://www2c.cdc.gov/podcasts/player.asp?f=8628220 Emerging Infectious Diseases Journal Podcasts Zombies—A Pop Culture Resource for Public Health Awareness Reginald Tucker reads an abridged version of the Emerging Infectious Diseases Another Dimension, Zombies— A Pop Culture Resource for Public Health Awareness. http://www2c.cdc.gov/podcasts/player.asp?f=8628220 Emerging Infectious Diseases Journal Podcasts Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014 416 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 3, March 2014
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Bell’s Palsy Onboard: A Case Report of Widespread Disease in a Special Setting
International journal of travel medicine and global health
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Abstract Introduction: Cruises are one of the major travel industries worldwide, and various medical issues can happen onboard. This is a case report of peripheral facial nerve palsy in an older man with diabetes treated onboard a world cruise ship. Case Presentation: A 78-year-old man presented to the infirmary due to difficulty closing his left eye during a 105-day world voyage. The patient was in good general health and had a history of hypertension and diabetes mellitus (treated by dietary intervention). He was diagnosed with severe Bell’s palsy by history and physical examination and treated with prednisolone and valacyclovir without any complications and a near-complete recovery. The possibility of misdiagnosis and deterioration of diabetes mellitus due to the corticosteroid treatment was explained to the patient. Conclusion: When it comes to medical management in a resource-limited setting like cruise ships, passenger physicians trained in a wide variety of illnesses are crucial. It is essential to explain to patients the prognosis and expected outcome of the medical condition. Emergency medical evacuation may be warranted in some instances. Keywords: Aged, Diabetes Mellitus Type 2, Emergency Medicine, Facial Paralysis, Oceans, Seas Citation: Aoki Y. Bell’s palsy onboard: a case report of widespread disease in a special setting. Int J Travel Med Glob Health. 2021;9(4):191-193. doi:10 34172/ijtmgh 2021 31 Citation: Aoki Y. Bell’s palsy onboard: a case report of widespread disease in a special setting. Int J Travel Med Glob Health. 2021;9(4):191-193. doi:10.34172/ijtmgh.2021.31. difficulty closing his left eye. The patient and his wife were on a 105-day world voyage. He was on candesartan for hypertension and only dietary intervention for diabetes mellitus. A “fit to travel” letter was provided by his primary care physician before he had embarked. On the day of embarkation (day 1) from Japan, he noticed dryness and redness in his left eye, and on day 3, he consulted the ship’s infirmary. At that time, the ship would be at sea for 8 d to the next port of call. The patient was in an excellent general condition, with a blood pressure of 130/68 mm Hg, heart rate of 82 beats/min, oxygen saturation of 96% on room air, respiratory rate of 16 breaths/min, and body temperature of 36.6℃. On physical examination, the patient’s face was asymmetric. Other than the facial nerve paralysis that had resulted in the loss of forehead movement, there were no cranial nerve deficits. TMGH IInternational Journal of Travel Medicine and Glob J 10.34172/ijtmgh.2021.31 Case Report Open Access Copyright © 2021 The Author(s). 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. Introduction Cruises are one of the significant travel industries globally, although they are currently suspended due to the emergence of the coronavirus disease 2019 (COVID-19). Various medical issues can happen onboard, which are most commonly outbreaks of respiratory infections and gastroenteritis.1 The need to revise infection control regulations onboard ships are even more critical now with COVID-19. In addition, other illnesses unrelated to infectious diseases can also occur, such as cardiovascular and cerebrovascular events, which sometimes require medical evacuation.2 Because of this wide range of medical issues, onboard physicians, crew members, and maybe even passengers are required to provide appropriate first aid for all kinds of emergencies. Even though most patients visiting onboard clinics do not have severe conditions, some passengers may develop acute diseases due to their underlying diseases, as many passengers on cruise ships are older adults.3 This is a case report of peripheral facial nerve palsy in an elderly male patient treated onboard a world cruise ship before the start of the COVID-19 pandemic. Abstract No paralysis in the upper and lower extremities was noted, neither was ataxia. The tympanic membrane was transparent bilaterally, and there were no bullous eruptions around the auricle. The patient did not report any hearing loss. The probability of Lyme disease was low based on its geographic distribution. Unfortunately, the ship did not have any diagnostic imaging facilities. The patient was subsequently diagnosed with severe Bell’s palsy because of the difference between the left and right sides of Bell’s Palsy Onboard: A Case Report of Widespread Disease in a Special Setting epartment of Emergency and Critical Care Medicine, Aizawa Hospital, Nagano, Japan 1Department of Emergency and Critical Care Medicine, Aizawa Hospital, Nagano, Japan Corresponding Author: Yoshihiro Aoki, MD, Department of Emergency and Critical Care Medicine, Aizawa Hospital, 2-5-1 Honjo, Matsumoto, Nagano 390-8510, Japan. Tel: +81-263-33-8600, Fax: +81-263-32-6763, Email: yaoki-hki@umin.ac.jp http://ijtmgh.com Int J Travel Med Glob Health. 2021 Dec;9(4):191-193 doi 10.34172/ijtmgh.2021.31 TMGH IInternational Journal of Travel Medicine and Global Health J Case Report Open Access http://ijtmgh.com Int J Travel Med Glob Health. 2021 Dec;9(4):191-193 doi 10.34172/ijtmgh.2021.31 TMGH IInternational Journal of Travel Medicine and Global Health J Case Report Open Access http://ijtmgh.com Int J Travel Med Glob Health. 2021 Dec;9(4):191-193 Discussion We present a case of peripheral facial palsy in an elderly male patient with diabetes onboard. Bell’s palsy is a widespread disease that emergency physicians frequently encounter in the emergency department on the land. Although considering developments in maritime travel, experienced medical providers on board must have diagnosed and treated patients with Bell’s palsy in each setting, which has been rarely reported in the literature to date. Corticosteroids and antivirals are usually available on board; therefore, patients can be sufficiently managed. However, treating patients with Bell’s palsy, especially on board a cruise ship, in a challenging setting with limited resources includes some important points to note. p g y p p The cruise ship physician must be capable of responding to various illnesses and make appropriate decisions regarding the transfer of patients for further medical treatment; thus, it is necessary for the physician to receive training in a wide range of illnesses. Broad experience in emergency and general medicine, good communication skills, and previous cruise experience are useful requirements.11 To tackle challenges in management, access to medical information online is crucial. In our case, it was possible to search relevant articles, which greatly affected the treatment. Reports describing misdiagnosed cases can also contribute to the accumulation of knowledge of the disease. Using Bell’s palsy as an example, a recent literature review revealed that in cases of facial palsy, the presence of malignant or benign tumors, sarcoidosis, infections, neurodegeneration, and cranial hemorrhage were sometimes overlooked because of the rare diseases nature, false-negative or misinterpreted neuroimaging studies, or a failure to recognize the physical signs.12 During the patient’s initial consultation, the ship was supposed to be at sea for an additional 8 d before moving to the subsequent scheduled port. In the clinical setting, Bell’s palsy can be identified by emergency physicians with a low degree of misdiagnosis,4 in most cases by history and physical examination alone.5 However, differential diagnoses include otitis media, temporal bone fracture, postoperative damage, neoplasm, sarcoidosis, herpes zoster viral infection, and cerebrovascular accident.6 Accordingly, computed tomography and magnetic resonance imaging should be performed if a cause related to the central nervous system, such as a cerebrovascular disorder or a brain tumor, is suspected. Therefore, if upper motor neuron disease or cranial nerve involvements other than the facial nerve are suspected, an emergency evacuation may be required for further investigation. Conclusion A case of a male diabetes patient with Bell’s palsy occurred onboard during a long-term cruise. A passenger physician onboard should be aware that Bell’s palsy may be encountered during a long-term cruise. When it comes to medical management in a resource-limited setting like cruise ships, physicians trained in a wide variety of illnesses are crucial. It is essential to explain to patients the prognosis and expected outcome of the medical condition. Emergency medical evacuation may be warranted in some instances. Discussion In addition, this raise concerns regarding the impact on the entire shipping route, transportation, and cost. In the case of onboard medical treatment, however, patient disposition should always be determined based on what is best for the patient. In the present case, the medical history and physical examination were compatible with a diagnosis of Bell’s palsy. However, the potential of missing another diagnosis, such as a neoplasm, and the implications of delayed definitive management were explained to the patient because a previous study had suggested that diabetes in older adults is a risk factor for the misdiagnosis of Bell’s palsy.4 Notably, COVID‑19 has recently been reported to be associated with Furthermore, incorporating remote medical staff or telemedical advice is essential for safe cruise travel, which creates a system that allows for consultation with specialists, such as otorhinolaryngologists. Case Presentation A 78-year-old man presented to the infirmary onboard due to Aoki the face at rest and the inability to close the eye despite the maximum effort completely. Bell’s palsy.7 In the relevant situations, physicians should be aware that Bell’s palsy can be a sign of COVID-19, although the current case occurred before COVID-19 emerged. y Treatment with prednisolone 60 mg/d and valacyclovir 3000 mg/day was initiated. The symptoms began to improve, and on the fifth day of treatment, prednisolone was gradually tapered and discontinued on the tenth day. Valacyclovir was also discontinued after seven days. The patient’s blood glucose level temporarily increased to 200–250 mg/ dL, but no alterations in the level of consciousness were observed. On the 71st day of the voyage, the facial paralysis had almost completely resolved. The patient eventually completed the cruise travel and disembarked with a referral letter to an otorhinolaryngologist on the land for a follow-up consultation. However, the outcome and final diagnosis are unknown as no reply was received to the referral letter. About not only diagnosis but also treatment, diabetes mellitus should be managed carefully. Treatment of Bell’s palsy with corticosteroids within three days of symptom onset has been associated with an increased recovery rate at 3 and 9 months.8 Thus, passenger physicians should initiate appropriate treatment immediately to improve long-term recovery. Regarding adverse events, glucocorticoids can induce diabetes mellitus and exacerbate hyperglycemia in patients with underlying diabetes.9 Notably, cruise travel generally serves rich foods to passengers, which can cause deterioration of blood sugar control in diabetic patients. In this case, the patient was informed that the treatment might affect glycemic control and potentially result in a diabetic coma. Although the patient’s diabetes was well controlled without any medications or insulin, preboarding management of underlying diseases is particularly important in a resource- limited cruise ship setting that includes many older people.10 International Journal of Travel Medicine and Global Health. 2021;9(4):191-193 192 Funding/Support None. 9. Perez A, Jansen-Chaparro S, Saigi I, Bernal-Lopez MR, Miñambres I, Gomez-Huelgas R. Glucocorticoid-induced hyperglycemia. J Diabetes. 2014;6(1):9-20. doi:10.1111/1753- 0407.12090. Conflict of Interest Disclosures Conflict of Interest Disclosures The authors have no conflicts of interest to declare. 6. Phan NT, Panizza B, Wallwork B. A general practice approach to Bell’s palsy. Aust Fam Physician. 2016;45(11):794-797. International Journal of Travel Medicine and Global Health. 2021;9(4):191-193 193 Ethical Approval 7. Tamaki A, Cabrera CI, Li S, et al. Incidence of Bell’s palsy in patients with COVID-19. JAMA Otolaryngol Head Neck Surg. 2021;147(8):767-768. doi:10.1001/jamaoto.2021.1266. Informed consent was obtained from the patient for the publication of this manuscript. Informed consent was obtained from the patient for the publication of this manuscript. 8. Sullivan FM, Swan IR, Donnan PT, et al. Early treatment with prednisolone or acyclovir in Bell’s palsy. N Engl J Med. 2007;357(16):1598-1607. doi:10.1056/NEJMoa072006. Author’s Contributions The author managed the patient on board under employment by the Japanese travel agency that chartered the cruise ship described in this manuscript, reviewed the clinical chart, perused the potentially International Journal of Travel Medicine and Global Health. 2021;9(4):191-193 2 Bell’s Palsy Onboard Bell’s palsy in the emergency department. Ann Emerg Med. 2014;63(4):428-434. doi:10.1016/j.annemergmed.2013.06.022. Bell’s palsy in the emergency department. Ann Emerg Med. 2014;63(4):428-434. doi:10.1016/j.annemergmed.2013.06.022. relevant literature, and drafted the manuscript. The author approved the submission of the report and took full responsibility for its content. 5. Garro A, Nigrovic LE. Managing peripheral facial palsy. Ann Emerg Med. 2018;71(5):618-624. doi:10.1016/j. annemergmed.2017.08.039. References 1. Pavli A, Maltezou HC, Papadakis A, et al. Respiratory infections and gastrointestinal illness on a cruise ship: a three-year prospective study. Travel Med Infect Dis. 2016;14(4):389-397. doi:10.1016/j.tmaid.2016.05.019. 10. Aoki Y, Amaya Dimas LDC, Hatachi T, et al. Association between underlying disease and serious events on a world cruise ship: a prospective cohort study. Travel Med Infect Dis. 2021;41:102052. doi:10.1016/j.tmaid.2021.102052. 2. Peake DE, Gray CL, Ludwig MR, Hill CD. Descriptive epidemiology of injury and illness among cruise ship passengers. Ann Emerg Med. 1999;33(1):67-72. doi:10.1016/ s0196-0644(99)70419-1. 11. Dahl E. Medical practice during a world cruise: a descriptive epidemiological study of injury and illness among passengers and crew. Int Marit Health. 2005;56(1-4):115-128. 3. Miller JM, Tam TW, Maloney S, et al. Cruise ships: high-risk passengers and the global spread of new influenza viruses. Clin Infect Dis. 2000;31(2):433-438. doi:10.1086/313974. 12. Bacorn C, Fong NST, Lin LK. Misdiagnosis of Bell’s palsy: case series and literature review. Clin Case Rep. 2020;8(7):1185- 1191. doi:10.1002/ccr3.2832. 4. Fahimi J, Navi BB, Kamel H. Potential misdiagnoses of
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Two new proofs of the fact that triangle groups are distinguished by their finite quotients
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NEW ZEALAND JOURNAL OF MATHEMATICS Volume 52 (2022), 827–844 https://doi.org/10.53733/193 NEW ZEALAND JOURNAL OF MATHEMATICS Volume 52 (2022), 827–844 https://doi.org/10.53733/193 NEW ZEALAND JOURNAL OF MATHEMATICS Volume 52 (2022), 827–844 https://doi.org/10.53733/193 TWO NEW PROOFS OF THE FACT THAT TRIANGLE GROUPS ARE DISTINGUISHED BY THEIR FINITE QUOTIENTS Marston Conder (Received 14 December, 2021) Dedicated to the memory of my good friend and colleague Sir Vaughan Jones Abstract. In a 2016 paper by Alan Reid, Martin Bridson and the author, it was shown using the theory of profinite groups that if Γ is a finitely-generated Fuchsian group and Σ is a lattice in a connected Lie group, such that Γ and Σ have exactly the same finite quotients, then Γ is isomorphic to Σ. As a consequence, two triangle groups ∆(r, s, t) and ∆(u, v, w) have the same finite quotients if and only if (u, v, w) is a permutation of (r, s, t). A direct proof of this property of triangle groups was given in the final section of that paper, with the purpose of exhibiting explicit finite quotients that can distinguish one triangle group from another. Unfortunately, part of the latter direct proof was flawed. In this paper two new direct proofs are given, one being a cor- rected version using the same approach as before (involving direct products of small quotients), and the other being a shorter one that uses the same prelim- inary observations as in the earlier version but then takes a different direction (involving further use of the ‘Macbeath trick’). 1. Introduction For any positive integers r, s and t, the ordinary (r, s, t) triangle group is the abstract group ∆(r, s, t) with presentation ⟨x, y, z | xr = ys = zt = xyz = 1 ⟩. In a recent paper [2], it was shown that if Γ is a finitely-generated Fuchsian group and Σ is a lattice in a connected Lie group, such that Γ and Σ have exactly the same finite quotients, then Γ is isomorphic to Σ. As a consequence, two triangle groups ∆(r, s, t) and ∆(u, v, w) have the same finite quotients if and only if (u, v, w) is a permutation of (r, s, t). The main theorems in the first seven sections of [2], including these, were proved using the theory of profinite groups, without reference to explicit finite quotients. A second proof of the fact concerning triangle groups was given in the final section of [2], with the purpose of exhibiting explicit finite quotients that can distinguish one triangle group from another. Unfortunately, this second proof (given as the proof of Theorem 8.1 in [2]) was flawed. In particular, Lemma 8.11 is incorrect, even when restricted to triples (r, s, t) and (u, v, w) that survive Lemmas 8.3, 8.4, 8.5 and 8.7, Proposition 8.6 and Corollaries 8.8 and 8.10, as it fails for the triples (15, 105, 126) and (21, 30, 315) with (q2, q2) = (7, 45), as well as for similar triples. Also there were gaps in the argument used in the proof of Theorem 8.1. The situation is somewhat more complicated than was indicated. In particular, the use of direct products of two quotients each isomorphic to PSL(2, p) for some prime p did not cover all possibilities remaining after simpler methods were applied. Please note, however, that the main findings of [2] are unaffected by these flaws, as Section 8 of [2] is independent of the earlier sections. 828 MARSTON CONDER Here we must sincerely thank Frankie Chan, who pointed out the problems with Lemma 8.11, and provided an effective version of Theorem 8.1 of [2] in his recent PhD thesis [3], while developing an algorithm for distinguishing finite quotients between cocompact triangle groups and lattices of constant-curvature symmetric 2-spaces, with specific attention paid to the case where these lattices are Fuchsian groups. 1. Introduction In this paper we give two new direct proofs of Theorem 8.1 of [2], without requiring Lemma 8.11. The first one is rather intricate, showing how the approach involving direct products of quotients isomorphic to PSL(2, p) for some prime p can be undertaken correctly and extended in some exceptional cases (by using quotients that are direct products of the form PSL(2, p1) × PSL(2, p2) × A where A is cyclic, in those cases). The second proof is quite a lot shorter, using certain smooth quotients of triangle groups ∆(k, l, m), where k, l and m divide either r, s and t, or u, v and w (in some order). This is an approach we considered earlier and were able to use successfully to deal with the vast majority of triple pairs, but completed only with the help of a key observation made by Frankie Chan in [3] for the remaining pairs, and again we owe a debt of gratitude to him for that. Here we note that both proofs show that any two non-isomorphic hyperbolic triangle groups ∆(r, s, t) and ∆(u, v, w) can be distinguished by finite quotients that are abelian, dihedral, isomorphic to PSL(2, p) for some prime p, or a direct product PSL(2, p1) × PSL(2, p2) × A where p1 and p2 are distinct primes and A is cyclic (or trivial), or an extension of a homocyclic abelian group by one of the preceding groups. Before giving the two new proofs, we repeat and extend some of the background to this topic, in order to make the paper self-contained. DISTINGUISHING TRIANGLE GROUPS canonical generators of ∆(k, l, m), and has torsion-free kernel. For any hyperbolic triple (k, l, m), the set of (k, l, m)-generated groups is non-empty because of the residual finiteness of the hyperbolic triangle group ∆(k, l, m), but in most cases ∆(k, l, m) can also have ‘non-smooth’ quotients, in which the orders of the canonical generators of ∆(k, l, m) are not preserved. ( ) We will make use of the following important but relatively straightforward ob- servation, the proof of which we leave as an exercise for the reader: Lemma 2.1. For any integers k, l and m, all greater than 1, let ∆be the triangle group ∆(k, l, m). Then the abelianisation ∆/∆′ = ∆/[∆, ∆] of ∆is isomorphic to the direct product Ce × Cd, where e = lcm(gcd(k, l), gcd(k, m), gcd(l, m)) and d = gcd(k, l, m), and also de = klm lcm(k, l, m). Moreover, ∆/[∆, ∆] is (k′, l′, m′)- generated, where k′ = gcd(k, lm), l′ = gcd(l, km) and m′ = gcd(m, kl). Also a theorem below by Macbeath [6] on triangle-generation of the finite simple groups PSL(2, q) is critical to this work: Theorem 2.2. Let (k, l, m) be any hyperbolic triple other than (2, 5, 5), (3, 4, 4), (3, 3, 5), (3, 5, 5) or (5, 5, 5). Then for any given odd prime p, the group PSL(2, pf) is (k, l, m)-generated if and only if pf is the smallest power of p for which PSL(2, pf) contains elements of orders k, l and m. The triples (2, 5, 5), (3, 4, 4), (3, 3, 5), (3, 5, 5) and (5, 5, 5), together with the spherical triples and the triple (3, 3, 3), were called exceptional by Macbeath. Note that the spherical group ∆(2, 3, 5) ∼= A5 ∼= PSL(2, 5) is also (2, 5, 5)-, (3, 3, 5)-, (3, 5, 5)- and (5, 5, 5)-generated, while the spherical group ∆(2, 3, 4) ∼= S4 is also (3, 4, 4)-generated. We also make use of the fact that if the finite group G is (k, l, m)-generated, then G is a group of conformal automorphisms of a compact Riemann surface S of genus g, where 2 −2g = |G| 1 k + 1 l + 1 m −1  as a consequence of the Riemann-Hurwitz formula. 2. Further Background Each triangle group ∆(r, s, t) is called spherical, Euclidean or hyperbolic accord- ing to whether the quantity 1/r + 1/s + 1/t is greater than, equal to or less than 1, respectively. Note that ∆(r, s, t) is isomorphic to ∆(u, v, w) if and only if the triple (u, v, w) is a permutation of the triple (r, s, t). ( ) ( ) The spherical triangle groups are ∆(1, n, n), ∆(2, 2, n), ∆(2, 3, 3), ∆(2, 3, 4) and ∆(2, 3, 5), which are isomorphic to Cn (cyclic), Dn (dihedral of order 2n), A4, S4 and A5, respectively. The Euclidean triangle groups are ∆(2, 3, 6), ∆(2, 4, 4) and ∆(3, 3, 3), each which is an extension of a free abelian group of rank 2 by a cyclic group (C6, C4 and C3, respectively). In particular, the spherical triangle groups are finite, while the Euclidean triangle groups are infinite but soluble. See [4] for further details. In contrast, all hyperbolic triangle groups are infinite and insoluble, and have a wealth of finite quotients (see [5] for example). The latter categorisation makes the spherical and Euclidean triangle groups easy to distinguish from others by their finite quotients, and so we will focus our attention on the hyperbolic ones, namely those with 1/r + 1/s + 1/t < 1. As in [2], we define a finite group G to be (k, l, m)-generated if G can be generated by elements a, b and c of (precise) orders k, l and m such that abc = 1. In this case we say that G is a smooth quotient of the triangle group ∆(k, l, m), noting that the corresponding epimorphism from ∆(k, l, m) to G preserves the orders of the 829 DISTINGUISHING TRIANGLE GROUPS DISTINGUISHING TRIANGLE GROUPS The kernel K of the correspond- ing smooth homomorphism from ∆(k, l, m) onto G is the fundamental group of S, and is itself a Fuchsian group, with signature (2g; −). In particular, if g ≥1 then K is generated by 2g elements a1, b1, . . . , ag, bg subject to a single defining relation [a1, b1] . . . [ag, bg] = 1. Now for any positive integer n, the subgroup K′K(n) gener- ated by the derived subgroup K′ and the nth powers of all elements of K is charac- teristic in K and hence normal in ∆(k, l, m), and the quotient ∆(k, l, m)/K′K(n) is then isomorphic to an extension by G of an abelian subgroup K/K′K(n) of rank 2g and exponent n (and order n2g). This observation is often known as ‘the Macbeath trick’. as a consequence of the Riemann-Hurwitz formula. The kernel K of the correspond- ing smooth homomorphism from ∆(k, l, m) onto G is the fundamental group of S, and is itself a Fuchsian group, with signature (2g; −). In particular, if g ≥1 then K is generated by 2g elements a1, b1, . . . , ag, bg subject to a single defining relation [a1, b1] . . . [ag, bg] = 1. Now for any positive integer n, the subgroup K′K(n) gener- ated by the derived subgroup K′ and the nth powers of all elements of K is charac- teristic in K and hence normal in ∆(k, l, m), and the quotient ∆(k, l, m)/K′K(n) is then isomorphic to an extension by G of an abelian subgroup K/K′K(n) of rank 2g and exponent n (and order n2g). This observation is often known as ‘the Macbeath trick’. Hence for any (k, l, m)-generated group G, we can construct an infinite family of smooth quotients of ∆(k, l, m), to help distinguish ∆(k, l, m) from other triangle groups. In what follows, we will also require some information about the groups PSL(2, p), for p prime. When p is odd, the orders of the elements of PSL(2, p) are precisely the divisors of p, p−1 2 and p+1 2 (see [7, Chapter 3.6] for example). The integers p, p−1 2 830 MARSTON CONDER and p+1 2 are pairwise coprime, so the order of any non-trivial element of PSL(2, p) divides exactly one of them. Corollary 2.3. (a) If (k, l, m) is a hyperbolic triple and p is an odd prime, then the group PSL(2, p) is (k, l, m)-generated if and only if every member of the L2-set of the triple (k, l, m) is equal to p or a divisor of p±1 2 . (b) Let (k, l, m) be any triple of integers greater than 1. Then for every integer q > 3 that does not divide any of the members of the L2-set of (k, l, m), there exists a smooth finite quotient G of the (k, l, m) triangle group such that G has no element of order q. Similarly, for every integer q > 1 that is coprime to 6 and to every member of the L2-set of (k, l, m), there exists a smooth finite quotient G of the (k, l, m) triangle group such that G has no non-trivial element of order dividing q. Moreover, when the triple (k, l, m) is hyperbolic, in both cases G can be taken as PSL(2, p) for some prime p > 5. (c) If two triples of integers greater than 1 have the same least common multiple but different L2-sets, then the corresponding triangle groups have different sets of smooth quotients. Proof. Part (a) is an immediate consequence of Theorem 2.2. ( ) q Both cases of part (b) are easy for all non-hyperbolic triples and exceptional triples: we can take G = Dm for (k, l, m) = (2, 2, m) whenever m ≥2, or G = A4 for (k, l, m) = (2, 3, 3), or G = S4 for (k, l, m) = (2, 3, 4) or (3, 4, 4), or G = A5 for (k, l, m) = (2, 3, 5), (2, 5, 5), (3, 3, 5), (3, 5, 5) or (5, 5, 5), or G = C6 for (k, l, m) = (2, 3, 6), or G = C4 for (k, l, m) = (2, 4, 4), or G = C3 for (k, l, m) = (3, 3, 3). DISTINGUISHING TRIANGLE GROUPS Next, define the L2-set of a triple (k, l, m) to be the (unique) set of pairwise co- prime positive integers whose least common multiple is the same as that of {k, l, m} and which has the property that each of k, l and m divides exactly one member of that set. For example, if k, l and m are themselves pairwise coprime, then the L2- set of the triple (k, l, m) is just {k, l, m}, while if gcd(k, lm) = 1 but gcd(l, m) > 1 then its L2-set is {k, lcm(l, m))}, and if gcd(k, l) > 1 and gcd(l, m) > 1 then its L2- set is {lcm(k, l, m))}. Note that every maximal prime-power divisor of lcm(k, l, m) divides exactly one member of the L2-set. Accordingly, we have the following corollary of Macbeath’s theorem (Theo- rem 2.2): Corollary 2.3. For any non-exceptional hyperbolic triple (k, l, m), we can take G = PSL(2, p), where p is a prime such that p ≡±1 modulo twice each of the members of the L2-set of (k, l, m), but p ̸≡±1 modulo 2q in the first case, and p ̸≡±1 modulo h for every prime divisor h of q in the second case. In both cases, the existence of such a prime p is guaranteed by the Chinese Remainder Theorem and Dirichlet’s theorem on primes in arithmetic progression. Finally, for part (c), the L2-set of one of the two triples must contain an integer q > 3 that does not divide any of the members of the L2-set of the other triple, and then the assertion follows from part (b). □ DISTINGUISHING TRIANGLE GROUPS 831 3. Main Theorem and Preliminary Steps for its New Proofs Parts (a) to (c) follow immediately from Lemma 2. ( ) ( ) y For part (d), we may suppose without loss of generality that 1 r + 1 s + 1 t ≤ 1 u + 1 v + 1 w, and let G be any (r, s, t)-generated finite quotient of Γ. Then G is also a quotient of Σ. So now let u′, v′ and w′ be divisors of u, v and w (respectively) chosen such that G is (u′, v′, w′)-generated, and 1 u′ + 1 v′ + 1 w′ is as small as possible subject to those conditions. Then in particular, 1 u′ + 1 v′ + 1 w′ ≥1 u + 1 v + 1 w ≥1 r + 1 s + 1 t . Next, for any n coprime to |G|, the largest quotient of Γ that is an extension of an abelian group of exponent n by G has order n2g|G|, where 2−2g = |G|( 1 r + 1 s + 1 t −1), by comments made in the previous section. On the other hand, the largest quotient of Σ that is an extension of an abelian group of exponent n by G is a smooth quotient of the (u′, v′, w′) triangle group and so has order n2g′|G|, where 2−2g′ = |G|( 1 u′ + 1 v′ + 1 w′ −1). Since Γ and Σ have the same quotients, we find that g′ = g, so 1 u′ + 1 v′ + 1 w′ = 1 r + 1 s + 1 t . The final observation in the previous paragraph now gives us 1 u + 1 v + 1 w = 1 r + 1 s + 1 t , as required. ( ) ( ) For part (d), we may suppose without loss of generality that 1 r + 1 s + 1 t ≤ 1 u + 1 v + 1 w, and let G be any (r, s, t)-generated finite quotient of Γ. Then G is also a quotient of Σ. So now let u′, v′ and w′ be divisors of u, v and w (respectively) chosen such that G is (u′, v′, w′)-generated, and 1 u′ + 1 v′ + 1 w′ is as small as possible subject to those conditions. 3. Main Theorem and Preliminary Steps for its New Proofs We can now begin to prove the following, in which we use the notation (r, s, t) ≃ (u, v, w) to mean that (r, s, t) is a permutation of (u, v, w). Theorem 3.1. If Γ = ∆(r, s, t) and Σ = ∆(u, v, w) are triangle groups having exactly the same finite quotients, then (r, s, t) ≃(u, v, w) and so Γ ∼= Σ. As a first step, we may suppose that (r, s, t) and (u, v, w) are hyperbolic triples. Next, we present a key observation that generalises part of Lemma 8.4 of [2], followed by combination of the remainder of Lemmas 8.3 to 8.5 and 8.7 of [2] and some further helpful properties. Lemma 3.2. Under the given assumptions, Γ = ∆(r, s, t) and Σ = ∆(u, v, w) have the same (k, l, m)-generated quotients, for every triple (k, l, m) of integers greater than 1. Proof. This follows easily from the assumption that Γ and Σ have exactly the same finite quotients. □ Lemma 3.3. Under the given assumptions, Lemma 3.3. Under the given assumptions, Lemma 3.3. Under the given assumptions, Lemma 3.3. 3. Main Theorem and Preliminary Steps for its New Proofs Under the given assumptions, (a) gcd(r, s, t) = gcd(u, v, w), (b) rst lcm(r, s, t) = uvw lcm(u, v, w), (c) lcm(gcd(r, s), gcd(r, t), gcd(s, t)) = lcm(gcd(u, v), gcd(u, w), gcd(v, w)), (d) 1 + 1 + 1 t = 1 + 1 + 1 , (c) lcm(gcd(r, s), gcd(r, t), gcd(s, t)) = lcm(gcd(u, v), gcd(u, w), gcd(v, w)) (d) 1 r + 1 s + 1 t = 1 u + 1 v + 1 w, (e) a finite group is (r, s, t)-generated if and only if it is (u, v, w)-generated, (f) rst = uvw, and lcm(r, s, t) = lcm(u, v, w), and rs + rt + st = uv + uw + vw, (g) v2(s −u)(w −s) = s2(v −r)(t −v), and similarly for every permutation of (r, s, t) and every permutation of (u, v, w), (f) rst = uvw, and lcm(r, s, t) = lcm(u, v, w), and rs + rt + st = uv + uw + vw, (f) rst = uvw, and lcm(r, s, t) = lcm(u, v, w), and rs + rt + st = uv + uw + vw, (g) v2(s −u)(w −s) = s2(v −r)(t −v), and similarly for every permutation of (r, s, t) and every permutation of (u, v, w), (g) v2(s −u)(w −s) = s2(v −r)(t −v), and similarly for every permutation of (r, s, t) and every permutation of (u, v, w), (g) v2(s −u)(w −s) = s2(v −r)(t −v), and similarly for every permutation of (r, s, t) and every permutation of (u, v, w), (h) if r ≤s ≤t and u ≤v ≤w, and (r, s, t) and (u, v, w) have no common entry, then either r < u ≤v < s ≤t < w or u < r ≤s < v ≤w < t, and (h) if r ≤s ≤t and u ≤v ≤w, and (r, s, t) and (u, v, w) have no common entry, then either r < u ≤v < s ≤t < w or u < r ≤s < v ≤w < t, and (i) the triples (r, s, t) and (u, v, w) have the same L2-set. (i) the triples (r, s, t) and (u, v, w) have the same L2-set. Proof. Parts (a) to (c) follow immediately from Lemma 2.1. Proof. 3. Main Theorem and Preliminary Steps for its New Proofs Then since (r, s, t) and (u, v, w) have no common entry, we have only the following five possibilities, with others ruled out by the identity rst = uvw: r ≤s < u ≤v ≤w < t, or r < u < s < v ≤w < t, or r < u ≤v < s ≤t < w, or r < u ≤v < s < w < t, or r < u ≤v ≤w < s ≤t. The first, second, fourth and fifth of these possibilities can be eliminated, however, as they imply v2(s −u)(w −s) < 0 < s2(v −r)(t −v), which contradicts part (g). The analogous argument works also when u < r. r ≤s < u ≤v ≤w < t, or r < u < s < v ≤w < t, or r < u ≤v < s ≤t < w, or r < u ≤v < s < w < t, or r < u ≤v ≤w < s ≤t. r ≤s < u ≤v ≤w < t, or r < u < s < v ≤w < t, or r < u ≤v < s ≤t < w, or r < u ≤v < s < w < t, or r < u ≤v ≤w < s ≤t. The first, second, fourth and fifth of these possibilities can be eliminated, however, as they imply v2(s −u)(w −s) < 0 < s2(v −r)(t −v), which contradicts part (g). The analogous argument works also when u < r. □ Finally, part (i) follows immediately from part (c) of Corollary 2.3. At this stage we have enough to prove what happens in certain special cases, some of which were covered by Lemma 8.6 and Corollaries 8.8 and 8.10 of [2]. Proposition 3.4. If u, v and w have divisors u′, v′ and w′, all greater than 1, such that at least one of r, s and t is coprime to each of 6, u′, v′ and w′, then there exists a finite group that is a quotient of ∆(u, v, w) but not a quotient of ∆(r, s, t). In particular, this holds when one of r, s and t is a power of some prime p > 3 such that none of u, v and w is a power of p. 3. Main Theorem and Preliminary Steps for its New Proofs Then in particular, 1 u′ + 1 v′ + 1 w′ ≥1 u + 1 v + 1 w ≥1 r + 1 s + 1 t . Next, for any n coprime to |G|, the largest quotient of Γ that is an extension of an abelian group of exponent n by G has order n2g|G|, where 2−2g = |G|( 1 r + 1 s + 1 t −1), by comments made in the previous section. On the other hand, the largest quotient of Σ that is an extension of an abelian group of exponent n by G is a smooth quotient of the (u′, v′, w′) triangle group and so has order n2g′|G|, where 2−2g′ = |G|( 1 u′ + 1 v′ + 1 w′ −1). Since Γ and Σ have the same quotients, we find that g′ = g, so 1 u′ + 1 v′ + 1 w′ = 1 r + 1 s + 1 t . The final observation in the previous paragraph now gives us 1 u + 1 v + 1 w = 1 r + 1 s + 1 t , as required. u v w u v w r s t Next, for any n coprime to |G|, the largest quotient of Γ that is an extension of an abelian group of exponent n by G has order n2g|G|, where 2−2g = |G|( 1 r + 1 s + 1 t −1), by comments made in the previous section. On the other hand, the largest quotient of Σ that is an extension of an abelian group of exponent n by G is a smooth quotient of the (u′, v′, w′) triangle group and so has order n2g′|G|, where 2−2g′ = |G|( 1 u′ + 1 v′ + 1 w′ −1). Since Γ and Σ have the same quotients, we find that g′ = g, so 1 u′ + 1 v′ + 1 w′ = 1 r + 1 s + 1 t . The final observation in the previous paragraph now gives us 1 u + 1 v + 1 w = 1 r + 1 s + 1 t , as required. 832 MARSTON CONDER The latter also implies that (u′, v′, w′) = (u, v, w), and hence that G is (u, v, w)- generated. 3. Main Theorem and Preliminary Steps for its New Proofs The converse holds by the same argument, with the roles of (r, s, t) and (u, v, w) reversed, and this proves (e). For part (f), let p be any prime divisor of rst, and let pα, pβ and pγ be the largest powers of p dividing r, s and t, ordered in such a way that α ≤β ≤γ. Then pα must be the largest power of p dividing gcd(r, s, t), while pβ is the largest power of p dividing lcm(gcd(r, s), gcd(r, t), gcd(s, t)), and pγ is the largest power of p dividing lcm(r, s, t). Also pα+β+γ is the largest power of p dividing rst, and so pα+β is the largest power of p dividing rst lcm(r,s,t). Furthermore, either γ = β, or pγ is the largest power of p dividing the denominator of 1 r + 1 s + 1 t = rs+rt+st rst when the latter is expressed in reduced form. (To verify the last claim, note that rs + rt + st is divisible by pα+β but not pα+β+1 when α ≤β < γ.) Hence the largest powers of p dividing r,s and t are determined by the quantities gcd(r, s, t), rst lcm(r,s,t) and 1 r + 1 s + 1 t . By parts (a), (b) and (d), these three quantities are the same for the triple (u, v, w), and hence the largest powers of p dividing u, v and w are equal to those for r, s and t, in some order. As this holds for all p, the stated identities follow easily. Part (g) follows from expanding and simplifying v2(s−u)(w−s)−s2(v−r)(t−v) using the identities rst = uvw and rs + rt + st = uv + uw + vw from part (f), and noting that those two identities are invariant under all permutations of (r, s, t) and/or (u, v, w). / ( ) To prove part (h), let us suppose that r < u, so that r = min{r, s, t, u, v, w}. 3. Main Theorem and Preliminary Steps for its New Proofs Then lcm(gcd(r, s), gcd(r, t), gcd(s, t)) is even, and therefore so is lcm(gcd(u, v), gcd(u, w), gcd(v, w)), and hence two or more of u, v and w are even. Also if all three of r, s and t are even, then gcd(r, s, t) is even, and then so is gcd(u, v, w), and hence all three of u, v and w are even. Now let m = max(r, s, t, u, v, w) if all three of r, s and t are even, or otherwise let m be the largest odd integer among r, s, t, u, v and w. Then the dihedral group Dm is a (2, 2, m)-generated quotient of Γ or Σ, and hence must also be a (2, 2, m)-generated quotient of the other. By definition of m, and the fact mentioned at the start of this paragraph, it follows that m appears in both triples (r, s, t) and (u, v, w), and hence by case (b) we know that (r, s, t) ≃(u, v, w). In case (d), suppose first that gcd(r, st) = 1. Then the L2-set of (r, s, t) is either {r, s, t} or {r, st}, and is the same as the L2-set of (u, v, w), by part (i) of Lemma 3.3. It follows that each of u, v and w divides r or st. If one of them divides r and the other two divide st, then since gcd(r, st) = 1, one of them is equal to r and then (r, s, t) ≃(u, v, w) by case (b). On the other hand, suppose two of them divide r, while the other one divides st and hence is coprime to the other two. Then A = lcm(gcd(u, v), gcd(u, w), gcd(v, w)) divides r, while B = lcm(gcd(r, s), gcd(r, t), gcd(s, t)) = gcd(s, t) which divides st, and then because gcd(r, st) = 1, and A = B (by part (c) of Lemma 3.3), we find A = B = 1, and so gcd(s, t) = 1. Thus r, s and t are pairwise coprime, and it follows that the L2-sets of (r, s, t) and (u, v, w) are both equal to {r, s, t}, and therefore (r, s, t) ≃(u, v, w). 3. Main Theorem and Preliminary Steps for its New Proofs First, case (a) follows from parts (a) and (f) of Lemma 3.3, because (2, 5, 5) is the only hyperbolic triple with rst = 50, and (3, 4, 4) is the only hyperbolic triple with rst = 48, lcm(r, s, t) = 12 and gcd(r, s, t) = 1, and (3, 3, 5) is the only hyperbolic triple with rst = 45, and (3, 5, 5) is the only hyperbolic triple with rst = 75, and finally, (5, 5, 5) is the only hyperbolic triple with rst = 125. Proof. First, case (a) follows from parts (a) and (f) of Lemma 3.3, because (2, 5, 5) is the only hyperbolic triple with rst = 50, and (3, 4, 4) is the only hyperbolic triple with rst = 48, lcm(r, s, t) = 12 and gcd(r, s, t) = 1, and (3, 3, 5) is the only hyperbolic triple with rst = 45, and (3, 5, 5) is the only hyperbolic triple with rst = 75, and finally, (5, 5, 5) is the only hyperbolic triple with rst = 125. ( ) For case (b), suppose for example that t = w. Then rs = rst t = uvw w = uv, and then since rs + (r + s)t = rs + rt + st = rst( 1 r + 1 s + 1 t ) = uvw( 1 u + 1 v + 1 w) = uv + uw + vw = uv + (u + v)w, we find that r + s = u + v. Hence r and s are the zeroes of the same quadratic x2 −bx + c as u and v (namely with b = u + v and c = uv), so {r, s} = {u, v}, and then (r, s, t) ≃(u, v, w). The same argument works for all other coincidences between entries of (r, s, t) and (u, v, w). Case (c) follows from the fact that for every integer m ≥2, the only triangle group having the dihedral group Dm of order 2m as a smooth quotient is ∆(2, 2, m). Case (c) follows from the fact that for every integer m ≥2, the only triangle group having the dihedral group Dm of order 2m as a smooth quotient is ∆(2, 2, m). For suppose that two or more of r, s and t are even. 3. Main Theorem and Preliminary Steps for its New Proofs Also the analogous statements hold when (r, s, t) and (u, v, w) are interchanged. Proof. Suppose k ∈{r, s, t} is coprime to 6, u′, v′ and w′. Then by part (b) of Corollary 2.3, there exists a smooth finite quotient G of the triangle group ∆(u′, v′, w′) such that G has no non-trivial element of order dividing k, and so G is a quotient of ∆(u, v, w) but cannot be a quotient of ∆(r, s, t). The rest follows easily. □ 833 DISTINGUISHING TRIANGLE GROUPS Proposition 3.5. Theorem 3.1 holds in each of the following cases (or its equivalent form when r, s and t are permuted, or (r, s, t) and (u, v, w) are interchanged ): (a) (r, s, t) is one of the exceptional triples (2, 5, 5), (3, 4, 4), (3, 3, 5), (3, 5, 5), (5, 5, 5); Proposition 3.5. Theorem 3.1 holds in each of the following cases (or its equivalent form when r, s and t are permuted, or (r, s, t) and (u, v, w) are interchanged ): (a) (r, s, t) is one of the exceptional triples (2, 5, 5), (3, 4, 4), (3, 3, 5), (3, 5, 5), (5, 5, 5); Proposition 3.5. Theorem 3.1 holds in each of the following cases (or its equivalent form when r, s and t are permuted, or (r, s, t) and (u, v, w) are interchanged ): (a) (r, s, t) is one of the exceptional triples (2, 5, 5), (3, 4, 4), (3, 3, 5), (3, 5, 5), (5, 5, 5); (b) (r, s, t) and (u, v, w) have a common entry; (b) (r, s, t) and (u, v, w) have a common entry; (c) two or more of (r, s, t) are even; (d) one of r, s, t is coprime to each of the other two; (e) one of r, s, t is a power of 2; (e) one of r, s, t is a power of 2; (f) one or more of r, s, t, u, v and w is equal to gcd(r, s, t). Proof. 4. The First New Proof, using Direct Products For our first new proof of Theorem 3.1, we may suppose that (r, s, t) and (u, v, w) are non-exceptional hyperbolic triples for which Γ = ∆(r, s, t) and Σ = ∆(u, v, w) have exactly the same finite quotients, and hence satisfy the conclusions of Lemma 3.3 but not the principal hypothesis of Proposition 3.4, and do not satisfy any of the sufficient conditions (b) to (f) given in Proposition 3.5. In particular, we suppose that (r, s, t) and (u, v, w) have no entry in common, no entry that is a power of 2, no entry equal to gcd(r, s, t), and no more than one even entry each, and also that no element of one of the triples is coprime to each of the other two, and no element of one of the triples is coprime to 6 and to proper divisors of the elements of the other triple. Furthermore, we let M = lcm(r, s, t) = lcm(u, v, w), the maximal prime-power divisors of which will be a key to what follows. For interested readers, we also give an indication of the relative numbers of triple-pairs in each situation considered, among the set T of 542970 distinct triple- pairs {(r, s, t), (u, v, w)} satisfying the hypotheses two paragraphs above, with 2 ≤ r < u ≤v < s ≤t < w and rst = uvw ≤12000000d 3, where d = gcd(r, s, t) = gcd(u, v, w). (Here we note that dropping the condition given in Proposition 3.4 would add another 830030 triple-pairs, which gives an indication of the importance of that condition.) We consider four separate cases, which depend on the distribution of the maximal prime-power divisors of M among the divisors of the integers r, s, t, u, v and w, and we derive contradictions in all four cases. To do this, we consider a wider class of finite quotients of Γ and Σ, namely direct products of the form G = Q1 × Q2 or Q1 × Q2 × A, where each Qi is PSL(2, pi) for some prime pi, and A is abelian (indeed cyclic of order d = gcd(r, s, t)). 3. Main Theorem and Preliminary Steps for its New Proofs The other two possibilities gcd(s, rt) = 1 and gcd(t, sr) = 1 can be handled in the same way. 834 MARSTON CONDER Case (e) now follows immediately from cases (c) and (d). Case (e) now follows immediately from cases (c) and (d). ( ) ( ) ( ) Finally, for case (f), suppose that d = gcd(r, s, t) = r, say, with r ≤s ≤t and u ≤v ≤w. If (r, s, t) and (u, v, w) have no common entry, then r < u ≤v < s ≤ t < w by part (h) of Lemma 3.3, and then uvw = rst = dst, so st = uvw d , and then because t ≥s ≥v+1 it follows that rs+rt+st = d(s+t)+ uvw d ≥2d(v+1)+ uvw d . Now if u ≥3d, then rs + rt + st > uvw d ≥3vw > uv + uw + vw, while on the other hand, if u < 3d then u = 2d, and therefore 2d(v + 1) = u(v + 1) > uv and uvw d = 2vw ≥uw + vw, which together imply that rs + rt + st ≥2d(v + 1) + uvw d > uv + uw + vw, another contradiction. Thus (r, s, t) and (u, v, w) have a common entry, and case (b) applies. □ (d) Q1 × Q2 is not a smooth quotient of Σ = ∆(u, v, w). 4. The First New Proof, using Direct Products By swapping the triples (r, s, t) and (u, v, w) and/or re-ordering each one if nec- essary, we may suppose that q divides both t and w but divides none of r, s, u and v, and that t q < w q , noting that t ̸= w because (r, s, t) and (u, v, w) have no entry in common. When this happens, let m = q, let p be the prime divisor of m, so that m = pγ, say, and let m′ = M q . Then mm′ = M and gcd(m, m′) = 1, with m = q > 1 and also m′ ≥w q > 1. Furthermore, t ̸= p, for otherwise t = m = q, and so t is coprime to each of r and s. Next, let (r1, s1, t1) and (r2, s2, t2) be the triples defined for each x ∈{r, s, t} as follows: • x1 = x and x2 = p if x divides m, or • x1 = x and x2 = p if x divides m, or • x1 = x and x2 = p if x divides m, or • x1 = m and x2 = x m if x is a proper multiple of m (in which case x = t), or • x1 = x and x2 is a small prime divisor of x if x divides m′ (so p does not divide x), or nd x2 is a small prime divisor of x if x divides m • x1 = x and x2 is a small prime divisor of x if x divides m′ • x1 = x and x2 is a small prime (so p does not divide x), or (so p does not divide x), or (so p does not divide x), or • x1 = gcd(x, m′) and x2 = gcd(x, m) if 1 < gcd(x, m) < m and gcd(x, m′) > 1. Then clearly x1 > 1 and x2 > 1 and lcm(x1, x2) = x for all x ∈{r, s, t}. Moreover, each xi is a divisor of either m or m′, and in particular, t1 = m and t2 = t m or p, but on the other hand, none of r2, s2 and t2 is divisible by m. 4. The First New Proof, using Direct Products In the first three of our four cases, Q1 and Q2 will be determined by a ‘factorisa- tion’ of one of the triples, say (r, s, t), as a kind of product of two triples (r1, s1, t1) and (r2, s2, t2) of integers greater than 1, with the following properties: (a) lcm(r1, r2) = r and lcm(s1, s2) = s and lcm(t1, t2) = t, (b) Q1 and Q2 are smooth quotients of the triangle groups ∆(r1, s1, t1) and ∆(r2, s2, t2), respectively, so that Q1 × Q2 is a smooth quotient of Γ = ∆(r, s, t), but (c) at least one of u, v and w is not the order of some element of Q1 × Q2, and therefore (d) Q1 × Q2 is not a smooth quotient of Σ = ∆(u, v, w). 835 DISTINGUISHING TRIANGLE GROUPS In the fourth case, we do the same but using three triples (r1, s1, t1), (r2, s2, t2) and (r3, s3, t3), and a smooth abelian quotient A of ∆(r3, s3, t3), such that lcm(r1, r2, r3) = r and lcm(s1, s2, s3) = s and lcm(t1, t2, t3) = t, but at least one of u, v and w is not the order of some element of Q1 × Q2 × A. In this case Q1 × Q2 × A is a smooth quotient of Γ but not one of Σ. (In fact we take A = Cd and (r3, s3, t3) = (d, d, d), where d = gcd(r, s, t).) ( ) ) The first two cases cover the vast majority of the triple-pairs in our set T of ‘small’ triples, but each of them has a special sub-case which despite involving no triple-pairs from T , appeared to need special treatment. (It may be possible to simplify the proof for those two cases by a more thorough application of the condition rs + rt + st = uv + uw + vw.) Case (1): Suppose that some maximal prime-power divisor of M = lcm(r, s, t) greater than 3 divides just one of r, s and t, and hence also divides just one of u, v and w. In this case, let q be the largest such maximal prime-power divisor of M. 836 Now if w m can be found (r2, s2, t2)-ge then follows of order divi contradiction (For exam M = 2 · 27 (3, 270, 49) a can choose element of o 49 · 30 = 147 Case (1) of the L2-set proof. Assume t or l, and so not one of t m definition of (but not by another prim L so it conta r and s, say Next, bec that s is divi and s cannot to t, and the of u, v and w that v is div of k dividing s are k and Now assu f = gcd(s, t) be coprime is coprime t 836 MARSTON CONDER Now if w m divides no element of the L2-set of (r2, s2, t2), then primes p1 and p2 can be found such that Q1 = PSL(2, p1) and Q2 = PSL(2, p2) are (r1, s1, t1)- and (r2, s2, t2)-generated, but Q2 contains no element of order divisible by m or w m. It then follows that Q1 × Q2 is (r, s, t)-generated, but Q1 × Q2 contains no element of order divisible by m w m = w, and hence Q1 × Q2 cannot be (u, v, w)-generated, a contradiction. (For example, when (r, s, t) = (21, 270, 441) and (u, v, w) = (27, 63, 1470), with M = 2 · 27 · 5 · 49, we take m = q = 49 and m′ = 270, and then (r1, s1, t1) = (3, 270, 49) and (r2, s2, t2) = (7, 2, 9), with L2-sets {49, 270} and {2, 7, 9}, and we can choose p2 so that Q2 = PSL(2, p2) has elements of order 14 and 9 but no element of order m = 49 or w m = 30, and so Q1 × Q2 has no element of order 49 · 30 = 1470 = w.) Case (1) special sub-case: To complete case (1), we show that no element of the L2-set of (r2, s2, t2) is divisible by w m. This is the most intricate part of the proof. Assume the contrary. Then since w m divides neither pα nor t m, it must divide k or l, and so we may suppose that w = mk, where k is a prime divisor of m′ but not one of t m and hence not one of t. Also because k ∈L we may suppose from the definition of (r2, s2, t2) that at least one of r and s divides m′ and is divisible by k (but not by p). Moreover, if every x ∈{r, s} with this property were divisible by another prime divisor k′ of m′, then we could re-define x2 as k′, and thereby alter L so it contains no element divisible by k = w m. Hence we may suppose that one of r and s, say r, is a power of k. Thus (r, s, t) = (k, kλpαs′, mt′) or (kλ, kpαs′, mt′) for some positive integers s′ and t′, both coprime to k and m, and (u, v, w) = (pαu′, kλv′, km) or (u′, pαkλv′, km) for some positive integers u′ and v′, both coprime to k and m. Also k1+λpαs′mt′ = rst = uvw = pαk1+λu′v′m and therefore s′t′ = u′v′. 836 Now if w m can be found (r2, s2, t2)-ge then follows of order divi contradiction (For exam M = 2 · 27 (3, 270, 49) a can choose element of o 49 · 30 = 147 Case (1) of the L2-set proof. Assume t or l, and so not one of t m definition of (but not by another prim L so it conta r and s, say Next, bec that s is divi and s cannot to t, and the of u, v and w that v is div of k dividing s are k and Now assu f = gcd(s, t) be coprime is coprime t Next, because k is coprime to t, but r is not coprime to both s and t, we find that s is divisible by k as well. Also gcd(r, s) is divisible by k, so k is odd (because r and s cannot both be even), and gcd(u, v, w) = gcd(r, s, t) = 1 because r is coprime to t, and then since gcd(r, s) = k we find by part (c) of Lemma 3.3 that exactly two of u, v and w are divisible by k. Hence we may suppose without loss of generality that v is divisible by k, but u is not. In particular, if we let kλ be the largest power of k dividing M = lcm(r, s, t), then the k-part of v is kλ, and the k-parts of r and s are k and kλ in some order. Now assume for the moment that s is coprime to p (and hence to m), and let f = gcd(s, t). Then f > 1, because t is coprime to k and hence to r, and so cannot be coprime to s. But then f is odd (as it divides both s and t), so f ≥3, and f is coprime to p (as f divides s), and so km = w > t ≥mf ≥3m, which implies that k ≥5. Moreover, f must divide two of u, v and w, but is coprime to w, so f divides v, which then cannot be a power of k. Hence Proposition 3.4 applies to r, because r (= k or kλ) is coprime to 6 while none of u, v and w is a power of k. This contradiction shows that gcd(p, s) > 1, and puts us in case (b) of the possibilities for the set L. In particular, s must be divisible by pα, and so p is odd (because t is divisible by p as well), and also pα divides exactly one of u and v (because it divides s and t and w but not r). Thus (r, s, t) = (k, kλpαs′, mt′) or (kλ, kpαs′, mt′) for some positive integers s′ and t′, both coprime to k and m, and (u, v, w) = (pαu′, kλv′, km) or (u′, pαkλv′, km) for some positive integers u′ and v′, both coprime to k and m. 4. The First New Proof, using Direct Products (To see this, note that if x divides m, and p = x2 = m, then x = m = p and so t = x = p, but we showed above that t ̸= p.) Hence the L2-set of (r1, s1, t1) is {m, b} or {m, b, c} where b and c are divisors of m′, while the prime-power m = pγ divides no member of the L2-set of (r2, s2, t2). Hence the L2-set of (r1, s1, t1) is {m, b} or {m, b, c} where b and c are divisors of m′, while the prime-power m = pγ divides no member of the L2-set of (r2, s2, t2). ( ) In fact, the L2-set L of (r2, s2, t2) has one of the following forms, where α satisfies 1 ≤α < γ, and k and l are prime divisors of m′ : (a) {pα} or {pα t } if p divides r and s In fact, the L2-set L of (r2, s2, t2) has one of the following forms, where α satisfies 1 ≤α < γ, and k and l are prime divisors of m′ : (a) {pα} or {pα, t m}, if p divides r and s, (b) {pα, t m} or {pα, k} or {pα, t m, k}, if p divides just one of r and s, (c) { t m} or { t m, k} or { t m, k, l}, if p divides neither r nor s. (Note that in case (c), we cannot have t2 = p, again because t is not coprime to r and s.) 836 Now if w m can be found (r2, s2, t2)-ge then follows of order divi contradiction (For exam M = 2 · 27 (3, 270, 49) a can choose element of o 49 · 30 = 147 Case (1) of the L2-set proof. Assume t or l, and so not one of t m definition of (but not by another prim L so it conta r and s, say Next, bec that s is divi and s cannot to t, and the of u, v and w that v is div of k dividing s are k and Now assu f = gcd(s, t) be coprime is coprime t Also k1+λpαs′mt′ = rst = uvw = pαk1+λu′v′m and therefore s′t′ = u′v′. 837 DISTINGUISHING TRIANGLE GROUPS Moreover, gcd(r, s) = k and gcd(r, t) = 1 and gcd(s, t) = pα gcd(s′, t′), the least common multiple of which is kpα gcd(s′, t′), while gcd(u, v) = gcd(u′, v′), and either gcd(u, w) = pα and gcd(v, w) = k, or gcd(u, w) = 1 and gcd(v, w) = pα, with least common multiple kpα gcd(u′, tv). Hence by part (c) of Lemma 3.3 we find that gcd(s′, t′) = gcd(u′, v′). In particular, it follows that if gcd(s′, t′) = 1 then u is divisible by pα, for otherwise u = u′ which is coprime to each of v and w. We can now complete this sub-case by considering possibilities for s′ and t′. Case (i): Suppose s′t′ = 1. Then u′v′ = s′t′ = 1 and so s′ = t′ = u′ = v′ = 1, which gives (r, s, t) = (k, kλpα, m) and (u, v, w) = (pα, kλ, km) because u > 1, and then also λ > 1, because r ̸= v and u > 1. Next, dividing the identity rs+rt+st = uv+uw+vw by kpα gives kλ+pγ−α+kλ−1m = kλ−1+kλpγ−α+m, and it follows that kλ ≡kλ−1 mod pγ−α and pγ−α ≡m mod kλ−1, and therefore k ≡1 mod pγ−α and 1 ≡pα mod kλ−1. Accordingly, we find that pα > kλ−1 ≥k > pγ−α, which gives α > γ −α > 0, and so α > 1. Now we can define (u1, v1, w1) = (pα, kλ, m) and (u2, v2, w2) = (p, k, k), so that y1 > 1 and y2 > 1 and lcm(y1, y2) = y for all y ∈{u, v, w}, and the L2-sets of (u1, v1, w1) and (u2, v2, w2) are {kλ, m} and {k, p}, respectively. Hence there exist primes p1 and p2 such that Q1 = PSL(2, p1) and Q2 = PSL(2, p2) are (u1, v1, w1)- and (u2, v2, w2)-generated, respectively, but Q1 contains no element of order kλpα, and Q2 contains no element of order kλ or pα (since λ > 1 and α > 1). It follows that Q1 × Q2 is (u, v, w)-generated, but contains no element of order kλpα = s, so cannot be (r, s, t)-generated, a contradiction. Case (ii): Suppose s′t′ > 1. 836 Now if w m can be found (r2, s2, t2)-ge then follows of order divi contradiction (For exam M = 2 · 27 (3, 270, 49) a can choose element of o 49 · 30 = 147 Case (1) of the L2-set proof. Assume t or l, and so not one of t m definition of (but not by another prim L so it conta r and s, say Next, bec that s is divi and s cannot to t, and the of u, v and w that v is div of k dividing s are k and Now assu f = gcd(s, t) be coprime is coprime t Here the situation depends on whether k = 3 or k > 3. λ If k > 3, then v cannot be a proper multiple of kλ, for otherwise Proposition 3.4 would apply to r (= k or kλ), and therefore v = kλ (and v′ = 1), and pα divides u. Then since r ̸= v we find that (r, s, t) = (k, kλpαs′, mt′) while (u, v, w) = (pαs′t′, kλ, km), and λ > 1. If s′ = 1, then t′ > 1, and we can take (u1, v1, w1) = (pα, kλ, m) and (u2, v2, w2) = (t′, k, k), with L2-sets {kλ, m} and {k, t′}, and accordingly there exist primes p1 and p2 such that Q1 = PSL(2, p1) and Q2 = PSL(2, p2) are (u1, v1, w1)- and (u2, v2, w2)- generated, but Q1 has no element of order kλpα (even if α = 1), and Q2 has no element of order kλ or pα. It follows that Q1 × Q2 is (u, v, w)-generated, but con- tains no element of order kλpα = s, and so Q1 × Q2 cannot be (r, s, t)-generated, a contradiction. Hence s′ > 1. But now we can take (u1, v1, w1) = (pαs′t′, kλ, m) and (u2, v2, w2) = (p, k, k), as in case (i) above, with L2-sets {kλ, ms′t′} and {k, p}, and there exist primes p1 and p2 such that Q1 = PSL(2, p1) and Q2 = PSL(2, p2) are (u1, v1, w1)- and (u2, v2, w2)-generated, but Q1 has no element of order kλs′, and Q2 has no element of order kλ or pαs′, and again Q1 × Q2 is (u, v, w)-generated, but has no element of order kλpαs′ = s, so cannot be (r, s, t)-generated, another contradiction. On the other hand, suppose k = 3. Then p > 3, so s ≥kpα > 3, and since t < w = 3m, we have t = m or 2m, and then u′v′ = s′t′ = s′ or 2s′, with s′ coprime to 3 (= k) and p. Now if t = m = pγ, then t′ = 1, so u′v′ = s′t′ = s′ > 1. 836 Now if w m can be found (r2, s2, t2)-ge then follows of order divi contradiction (For exam M = 2 · 27 (3, 270, 49) a can choose element of o 49 · 30 = 147 Case (1) of the L2-set proof. Assume t or l, and so not one of t m definition of (but not by another prim L so it conta r and s, say Next, bec that s is divi and s cannot to t, and the of u, v and w that v is div of k dividing s are k and Now assu f = gcd(s, t) be coprime is coprime t Also if u ̸= pα, then none of u, v and w is a power of p, in which case Proposition 3.4 applies to t, and therefore u = pα (with u′ = 1) and v = kλv′ = kλs′. Accordingly, we have 838 MARSTON CONDER (r, s, t) = (3, 3λpαs′, m) or (3λ, 3pαs′, m), and (u, v, w) = (pα, 3λs′, 3m). Hence we can take (u1, v1, w1) = (pα, 3λ, m) and (u2, v2, w2) = (p, s′, 3), with L2-sets {3λ, m} and {3, p, s′}, and find there are primes p1 and p2 such that Q1 = PSL(2, p1) and Q2 = PSL(2, p2) are (u1, v1, w1)- and (u2, v2, w2)-generated, but Q1 has no element of order 3p, 3s′ or ps′, and Q2 has no element of order 3s′ or ps′. It follows that Q1 × Q2 has no element of order 3ps′ and hence no element of order s, so again Q1 × Q2 is (u, v, w)-generated but not (r, s, t)-generated, another contradiction. Q Q ( , , ) g ( , , ) g , Thus t = 2m = 2pγ, with t′ = 2, and then s is odd, so gcd(s′, t′) = 1, and it follows that u is divisible by pα, by observations made before case (i) above. Accordingly, we have (r, s, t) = (3, 3λpαs′, 2m) or (3λ, 3pαs′, 2m), where p > 3, and s′ is coprime to 2, 3 and p, while (u, v, w) = (pαu′, 3λv′, 3m) and 2s′ = u′v′. Also u′ is even, for otherwise u′ divides s′ and then u = pαu′ strictly divides s = 3λpαs′ or 3pαs′, so Proposition 3.4 applies to u. Hence we can write s′ as gh where g = gcd(s′, u′) and h = gcd(s′, v′), and then we have (r, s, t) = (3, 3λpαgh, 2m) or (3λ, 3pαgh, 2m) and (u, v, w) = (2pαg, 3λh, 3m). Finally, rs+rt+st = 3λ+1pαgh+6m+3λpα2ghm or 3λ+1pαgh+3λ2m+6pαghm, while uv + uw + vw = 3λpα2gh + 3mpα2g + 3λ+1mh, and in each case dividing by 3pα and then taking residues mod p gives 3λgh ≡3λ−12gh mod p, so p divides 3λ−1gh(3 −2) = 3λ−1gh, which is impossible. 836 Now if w m can be found (r2, s2, t2)-ge then follows of order divi contradiction (For exam M = 2 · 27 (3, 270, 49) a can choose element of o 49 · 30 = 147 Case (1) of the L2-set proof. Assume t or l, and so not one of t m definition of (but not by another prim L so it conta r and s, say Next, bec that s is divi and s cannot to t, and the of u, v and w that v is div of k dividing s are k and Now assu f = gcd(s, t) be coprime is coprime t Case (2): Suppose that some maximal prime-power divisor q of M = lcm(r, s, t) divides just one of r, s and t, and just one of u, v and w, but the largest such q is 2 or 3. Here we may suppose that q divides t and w, but divides none of r, s, u and v, while every maximal prime-power divisor of M greater than 3 divides at least two of r, s and t, and least two of u, v and w. Also neither t nor w can be equal to q, for otherwise case (d) of Proposition 3.5 would hold, and so each of them is a proper multiple of q. We claim that t and w are not divisible by the same maximal prime-power divisors of M. Assume the contrary, and that (say) t < w. Then w has a non-trivial prime-power divisor k ̸= q such that k does not divide t. If k > q then k cannot be a maximal prime-power divisor of M (for otherwise by assumption k would divide t), and so k strictly divides some maximal prime-power divisor of M, which then divides both u and v. But this implies that k divides all three of u, v and w, and so divides gcd(u, v, w) = gcd(r, s, t), a contradiction (again since k does not divide t). Hence the only possibility is k = 2, giving q = 3, and then (t, w) = (3c, 6c} for some odd c. Also one of r and s must be even, say s, so s = 2b for some b coprime to 6. Now 6rbc = rst = uvw = 6uvc, and so rb = uv, which is coprime to 6, and yet 2rb ≡rs ≡rs + rt + st ≡uv + uw + vw ≡uv ≡rb mod 3, from which it follows that 3 divides 2rb −rb = rb, a contradiction. Hence we may suppose that there exists a maximal prime-power divisor m of M that divides w but not t. Then since m does not divide t, it divides r and s, so cannot be even, and as it does not divide gcd(r, s, t) = gcd(u, v, w), it divides just one of u and v, say v. DISTINGUISHING TRIANGLE GROUPS DISTINGUISHING TRIANGLE GROUPS With this choice of m, again let m′ = M m , so mm′ = M and gcd(m, m′) = 1. This time m′ is divisible by w m and hence by q, and so gcd(t, m′) is divisible by q, giving gcd(t, m′) > 1. Now let p be the prime divisor of m, and let (r1, s1, t1) = (m, m, gcd(t, m′)), and let r2 = p or r m, depending on whether or not r = m, and let s2 = p or s m, depending on whether or not s = m, and let t2 be gcd(t, m) or the smallest prime divisor of t q, depending on whether or not gcd(t, m) > 1. Then x1 > 1 and x2 > 1 and lcm(x1, x2) = x for all x ∈{r, s, t}. Note also that r2 ̸= p and s2 ̸= p when m = p, because otherwise r or s is equal to p and then Proposition 3.4 applies to r or s. Next, the L2-set of (r1, s1, t1) is {m, gcd(t, m′)}, while the L2-set of (r2, s2, t2) contains no element divisible by m or q, and hence no element of order w m = q w qm, since none of r2, s2 and t2 is divisible by m or q. It follows that unless w m = 2 or 3, there exist primes p1 and p2 such that Q1 = PSL(2, p1) and Q2 = PSL(2, p2) are (r1, s1, t1)- and (r2, s2, t2)-generated, respectively, but Q1 contains no element of order mq, and Q2 contains no element of order divisible by m or w m. When that happens, Q1 × Q2 is (r, s, t)-generated but has no element of order m w m = w, and so Q1 × Q2 cannot be (u, v, w)-generated, a contradiction. DISTINGUISHING TRIANGLE GROUPS (For example, when (r, s, t) = (175, 1225, 1470) and (u, v, w) = (245, 3675, 350), with M = 2 · 3 · 25 · 49, we take q = 2, m = 25 and m′ = 294, and then (r1, s1, t1) = (25, 25, 294) and (r2, s2, t2) = (7, 49, 5), with L2-sets {25, 294} and {5, 49}, and we can choose p2 so that PSL(2, p2) has elements of order 5 and 49 but no element of order m = 25 or w m = 14.) Case (2) special sub-case: To complete case (2), we suppose that w m = 2 or 3. Then we have w = qm = 2m or 3m, while every maximal prime-power divisor of M not in {2, q, m} divides both u and v. So now let B be the product of those other maximal prime-power divisors of M. Then B is odd and divides both u and v, while q does not divide u or v, and since m divides at least two of u, v and w, we may suppose without loss of generality that v = mB and u = pαB for some α ≥0. It follows that (r, s, t) = (mr′, ms′, qpαt′) for some divisors r′, s′ and t′ of B, while (u, v, w) = (pαB, mB, qm). Then from qpαm2r′s′t′ = rst = uvw = qpαm2B2 we find that r′s′t′ = B2. Also each of r′, s′ and t′ must be greater than 1, since for example if r′ = 1 then s′t′ = B2 and so s′ = t′ = B, but then s = mB = v, which is impossible. Moreover, pα < m, for otherwise t = qpαt′ ≥qm = w. Case (2) special sub-case: To complete case (2), we suppose that w m = 2 or 3. Then we have w = qm = 2m or 3m, while every maximal prime-power divisor of M not in {2, q, m} divides both u and v. So now let B be the product of those other maximal prime-power divisors of M. 836 Now if w m can be found (r2, s2, t2)-ge then follows of order divi contradiction (For exam M = 2 · 27 (3, 270, 49) a can choose element of o 49 · 30 = 147 Case (1) of the L2-set proof. Assume t or l, and so not one of t m definition of (but not by another prim L so it conta r and s, say Next, bec that s is divi and s cannot to t, and the of u, v and w that v is div of k dividing s are k and Now assu f = gcd(s, t) be coprime is coprime t It follows that m ̸= 3, for otherwise q = 2 and then u is coprime to 6 while r, s and t are divisible by 3, 3 and 2, so Proposition 3.4 applies to u. Thus m is odd and m > 3. 839 DISTINGUISHING TRIANGLE GROUPS Then B is odd and divides both u and v, while q does not divide u or v, and since m divides at least two of u, v and w, we may suppose without loss of generality that v = mB and u = pαB for some α ≥0. It follows that (r, s, t) = (mr′, ms′, qpαt′) for some divisors r′, s′ and t′ of B, while (u, v, w) = (pαB, mB, qm). Then from qpαm2r′s′t′ = rst = uvw = qpαm2B2 we find that r′s′t′ = B2. Also each of r′, s′ and t′ must be greater than 1, since for example if r′ = 1 then s′t′ = B2 and so s′ = t′ = B, but then s = mB = v, which is impossible. Moreover, pα < m, for otherwise t = qpαt′ ≥qm = w. Now if α > 0, then we can take (u1, v1, w1) = (B, B, q) and (u2, v2, w2) = (pα, m, m), which have L2-sets {q, B} and {m} respectively, and then find primes p1 and p2 such that Q1 = PSL(2, p1) and Q2 = PSL(2, p2) are (u1, v1, w1)- and (u2, v2, w2)-generated, but so that Q1 has no element of order qt′, and Q2 has no element of order pαt′ or pαq. It then follows that Q1 × Q2 is (u, v, w)-generated, but has no element of order qpαt′ = t and so cannot be (r, s, t)-generated, a con- tradiction. Thus α = 0, which gives u = B and t = qt′. In particular, u = B is coprime to qm = w, and so gcd(r, s, t) = gcd(u, v, w) = 1. (It also follows that B is divisible by 3, for otherwise Proposition 3.4 applies to u, with r, s and t being divisible by m, m and q, and hence in particular, q = 2; but we do not need to know these things.) 840 MARSTON CONDER Next, consider the maximal prime-power divisors of M that divide B. Every such divisor k is coprime to at least one of r′, s′ and t′, because 1 = gcd(r, s, t) = gcd(r′, s′, t′), and so must divide exactly two of them. DISTINGUISHING TRIANGLE GROUPS It follows that there exist pairwise coprime odd positive integers f, g and h such that (r′, s′, t′) = (fg, fh, gh), namely f = gcd(r′, s′), g = gcd(r′, t′) and h = gcd(s′, t′), so that B = fgh. Moreover, g > 1, for otherwise we find that v = mB = mfgh = mfh = ms′ = s, and similarly h > 1. Also f > 1, for otherwise (r, s, t) = (mg, mh, qgh) and (u, v, w) = (gh, mgh, qm) and then since at least two of m, g and h are coprime to 3 and hence to 6, we find that Proposition 3.4 applies to r or s or u. But now it follows that we can take (r1, s1, t1) = (mf, mf, gh) and (r2, s2, t2) = (g, h, q), which have L2-sets {mf, gh} and {q, g, h} respectively, and then find primes p1 and p2 such that Q1 = PSL(2, p1) and Q2 = PSL(2, p2) are (r1, s1, t1)- and (r2, s2, t2)-generated, but Q1 has no element of order mfg or mfh, and Q2 has no element of order mf or gh. Then Q1 × Q2 is (r, s, t)-generated, but has no element of order mfgh = mB = v and so cannot be (u, v, w)-generated, a final contradiction. Remarks: Cases (1) and (2) considered above apply to 535695 and 6507 of the 542695 triple-pairs in the set T given earlier, leaving just 768 of those triple-pairs to be covered. For the remaining possibilities, we may suppose that every maximal prime-power divisor k of M divides two or more of r, s and t, and two or more of u, v and w. In particular, every such k is odd, and therefore M is odd, so each of r, s, t, u, v and w is odd. Now for the moment, assume that gcd(r, s, t) = gcd(u, v, w) = 1. Then each prime divisor k of r must be coprime to one of s and t, say s, and furthermore, if m = kλ is the maximum power of k dividing M, then m must divide r and t but be coprime to s. DISTINGUISHING TRIANGLE GROUPS The analogous argument works for each of s, t, u, v and w, and hence every one of r, s, t, u, v and w is a product of maximal prime-power divisors of M. Next, let x be the smallest one of r, s, t, u, v and w that is not divisible by 3, and suppose without loss of generality that x ∈{u, v, w}. Then x is coprime to 6, and cannot be a multiple of any of r, s and t, and so each of r, s and t is divisible by some odd prime that does not divide x, but in that case Proposition 3.4 applies to x, a contradiction. Thus gcd(r, s, t) = gcd(u, v, w) > 1, and in particular, no two of r, s and t are coprime, and the same holds for u, v and w. Case (3): Suppose that no maximal prime-power divisor q of M = lcm(r, s, t) divides just one of r, s and t (or just one of u, v and w), but that some such q divides exactly two of r, s and t, and is coprime to the third. In this case, q is coprime to gcd(r, s, t) = gcd(u, v, w), and hence also q divides two of u, v and w and is coprime to the third. By swapping the triples (r, s, t) and (u, v, w) and/or re-ordering each one if necessary, we may suppose that gcd(r, q) = gcd(u, q) = 1, so that s, t, v and w are the elements of {r, s, t, u, v, w} divisible by q. Also we may suppose also that w is the largest of these. Now let m = q and m′ = M q , and define the triples (r1, s1, t1) and (r2, s2, t2) as follows: • s1 = m and s2 = s m = gcd(s, m′), 841 DISTINGUISHING TRIANGLE GROUPS • t1 = t m = gcd(t, m′) and t2 = m, and • t1 = t m = gcd(t, m′) and t2 = m, and • r1 = gcd(r, t1) and r2 = gcd(r, s2). Clearly s1 = m > 1 and t2 = m > 1, and s = s1 s2 and t = t1 t2. DISTINGUISHING TRIANGLE GROUPS (For example, when (r, s, t) = (105, 585, 819) and (u, v, w) = (315, 117, 1365), with M = 9 · 5 · 7 · 13, we can take m = 13 and m′ = 315, and then (r1, s1, t1) = (21, 13, 63) and (r2, s2, t2) = (15, 45, 13), with L2-sets {13, 63} and {13, 45}, and we can choose p1 and p2 so that Q1 × Q2 has no element of order w = 13 · 105.) (For example, when (r, s, t) = (105, 585, 819) and (u, v, w) = (315, 117, 1365), with M = 9 · 5 · 7 · 13, we can take m = 13 and m′ = 315, and then (r1, s1, t1) = (21, 13, 63) and (r2, s2, t2) = (15, 45, 13), with L2-sets {13, 63} and {13, 45}, and we can choose p1 and p2 so that Q1 × Q2 has no element of order w = 13 · 105.) Remarks: This case applies to 766 of the 542695 triple-pairs in our set T , leaving just 768 −766 = 2 triple-pairs in T that need to be covered by case (4). A computation using Magma [1] shows that in these two cases, there is no direct product of the form PSL(2, p1) × PSL(2, p2) that is a smooth quotient of one of ∆(r, s, t) and ∆(u, v, w) but not the other, and thereby explains why we need to consider direct products of three quotients for some triple-pairs. Case (4): Suppose that every maximal prime-power divisor of M = lcm(r, s, t) divides exactly two of r, s and t but is not coprime to the third (and hence also divides exactly two of u, v and w but is not coprime to the third). In this case, every prime divisor of M divides all six of r, s, t, u, v and w, and hence divides d = gcd(r, s, t) = gcd(u, v, w) > 1. On the other hand, none of those six is equal to d, because otherwise case (j) of Proposition 3.5 applies. Also by swapping and/or re-ordering the triples (r, s, t) and (u, v, w), we may suppose that w = max({r, s, t, u, v, w}). DISTINGUISHING TRIANGLE GROUPS Also s2 = gcd(s, m′) > 1, for otherwise s = m and then gcd(r, s) = 1, which contradicts the observation made above that gcd(r, s, t) > 1. Similarly t1 = gcd(t, m′) > 1. Thus s = ms2 > m and t = mt1 > m, and so s ≥3m and t ≥3m, and it follows that w > 3m. Next, every maximal prime-power divisor of M divides s or t (or both) and hence every maximal prime-power divisor of r divides s m = s2 or t m = t1, and so lcm(r1, r2) = r. Moreover, r1 = gcd(r, t1) = gcd(r, t1m) = gcd(r, t) > 1, and similarly r2 = gcd(r, s2) > 1. y g ( ) The L2-set of (r1, s1, t1) is {m, t1} since r1 = gcd(r, t1) divides t1, which is coprime to m, and similarly, the L2-set of (r2, s2, t2) is {m, s2}. It follows that there exist primes p1 and p2 such that Q1 = PSL(2, p1) and Q2 = PSL(2, p2) are (r1, s1, t1)- and (r2, s2, t2)-generated, but each of Q1 and Q2 has no element of order k such that k divides M = mm′ and is strictly divisible by m. In particular, Q1 × Q2 is (r, s, t)-generated. But on the other hand, the only orders of elements of Q1 × Q2 that are divisors of M and strictly divisible by m are divisors of ms2 (= s) or t1m (= t), or perhaps 3m, and then because w is greater than 3m, s and t, it follows that Q1 ×Q2 has no element of order w, and so Q1 ×Q2 is not (u, v, w)-generated, a contradiction. DISTINGUISHING TRIANGLE GROUPS Now let D be the product of all maximal prime-power divisors of M that divide d, let X be the set of all maximal prime-power divisors of M that do not divide d, and let E be their product. Then D divides d, and M = DE with gcd(D, E) = 1. Also every q ∈X divides exactly two of r, s and t but does not divide the third, and divides exactly two of u, v and w but does not divide the third. Moreover, no 842 MARSTON CONDER q ∈X can be prime, for otherwise q divides d. Hence every q ∈X is of the form pα for some odd prime p and some α ≥2, and in particular, 3 ̸∈X. q ∈X can be prime, for otherwise q divides d. Hence every q ∈X is of the form pα for some odd prime p and some α ≥2, and in particular, 3 ̸∈X. q ∈X can be prime, for otherwise q divides d. Hence every q ∈X is of the form pα for some odd prime p and some α ≥2, and in particular, 3 ̸∈X. Also let X1 be the set of all q ∈X that divide r and s but not t, let X2 be the set of all q ∈X that divide r and t but not s, let X3 be the set of all q ∈X that divide s and t but not r, and let m1, m2 and m3 be the product of the members of X1, X2 and X3, respectively. Then E = m1m2m3, with m1, m2 and m3 pairwise coprime, and it is easy to see that r = lcm(m1m2, d) = m1m2r′ for some r′ dividing d, while s = lcm(m1m3, d) = m1m3s′ for some s′ dividing d, and t = lcm(m2m3, d) = m2m3t′ for some t′ dividing d. ( ) Next, let (r1, s1, t1) = (m1, m1, m3), (r2, s2, t2) = (m2, m3, m2) and (r3, s3, t3) = (d, d, d). Then xi > 1 for all x ∈{r, s, t} and all i, and lcm(r1, r2, r3) = lcm(m1m2, d) = r, and lcm(s1, s2, s3) = lcm(m1m3, d) = s, and lcm(t1, t2, t3) = lcm(m2m3, d) = t. DISTINGUISHING TRIANGLE GROUPS The L2-sets of (r1, s1, t1) and (r2, s2, t2) are {m1, m3} and {m2, m3}, and it follows that there exist primes p1 and p2 such that Q1 = PSL(2, p1) and Q2 = PSL(2, p2) are (r1, s1, t1)- and (r2, s2, t2)-generated respectively, but Q1 has no element of order k where k divides E but does not divide m1 or m3, and Q2 has no element of order k where k divides E but does not divide m2 or m3. Then since 3 ̸∈X, it follows that if Q1 × Q2 has an element of order k where k is a product of members of X, then k divides m1m2, m1m3 or m3m2. Now Q1 × Q2 is (m1m2, m1m3, m2m3)-generated, and hence if A = Cd, which is (d, d, d)-generated, then also Q1 × Q2 × A is (r, s, t)-generated. On the other hand, w = mw′ = lcm(m, d) for some product m of members of X and some divisor w′ of d, and as w d > x d for all x ∈{r, s, t}, we know that m cannot divide any of m1m2, m1m3 and m3m2, and it follows that Q1 × Q2 has no element of order m. Thus Q1×Q2×A has no element of order lcm(m, d) = w, and hence Q1×Q2×A cannot be (u, v, w)-generated, a final contradiction. ( ) g (Examples include the two triple-pairs {(28665, 266175, 621075), (47775, 53235, 18632 and {(47775, 266175, 372645), (53235, 143325, 621075)}, not covered by cases (1) and (2), with d = gcd(r, s, t) = 1365 = 3 · 5 · 7 · 13 and D = 1 and M = lcm(r, s, t) = 33 52 72 132 for both, and (m1, m2, m3) = (32, 72, 652) and (52, 72, 392) respectively.) ( ) (Examples include the two triple-pairs {(28665, 266175, 621075), (47775, 53235, 186322 and {(47775, 266175, 372645), (53235, 143325, 621075)}, not covered by cases (1) and (2), with d = gcd(r, s, t) = 1365 = 3 · 5 · 7 · 13 and D = 1 and M = lcm(r, s, t) = 33 52 72 132 for both, and (m1, m2, m3) = (32, 72, 652) and (52, 72, 392) respectively.) □ y ) This case completes the first new proof. □ This case completes the first new proof. DISTINGUISHING TRIANGLE GROUPS Now take (k, l, m) = (u, v, w p ). Then by our choice of q = pγ and the assumption that w is not coprime to both u and v, it follows that w p is not coprime to both u , ( , , ) { p }, p y q Next, let h be an odd prime such that h−1 2 or h+1 2 is divisible by M p , but not by q, and hence not by w or M. Then G = PSL(2, h) is a smooth quotient of the triangle group ∆(u, v, w p ), and accordingly, so is an extension Qn of C 2g n by G for any positive integer n, where 2 −2g = |G| 1 u + 1 v + p w −1  . In particular, every such Qn is a quotient of Σ = ∆(u, v, w), but has no element of order q (or w). On the other hand, G is also a quotient of Γ = ∆(r, s, t), but we can show that Qn is not, for every n > 1. For suppose the contrary. Then G = PSL(2, h) is (k, l, m)- generated for some divisors k, l and m of r, s and t, respectively, and then for any such triple (k, l, m), the third entry m must divide t p because G has no element of order q. Hence the largest conceivable value of f for which an extension of C 2f n by G is a smooth quotient of ∆(k, l, m) is given by 2 −2f = |G| 1 r + 1 s + p t −1  . But now since 1 r + 1 s + 1 t = 1 u + 1 v + 1 w and t < w, it follows that 2g −2f = (2−2f) −(2−2g) = |G| p t −1 t −p w + 1 w  = |G|(p −1)( 1 t −1 w) > 0, so f < g. Thus Qn cannot be a smooth quotient of ∆(k, l, m), and hence cannot be a quotient of ∆(r, s, t), a contradiction. Cases (3) and (4): Suppose that no maximal prime-power divisor of M = lcm(r, s, t) divides just one of r, s and t (or just one of u, v and w). DISTINGUISHING TRIANGLE GROUPS DISTINGUISHING TRIANGLE GROUPS Just as in the first new proof, the choice of triple (k, l, m) depends on the distri- bution of the maximal prime-power divisors of M among the divisors of r, s, t, u, v and w. The group Q in each case is an extension of an abelian normal subgroup N ∼= C 2g n by G = PSL(2, h), where n > 1 and h is a carefully chosen prime, and 2 −2g = |G| 1 k + 1 l + 1 m −1  as described shortly after Theorem 2.2 in Section 2. We proceed by considering two possibilities, each of which is a combination of two of the cases from the first new proof. Credit should go to Frankie Chan for the choice of (k, l, m) in the second possibility, even though it now seems obvious with the benefit of hindsight! Also the approach for both possibilities further underlines the value of the Macbeath trick. Just as in the first new proof, the choice of triple (k, l, m) depends on the distri- bution of the maximal prime-power divisors of M among the divisors of r, s, t, u, v and w. The group Q in each case is an extension of an abelian normal subgroup N ∼= C 2g n by G = PSL(2, h), where n > 1 and h is a carefully chosen prime, and 2 −2g = |G| 1 k + 1 l + 1 m −1  as described shortly after Theorem 2.2 in Section 2. k l m  We proceed by considering two possibilities, each of which is a combination of two of the cases from the first new proof. Credit should go to Frankie Chan for the choice of (k, l, m) in the second possibility, even though it now seems obvious with the benefit of hindsight! Also the approach for both possibilities further underlines the value of the Macbeath trick. Cases (1) and (2): Suppose that some maximal prime-power divisor of M = lcm(r, s, t) divides just one of r, s and t, and also so divides just one of u, v and w. ( ) Let q = pγ be the smallest such maximal prime-power divisor of M, and also suppose without loss of generality that q divides t and w, and that t < w. 5. The Second New Proof, using Divisor Triples Again here we may suppose that (r, s, t) and (u, v, w) are non-exceptional hy- perbolic triples for which Γ = ∆(r, s, t) and Σ = ∆(u, v, w) have exactly the same finite quotients, and hence satisfy the conclusions of Lemma 3.3, but not the princi- pal hypothesis of Proposition 3.4, and do not satisfy any of the sufficient conditions (b) to (f) given in Proposition 3.5. In particular, no element of one of the triples can be coprime to each of the other two, and it follows that the L2-sets of the triples (r, s, t) and (u, v, w) are both equal to {M}, where M = lcm(r, s, t) = lcm(u, v, w). The approach we take is to show that for some triple (k, l, m), consisting of respective divisors of either r, s and t, or u, v and w, there exists a (k, l, m)- generated group Q that is a quotient of one of just one of Γ and Σ, which contradicts Lemma 3.2. 843 M p is not divisible by q. DISTINGUISHING TRIANGLE GROUPS In this case, every maximal prime-power divisor of M divides two or all three of r, s and t, and at least one of them divides exactly two of r, s and t, since by assumption no entry of (r, s, t) is equal to gcd(r, s, t). The analogous properties hold also for the triple (u, v, w). Moreover, by the remarks following cases (1) and (2) in our first proof, we can suppose that gcd(r, s, t) = gcd(u, v, w) > 1. So let q = pγ be the smallest maximal prime-power divisor of M that divides exactly two of r, s and t, and suppose without loss of generality that q divides s, t, v and w, and r < u. So let q = pγ be the smallest maximal prime-power divisor of M that divides exactly two of r, s and t, and suppose without loss of generality that q divides s, t, v and w, and r < u. Now let (k, l, m) = (r, s p, t p). Then by our choice of q = pγ and because gcd(r, s, t) > 1, we find that the L2-set of (k, l, m) is { M p } once more, and that M p is not divisible by q. 844 MARSTON CONDER Again let h be an odd prime such that h−1 2 or h+1 2 is divisible by M p , but not by q, and hence not by v or w or M. Then G = PSL(2, h) is a smooth quotient of the triangle group ∆(r, s p, t p), and accordingly, so is an extension Qn of C 2g n by G for any positive integer n, where this time 2 −2g = |G| 1 r + p s + p t −1  . In particular, every such Qn is a quotient of Σ = ∆(r, s, t), but has no element of order q (or s or t). On the other hand, G is also a quotient of Γ = ∆(u, v, w), but we can show that Qn is not, for every n > 1. DISTINGUISHING TRIANGLE GROUPS For otherwise G = PSL(2, h) is (k, l, m)-generated for some divisors k, l and m of u, v and w, respectively, and then for any such triple (k, l, m), the second and third entries must divide v p and w p , as G has no element of order q. Hence the largest conceivable value of f for which an extension of C 2f n by G is a smooth quotient of ∆(k, l, m) is given by 2 −2f = |G| 1 u + p v + p w −1  . But now since p r + p s + p t = p u + p v + p w and r < u, it follows that 2g −2f = (2−2f) −(2−2g) = |G| 1 u −p u −1 r + p r  = |G|(p −1)( 1 r −1 u) > 0, so f < g. Thus Qn cannot be a smooth quotient of ∆(k, l, m), and hence cannot be a quotient of ∆(u, v, w), a contradiction. □ This completes the shorter proof. This completes the shorter proof. Acknowledgements The author is grateful to the N.Z. Marsden Fund (via grant UOA2030) for sup- porting research that helped produce this paper, and also acknowledges the help of the Magma system [1] in investigating examples and testing conjectures. Department of Mathematics University of Auckland Private Bag 92019 Auckland New Zealand m.conder@auckland.ac.nz References [1] W. Bosma, J. Cannon and C. Playoust, The Magma Algebra System I: The User Language, J. Symbolic Computation 24 (1997), 235–265. [ ] A [1] W. Bosma, J. Cannon and C. Playoust, The Magma Algebra System I: The User Language, J. Symbolic Computation 24 (1997), 235–265. [2] M.R. Bridson, M.D.E. Conder and A.W. Reid, Determining Fuchsian groups by their finite quotients, Israel J. Math. 214 (2016), 1–41. [2] M.R. Bridson, M.D.E. Conder and A.W. Reid, Determining Fuchsian groups by their finite quotients, Israel J. Math. 214 (2016), 1–41. [3] F. Chan, Finite Quotients of Triangle Groups, PhD Thesis, Purdue University, August 2021. [4] H.S.M. Coxeter and W.O.J. Moser, Generators and Relations for Discrete Groups, 4th ed., Springer, Berlin (1980). [4] H.S.M. Coxeter and W.O.J. Moser, Generators and Relations for Discrete Groups, 4th ed., Springer, Berlin (1980). [5] M. Larsen, A. Lubotzky and C. Marion, Deformation theory and finite simple quotients of triangle groups I, J. Eur. Math. Soc. (JEMS) 16 (2014), 1349–1375. [5] M. Larsen, A. Lubotzky and C. Marion, Deformation theory and finite simple quotients of triangle groups I, J. Eur. Math. Soc. (JEMS) 16 (2014), 1349–1375. f g g ( ) ( ) [6] A.M. Macbeath, Generators of the fractional linear groups, Proc. Sympos. Pure Math. XII, Amer. Math. Soc., (1969), 14–32. [6] A.M. Macbeath, Generators of the fractional linear groups, Proc. Sympos. Pure Math. XII, Amer. Math. Soc., (1969), 14–32. ( ) [7] M. Suzuki, Group Theory I, Grundlehren der mathematischen Wissen. 247, Springer-Verlag, (1982). ( ) [7] M. Suzuki, Group Theory I, Grundlehren der mathematischen Wissen. 247, Springer-Verlag, (1982). Department of Mathematics University of Auckland Private Bag 92019 Auckland New Zealand m.conder@auckland.ac.nz
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A novel SPAST gene mutation identified in a Chinese family with hereditary spastic paraplegia
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© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yu et al. BMC Medical Genetics (2020) 21:123 https://doi.org/10.1186/s12881-020-01053-7 Yu et al. BMC Medical Genetics (2020) 21:123 https://doi.org/10.1186/s12881-020-01053-7 Open Access Abstract Background: Hereditary spastic paraplegia is a heterogeneous group of clinically and genetically neurodegenerative diseases characterized by progressive gait disorder. Hereditary spastic paraplegia can be inherited in various ways, and all modes of inheritance are associated with multiple genes or loci. At present, more than 76 disease-causing loci have been identified in hereditary spastic paraplegia patients. Here, we report a novel mutation in SPAST gene associated with hereditary spastic paraplegia in a Chinese family, further enriching the hereditary spastic paraplegia spectrum. Methods: Whole genomic DNA was extracted from peripheral blood of the 15 subjects from a Chinese family using DNA Isolation Kit. The Whole Exome Sequencing of the proband was analyzed and the result was identified in the rest individuals. RaptorX prediction tool and Protein Variation Effect Analyzer were used to predict the effects of the mutation on protein tertiary structure and function. Results: Spastic paraplegia has been inherited across at least four generations in this family, during which only four HSP patients were alive. The results obtained by analyzing the Whole Exome Sequencing of the proband exhibited a novel disease-associated in-frame deletion in the SPAST gene, and this mutation also existed in the rest three HSP patients in this family. This in-frame deletion consists of three nucleotides deletion (c.1710_1712delGAA) within the exon 16, resulting in lysine deficiency at the position 570 of the protein (p.K570del). This novel mutation was also predicted to result in the synthesis of misfolded SPAST protein and have the deleterious effect on the function of SPAST protein. Conclusion: In this case, we reported a novel mutation in the known SPAST gene that segregated with HSP disease, which can be inherited in each generation. Simultaneously, this novel discovery significantly enriches the mutation spectrum, which provides an opportunity for further investigation of genetic pathogenesis of HSP. Keywords: Hereditary spastic paraplegia, Gait disorder, Whole exome sequencing, SPAST gene, In-frame deletion Keywords: Hereditary spastic paraplegia, Gait disorder, Whole exome sequencing, SPAST gene, In-fram spasticity and pyramidal signs of the lower limbs are the prominent features of HSP, which can be well explained by the fact that the innervated function of the longest fibers to- ward to the lower extremity is prone to be affected [2]. A novel SPAST gene mutation identified in a Chinese family with hereditary spastic paraplegia Weiwei Yu, Haiqiang Jin, Jianwen Deng, Ding Nan and Yining Huang* Weiwei Yu, Haiqiang Jin, Jianwen Deng, Ding Nan and Yining Huang* * Correspondence: ynhuang@bjmu.edu.cn Department of Neurology, Peking University First Hospital, 8 Xishiku Street Xicheng District, Beijing 100034, China Background Hereditary spastic paraplegia (HSP), also called familial spas- tic paraparesis or Strümpell-Lorrain disease, is a group of neurodegenerative and inherited heterogeneous neurological disorders characterized by a length-dependent distal axonal degeneration of the corticospinal tracts [1]. The progressive According to whether accompanied by additional neurological or psychiatric symptoms such as ataxia, mental and cognitive changes, extrapyramidal signs, vis- ual dysfunction or epilepsy, or with extra neurological signs, the diseases can be categorized into either pure ual dysfu signs, th © The Author(s). 2020 Open Access This article is licensed which permits use, sharing, adaptation, distribution and rep appropriate credit to the original author(s) and the source, changes were made. The images or other third party mater licence, unless indicated otherwise in a credit line to the m licence and your intended use is not permitted by statutory permission directly from the copyright holder. To view a co The Creative Commons Public Domain Dedication waiver (h data made available in this article, unless otherwise stated i * Correspondence: ynhuang@bjmu.edu.cn Department of Neurology, Peking University First Hospital, 8 Xishiku Street Xicheng District, Beijing 100034, China * Correspondence: ynhuang@bjmu.edu.cn Department of Neurology, Peking University First Hospital, 8 Xishiku Street Xicheng District, Beijing 100034, China Libraries preparation and amplification DNA libraries were established with KAPA Library Prep- aration Kit (Kapa Biosystems, KR0453) following the manufacturer’s instructions, which mainly contains three major procedures: end-repair of fragmented DNA, A- tailing, adapter ligation and amplification [14]. Purifica- tions between procedures were achieved using Agen- court AMPure XP beads. After the ligation reaction with beads, 50 μl ligation was totally resuspended in 45 μl PEG/NaCl SPRI® Solution and then incubated at 37 °C for 2 min. Subsequently, the captured beads via a magnet were incubated until the liquid was clear. The beads were washed for three times using 200 μl 80% ethanol after discarding the clear supernatant and then was dried at room temperature (RT). Eventually, the beads cap- tured on a magnet were thoroughly resuspended in 25 μl water and incubated for 2 min at RT until the liquid was clear to be proceed with library amplification. g yp p yp Based on the distinguishably inherited trait, HSP can be classified into as autosomal dominant, autosomal reces- sive, X-linked, mitochondrial, or de novo [6]. Meanwhile, autosomal dominant HSP is the most common mode that accounts for approximately 70% of all HSP patients [7]. All modes of inheritance are associated with multiple genes or loci. Until now, there have been at least 76 spas- tic paraplegia associated with loci and more than 59 corre- sponding spastic paraplegia genes (SPG) have been identified [8]. The SPG4/ SPAST gene comprising 17 exons, identified as the 90-kb genomic region on chromo- some 2 (2p22.3, [9], has been reported to be the most fre- quent cause of HSP and accounts for approximately 40% of pure autosomal dominant HSP and 10% of sporadic cases [10, 11]. Over 500 mutations have been identified in the SPAST gene. Generally, SPAST gene mutations have a tendency to cause pure HSP [12] and are more common in males than females [5]. Here, we report a novel SPAST gene mutation site (c.1710_1712delGAA) that presented in a Chinese family with HSP, significantly enriching the mutation spectrum of HSP gene. Libraries amplification was fulfilled by polymerase chain reaction (PCR) under the following 25 μl reaction system: 12.5 μl 2× KAPA HiFi HotStart ReadyMix, 1 μl 5 μM each primer, 10 μl captured library beads suspen- sion and 1.5 μl water. Libraries preparation and amplification PCR amplification program was set up: 98 °C 2 min; 98 °C 30 s; 65 °C 30 s; 72 °C 30 s, 13 cycles; and a final step at 72 °C for 4 min. Subsequently, repeat the steps of washing and resuspending the beads as described above. The amplified libraries that were prepared for array capture were assessed with Qubit dsDNA HS Assay kit (Invitrogen, Q32851). Array capture and sequencing y p q g Array capture was performed via the Agilent SureSe- lectXT2 Target Enrichment System as previously de- scribed [14]. Briefly, array hybridization was captured by mixing the pooled libraries with a buffer solution and oligo-blockers, which was incubated for 24 h at 65 °C. The hybridized library molecules were performed with Dyna- beads® MyOne™Streptavidin T1 (Invitrogen, #65601). The captured library was amplified as following: 21 μl 2× KAPA HiFi HotStart ReadyMix, 1 μl 5 μM primer, 20 μl captured library beads suspension. PCR amplification pro- gram was 98 °C 2 min; 98 °C 30 s; 65 °C 30 s; 72 °C 30 s, 13 cycles and a final step at 72 °C for 4 min. Purifications be- tween procedures were conducted using Agencourt AMPure XP beads and the libraries were evaluated with Qubit dsDNA HS Assay kit (Invitrogen, Q32851). Finally, DNA libraries of the proband were analyzed by whole ex- ome sequencing (WES). WES was carried out on the HiSeq2500 platform as paired-end 200-bp reads. Illumina Yu et al. BMC Medical Genetics (2020) 21:123 Page 2 of 7 Yu et al. BMC Medical Genetics (2020) 21:123 Qubit fluorometer (Invitrogen, Q33216) using Qubit dsDNA HS Assay Kit (Invitrogen, Q32851). Meanwhile, 1% agarose gel electrophoresis was performed for quality control of each DNA sample. Qubit fluorometer (Invitrogen, Q33216) using Qubit dsDNA HS Assay Kit (Invitrogen, Q32851). Meanwhile, 1% agarose gel electrophoresis was performed for quality control of each DNA sample. HSP (pHSP) or complicated HSP (cHSP) [3, 4]. The on- set age of HSP exhibits a wide range from childhood to over 70 years old depending on the underlying genetic defect, even in the family with a same mutation [5]. Therefore, it is hard to explain the interaction between the genotype and the phenotype of HSP. Subjects In this study, we recruited 15 subjects (female: male ra- tio is about 1:1; age range: 3–63 years) in total from a Chinese family with HSP. This family was enrolled in our study on the basis of the following criteria: (1) Based on Harding’s criteria [4], the proband, a 63-years-old fe- male, was diagnosed with HSP; (2) The family of the proband had at least four affected relatives with HSP; (3) The family was potentially informative for designing a study to investigate the genetic mutations. Total of 15 individuals were performed with neurologic examin- ation, four of which had the same clinical manifestations and the rest were all asymptomatic. The study was con- ducted according to the Declaration of Helsinki and was authorized by the Ethics Committee of Peking University First Hospital. The related written informed consents for publication of details and images were obtained from all the participants and the legal guardian of the patient aged 11 years in our study. Clinical characteristics The results obtained from routine laboratory tests, electro- myography, cranial and cervical MRIs did not reveal any obvious pathognomonic alteration. For her previous his- tory, the proband had ever received some treatment on rheumatoid arthritis because of the pain in both hips and knees 26 years ago, but the uncomfortable symptom was not getting better. 9 years ago, a traumatic injury on her back further aggravated her discomfort though the cranial and cervical MRIs were both normal at that time. With regard to the proband’s family history (Fig. 1a), her parents were deceased, but her father had similar symptoms. In addition, she had three brothers and two sisters. One of her brothers who had similar symptoms has passed away and the rest were all asymptomatic. De- tails about her symptomatic father and brother are not clear. Physical examination gave the below results. III:9 aged 32 years had an abnormal spine physiological curvature. The shoulders of IV:9 aged 11 years were not equal. The (III:1) aged 46 years, III:9 and IV:9 showed brisk deep tendon reflexes in all four limbs and positive In-silico predictions Effects of the novel mutation on SPAST tertiary struc- ture were predicted by RaptorX prediction tool [15]. Additionally, PROVEAN (Protein Variation Effect Analyzer) [16], a new algorithm, is also adopted to pre- dict whether the mutation has an functional impact on the SPAST protein sequence variations. Clinical features of the four affected individuals in the family have been summarized in Table 1, and their clin- ical commonalities and personalities are exhibited re- spectively. The four symptomatic patients have the different degrees of disability. The disability score was evaluated according to a four-point scale (1: normal, 2: able to walk but not run, 3: need the help of a walking aid or support, 4: walk on wheelchair) [17]. In addition, the onset age ranges from 3 to 30 years old, although they have the same mutation in the exon 16. DNA extraction h l Whole genomic DNA was extracted from peripheral blood of the 15 family members using DNA Isolation Kit (Bioteke, AU1802) as previously described [13]. Con- centrations of each DNA sample were measured on a Page 3 of 7 Page 3 of 7 Yu et al. BMC Medical Genetics (2020) 21:123 Yu et al. BMC Medical Genetics (2020) 21:123 Babinski signs. All the three symptomatic patients all could not run and squat since young age. Despite their motor symptoms, the proband’s nephew(III:1) and the third daughter (III:9) have frequent urge to urinate and to have bowel functions at the age of 40 years and 27 years. Sequence Control Software (SCS) was used to evaluate the sequencing data, thus removing adapter sequences in the raw data and discarding low-quality sequencing reads. Conventional Sanger sequencing of the SPAST gene was further performed in 15 individuals from the Chinese family. The proband’s the third daughter (III:9) and grandso- n(IV:9) suffer from other disease except HSP. The III:9 patient who was diagnosed with pulmonary hypertension has been suffering from chest tightness and shortness of breath for 3 years, losing the ability to work. Similarly, the echocardiography of IV:9 aged 11 years showed mild reflux at mitral valve and tricuspid valve. Clinical characteristics The proband (II:2) (Fig. 1a), a 63-years-old female, pre- sented to the outpatient clinic in our hospital due to her progressive difficulty walking caused by moderate spasti- city of the lower limbs for 24 years. She has felt un- known gait disorder as early as in 1992. For the last 11 years, this gait disorder has gotten worse, especially in cold weather. And she was confined to a wheelchair at the age of 52. In the last year, the proband suffered from the frequently urged to urinating and bowel functions. Physical examination showed that she had brisk deep tendon reflexes in all four limbs, simultaneously accom- panied with obvious corticospinal tract signs (Babinski’s signs was positive), and decreased sense of pain, light touch and vibration in the lower limbs characterized with stocking pattern-distributed sensory loss. Muscle strength of the upper limbs was normal, while both the extensors and flexors in the lower limbs were 3/5. The results obtained from routine laboratory tests, electro- myography, cranial and cervical MRIs did not reveal any obvious pathognomonic alteration. For her previous his- tory, the proband had ever received some treatment on rheumatoid arthritis because of the pain in both hips and knees 26 years ago, but the uncomfortable symptom was not getting better. 9 years ago, a traumatic injury on her back further aggravated her discomfort though the cranial and cervical MRIs were both normal at that time. With d t th b d’ f il hi t (Fi 1 ) sented to the outpatient clinic in our hospital due to her progressive difficulty walking caused by moderate spasti- city of the lower limbs for 24 years. She has felt un- known gait disorder as early as in 1992. For the last 11 years, this gait disorder has gotten worse, especially in cold weather. And she was confined to a wheelchair at the age of 52. In the last year, the proband suffered from the frequently urged to urinating and bowel functions. Physical examination showed that she had brisk deep tendon reflexes in all four limbs, simultaneously accom- panied with obvious corticospinal tract signs (Babinski’s signs was positive), and decreased sense of pain, light touch and vibration in the lower limbs characterized with stocking pattern-distributed sensory loss. Muscle strength of the upper limbs was normal, while both the extensors and flexors in the lower limbs were 3/5. Genetic findings and prediction results of protein structure and function by different methods The Exome Sequencing analysis of the proband exhib- ited a novel disease-associated mutation in exon 16 of the already known disease-associated SPAST gene, and the in-frame deletion was identified in the three affected family members (Fig. 1b II:2, III:1,III:9,IV:9). It is an in- frame deletion mutation in the heterozygous state: the GAA nucleotides deletion at codon 1710–1712 position and the circled nucleotide represents the codon 1710 position, where the mutation starts. (Fig. 1b II:2, III:1, III:9,IV:9). According to Human Gene Mutation Data- base (HGMDpro), the pathogenic mutation site c.1710_ 1712delGAA has not been reported until now. There- fore, it is a novel mutation. And there are no mutations were identified when analysis of other genes associated with HSP was performed: PLP1, L1CAM, SPG11, SPG7, ATL1 and so on. While the rest asymptomatic family members had no mutations at this site (Fig. 1b III:5). As highlighted in Fig. 1b, the results of RaptorX pre- diction showed that this new-found mutation we re- ported resulted in the synthesis of misfolded protein (Fig. 2b) in comparison to native one (Fig. 2a). We per- formed a protein sequence alignment across species showing the area of this in-frame amino acid deletion and the surrounding residues (https://www.uniprot.org/ align/A20200502216DA2B77BFBD2E6699CA9B6D1C41 EB2087CC0O). The result revealed that the spastin pro- tein sequence across species is highly conserved at the position 570 of the protein (red frame) (Fig. 2c). With regard to the proband’s family history (Fig. 1a), her parents were deceased, but her father had similar symptoms. In addition, she had three brothers and two sisters. One of her brothers who had similar symptoms has passed away and the rest were all asymptomatic. De- tails about her symptomatic father and brother are not clear. Physical examination gave the below results. III:9 aged 32 years had an abnormal spine physiological curvature. The shoulders of IV:9 aged 11 years were not equal. The (III:1) aged 46 years, III:9 and IV:9 showed brisk deep tendon reflexes in all four limbs and positive Yu et al. BMC Medical Genetics (2020) 21:123 Page 4 of 7 Fig. 1 Pedigree of the investigated HSP family harbouring a novel SPAST gene mutation. a: The black arrow indicates the proband II:6. Squares indicate male, circles indicate females. Individuals affected with HSP are represented by black filled, while Healthy members are indicated by empty symbols. Slashes indicate already dead. Genetic findings and prediction results of protein structure and function by different methods b: Detection of the mutation of SPAST gene in a Chinese family. Sequence analysis revealed a newly identified in-frame deletion mutation in a heterozygous form in four affected individuals (II:2,III:1,III:9 and IV:9) within the family. The exon16 consists three nucleotides deletion(c 1710 1712delGAA) III:5 is the representative wild type sequences of the investigated healthy Fig. 1 Pedigree of the investigated HSP family harbouring a novel SPAST gene mutation. a: The black arrow indicates the proband II:6. Squares indicate male, circles indicate females. Individuals affected with HSP are represented by black filled, while Healthy members are indicated by empty symbols. Slashes indicate already dead. b: Detection of the mutation of SPAST gene in a Chinese family. Sequence analysis revealed a newly identified in-frame deletion mutation in a heterozygous form in four affected individuals (II:2,III:1,III:9 and IV:9) within the family. The exon16 consists three nucleotides deletion(c.1710_1712delGAA). III:5 is the representative wild type sequences of the investigated healthy family members Therefore, the lysine deficiency at the position 570 of the protein has a significant impact on the function. Additionally, the result of PROVEAN demonstrated that the mutation site c.1710_1712delGAA has an functional impact on the SPAST protein sequence variations. Given a list of genomic coordinates and variants (232,372,308, Therefore, the lysine deficiency at the position 570 of the protein has a significant impact on the function. Additionally, the result of PROVEAN demonstrated that the mutation site c.1710_1712delGAA has an functional impact on the SPAST protein sequence variations. Given a list of genomic coordinates and variants (232,372,308, AGAA,A), the amino acid change(p.K570del) can be quickly determined and PROVEAN score is computed to be −11.55, which is significantly lower than the score threshold (cutoff = −2.5). The deletion variant was pre- dicted as deleterious (Table 2). According to American College of Medical Genetics and Genomics (ACMG) Yu et al. Genetic findings and prediction results of protein structure and function by different methods BMC Medical Genetics (2020) 21:123 Page 5 of 7 Table 1 Clinical features of the affected individuals within the family Individual ID II:6 III:1 III:9 IV:9 Sex F M F M Age at onset (years)a early 30s early 10s early 10s 3 Age at examination (years) 63 46 32 10 Disease duration (years) > 33 > 36 > 22 7 Disability score b 4 2 2 2 Lower limb hyperreflexia + + + + Lower limb spasticity + + + + Lower limb pyramidal weakness – – – – Babinski sign + + + + Upper limb hyperreflexia + + + + Upper limb spasticity – – – – Sphincter disturbances + + + – Scoliosis – – + – Pes cavus + – – + Sensory deficits + – – – Mental retardation – – – – concomitant diseases – – Pulmonary hypertension Reflux at mitral and tricuspid valve + indicates the presence of a feature, −indicates the absence of a feature, respectively a: Age at onset was calculated approximately when appeared to have difficulty in walking first b: Disability stages: 1: normal, 2: able to walk alone but not run, 3: need the help of a walking aid or support, 4: wheelchair user Table 1 Clinical features of the affected individuals within the family + indicates the presence of a feature, −indicates the absence of a feature, respectively a: Age at onset was calculated approximately when appeared to have difficulty in walking first b: Disability stages: 1: normal, 2: able to walk alone but not run, 3: need the help of a walking aid or support, 4: wheelchair user criteria [18], we score this variant as likely pathogenic PM1, PM2, PM4, PP3. associated in-frame deletion in the SPAST gene. This mutation consists of three nucleotides deletions (c.1710_ 1712delGAA) within the exon 16. The onset age of pa- tients in this family is highly variable, which is accord- ance with the previous study [19]. However, the onset age of affected individuals in our study is obviously earl- ier than previous studies [20], which promotes us to consider the existence of the new mutation site c.1710_ Discussion According to the family history, accurate description of the clinical phenotype as well as cerebral and spinal MRI, we diagnosed the proband with HSP. Analyses of the Exome Sequencing revealed a novel disease- Fig. 2 Tertiary structure alteration prediction of SPAST by RaptorX tool. a. The tertiary structure of native protein. b. Tertiary structure of p.K570del affected protein. The novel mutation we reported resulted in the synthesis of misfolded protein. c. The spastin protein sequence alignment across species showing the area of this in-frame amino acid deletion (red frame) and the surrounding residues Fig. 2 Tertiary structure alteration prediction of SPAST by RaptorX tool. a. The tertiary structure of native protein. b. Tertiary structure of p.K570del affected protein. The novel mutation we reported resulted in the synthesis of misfolded protein. c. The spastin protein sequence alignment across species showing the area of this in-frame amino acid deletion (red frame) and the surrounding residues Fig. 2 Tertiary structure alteration prediction of SPAST by RaptorX tool. a. The tertiary structure of native protein. b. Tertiary structure of p.K570del affected protein. The novel mutation we reported resulted in the synthesis of misfolded protein. c. The spastin protein sequence alignment across species showing the area of this in-frame amino acid deletion (red frame) and the surrounding residues Yu et al. BMC Medical Genetics (2020) 21:123 Page 6 of 7 Yu et al. BMC Medical Genetics (2020) 21:123 Table 2 The genome variants results are represented as PROVEAN scores and predictions Table 2 The genome variants results are represented as PROVEAN scores and predictions 1712delGAA. Additionally, affected individuals in our study presented clinical features of the pHSP, which is consistent with Orlacchio A’ s study [21]. patient cell lines and the related verification on which one molecular mechanisms above of SPAST gene mu- tants in our study cannot be realized. Haploinsufficiency is the prevalent mechanism at present. Using the hap- loinsufficiency model of HSP-SPG4 with a 50% decrease in active spastin levels has shown a lower MT severing [31, 32]. Another hypothesis is mainly that mutated spastin can form defective heterohexamers with wild- type (WT) spastin, and exert a dominant-negative effect. However, there is still considerable debate about the lat- ter one hypothesis. It is unclear whether the mutant M87 can effectively impair the enzymatic activity of WT spastin [33]. Funding g No grant was provided for this analysis and this study. Authors’ contributions YN H and WW Y designed the work and analyzed the clinical data. HQ J and JW D performed the neurologic examination and interpreted the clinical data related to the HSP. D N collected the material of these family members and analyzed the genetic data. WW Y was a major contributor to writing the manuscript. All authors contributed toward drafting and critically revising the paper, gave final approval of the version to be published. Acknowledgements We would like to thank all members of the family participating in the study for agreeing to publish their available clinical data in medical journals. Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki and the Ethics Committee of Peking University First Hospital (PKUFH-2019-181). Written informed consents were obtained from all of the participants of this family in the study. The written informed consent of the boy aged 11 years in our study was obtained from his parents. Abbreviations HSP H d HSP: Hereditary spastic paraplegia; SPG: Spastic paraplegia genes; CT: Computed tomography; MRI: Magnetic resonance imaging; SCS: Sequence Control Software; NGS: Next Generation Sequence; HGMDpro: Human Gene Mutation Database; MIT: Microtubule interacting and trafficking; MT: Microtubule; WT: Wild- type; ER: Reticulum membrane; CK2: Casein kinase 2; FAT: Fast axonal transport; PROVEAN: Protein Variation Effect Analyzer; ACMG: American College of Medical Genetics and Genomics; PCR: Polymerase chain reaction; RT: Room temperature Availability of data and materials The datasets generated and/or analyzed during the current study are not publicly available in order to protect participant confidentiality. Discussion Thus, further study should ascertain the role of causative genes to help better understand the re- lationship between genotypes and phenotypes. The SPAST gene has 17 coding exons and encodes the protein spastin, a member of the AAA ATPase protein family. The protein spastin contains two main structural domains: the microtubule interacting and trafficking (MIT) domain in the N- terminus and the catalytic AAA domain at the C-terminus, in which the former mainly regulates microtubule organization and the later focus on ATPase activity associated with various cellular activities [22]. The two main domains are both essential to accom- plish the main known function of spastin: microtubule (MT) severing [23]. More than 200 different mutations lo- cated in sites within the AAA region have been identified in patients with HSP-SPG4 [24]. Therefore, it is believed that some mutated spastins may result in insufficient microtubule-severing activity by dominant-negative fash- ion. Additionally, another prevalent hypothesis is neuro- toxicity of mutant spastin proteins. The SPAST gene presents two translation initiation codons, which allows to synthesis two spastin isoforms: a full-length isoform called M1(616 amino acid) and a slightly shorter isoform called M87(530 amino acid) that lacks the first 87 amino acid [25]. Studies on rodents show that M87 is more abundant in various tissues, whereas M1 is only appreciably detected in brain and spinal cord [26]. Besides that, axonal trans- port and neurite growth are not affected by the mutated M87 [27]. However, Mutant spastin proteins can form de- fective heterohexamers with wild-type (WT) spastin, and simultaneously produce toxic effect when presented as the tissue-specific M1 isoform [28, 29]. Even though M87 likely harbors the same AAA mutations as the M1 iso- form, it is somehow degraded more effectively than mu- tated M1 in a dominant-negative scenario [30], thus possessing a lower toxicity. Conclusion In Our study, a novel mutation in SPAST gene was found in a Chinese family with multiple affected family members, which significantly enrich the mutation spectrum of HSP. This novel mutation has been inher- ited at least four generations according to the related in- vestigation, further emphasizing the closed interaction between the phenotypic and genetic heterogeneity of HSP. However, some shortcomings are still existed. For example, we haven’t performed functional laboratory studies about the novel mutation due to the unavailable Competing interests h h h 21. Orlacchio A, Patrono C, Borreca A, Babalini C, Bernardi G, Kawarai T. Spastic paraplegia in Romania: high prevalence of SPG4 mutations. J Neurol Neurosurg Psychiatry. 2008;79(5):606–7. Received: 23 April 2020 Accepted: 19 May 2020 Received: 23 April 2020 Accepted: 19 May 2020 References Fei QZ, Tang WG, Rong TY, Tang HD, Liu JR, Guo ZL, Fu Y, Xiao Q, Wang XJ, He SB, et al. 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Increasing JAK/STAT Signaling Function of Infant CD4+ T Cells during the First Year of Life
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I Myra Grace dela Peña-Ponce1, Jennifer Rodriguez-Nieves1, Janice Bernhardt2, Ryan Tuck1, Neelima Choudhary1, Michael Mengual1, Katie R. Mollan3, Michael G. Hudgens4, Sigal Peter-Wohl2 and Kristina De Paris1* 1 Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA, 2 Division of Neonatal Perinatal Medicine, Department of Pediatrics, School of Medicine, University of North Carolina, Chapel Hill, NC, USA, 3 Lineberger Cancer Center, Center for AIDS Research, University of North Carolina, Chapel Hill, NC, USA, 4 Gillings School of Global Public Health, Center for AIDS Research, University of North Carolina, Chapel Hill, NC, USA Most infant deaths occur in the first year of life. Yet, our knowledge of immune development during this period is scarce and derived from cord blood (CB) only. To more effectively combat pediatric diseases, a deeper understanding of the kinetics and the factors that regulate the maturation of immune functions in early life is needed. Increased disease susceptibility of infants is generally attributed to T helper 2-biased immune responses. The differentiation of CD4+ T cells along a specific T helper cell lineage is dependent on the pathogen type, and on costimulatory and cytokine signals provided by antigen-presenting cells. Cytokines also regulate many other aspects of the host immune response. Therefore, toward the goal of increasing our knowledge of early immune development, we defined the temporal development of the Janus kinase (JAK)/ signal transducers and activators of transcription (STAT) signaling function of CD4+ T cells using cross-sectional blood samples from healthy infants ages 0 (birth) to 14 months. We specifically focused on cytokines important in T cell differentiation (IFN-γ, IL-12, and IL-4) or in T cell survival and expansion (IL-2 and IL-7) in infant CD4+ T cells. Independent of the cytokine tested, JAK/STAT signaling in infant compared to adult CD4+ T cells was impaired at birth, but increased during the first year, with the most pronounced changes occurring in the first 6 months. The relative change in JAK/STAT signaling of infant CD4+ T cells with age was distinct for each cytokine tested. Thus, while about 60% of CB CD4+ T cells could efficiently activate STAT6 in response to IL-4, less than 5% of CB CD4+ T cells were able to activate the JAK/STAT pathway in response to IFN-γ, IL-12 or IL-2. I By 4–6 months of age, the activation of the cytokine-specific STAT molecules was comparable to adults in response to IL-4 and IFN-γ, while IL-2- and IL-12-induced STAT activation remained below adult levels even at 1 year. These results suggest that common developmental and cytokine-specific factors regulate the maturation of the JAK/STAT signaling function in CD4+ T cells during the first year of life. Original Research published: 21 February 2017 doi: 10.3389/fped.2017.00015 Keywords: infant CD4+ T cell development, cytokines, JAK/STAT signaling, STAT activation, age-dependent changes Edited by: Claudio Pignata, University of Naples Federico II, Italy University of Naples Federico II, Italy Reviewed by: Lisa Renee Forbes, Baylor College of Medicine, USA Raffaele Badolato, University of Brescia, Italy *Correspondence: Kristina De Paris abelk@med.unc.edu Reviewed by: Lisa Renee Forbes, Baylor College of Medicine, USA Raffaele Badolato, University of Brescia, Italy *Correspondence: Kristina De Paris abelk@med.unc.edu Specialty section: This article was submitted to Pediatric Immunology, a section of the journal Frontiers in Pediatrics Received: 12 September 2016 Accepted: 20 January 2017 Published: 21 February 2017 INTRODUCTION Cytokines mediate their specific function through the activa- tion of distinct tyrosine kinases after receptor ligation. Cytokines binding to their receptors cause receptor dimerization and activation of Janus (JAK) kinases that can then recruit specific transcription factors, the signal transducers and activators of transcription (STAT) (52, 53). This signaling pathway was first described in response to interferons (53). There are four members in the JAK kinase family and seven mammalian STATs. Therefore, the specificity of cytokine function is achieved in a stepwise fash- ion, through activation of distinct JAK kinases, the subsequent formation of specific homo- or heterodimers of activated STATs, and finally the regulated expression of distinct genes upon trans- location of the STAT molecules to the nucleus (52, 53). Every day, 17,000 children under the age of 5 years die with close to 50% of these deaths being caused by infectious diseases (1). Most infant deaths occur in the first year of life (1), at a time when the infant has to simultaneously acquire the ability to tolerate normal commensal flora and to recognize pathogenic stimuli. To adapt and overcome this challenge, the infant immune response is qualitatively distinct from that of adults. Yet, most of our knowl- edge of infant immunity is derived from CB analysis only. Thus, an important gap remains in our understanding of the kinetics and factors that drive and regulate the functional maturation of infant immune cells postnatally.h The development of the infant’s immune system is shaped by many different factors that range from genetic predispositions to nutrition, pathogen exposure, environmental stimuli, and others [reviewed in Ref. (2)]. It would be an insurmountable task to address all these factors adequately in a single study. The current study focused on CD4+ T cells because they play a key role in innate, in cellular, and in humoral adaptive immunity by secreting cytokines that (i) activate monocytes/macrophages and dendritic cells, (ii) promote effector function of CD8+ T cells, and (iii) influence antibody maturation and isotype switching (3–10). Increased susceptibility and severity of infectious diseases in infants has been attributed to impaired CD4+ T helper (Th) 1 responses. INTRODUCTION The bias of infant CD4+ T cells toward T helper 2 (Th2) responses was suggested more than 25 years ago in mice (11), and human studies confirmed reduced IFN-γ production by infant CD4+ T cells (12–15).h Considering the key role of cytokines in many aspect of host immunity and disease, the current study defined the temporal development of JAK/STAT signaling function of CD4+ T cells in response to cytokines important in Th cell differentiation (IFN-γ, IL-12, and IL-4) and in T cell expansion (IL-2 and IL-7) using cross-sectional blood samples from healthy infants ages 0 (birth) to 14 months. Our analysis was focused on key components of the JAK/STAT signaling pathway: (i) cytokine receptor expression, (ii) JAK activation, and (iii) the activation of cytokine-specific STAT molecules. IFN-γ and IL-12 signal through JAK1/JAK2 and JAK2/Tyk2, respectively, while IL-4, IL-2, and IL-7, as members of the common gamma chain (γc) cytokine family, all signal through JAK1 and JAK3 [reviewed in Ref. (54)]. As the JAK1 kinase is common to all of the signaling pathways, the activation of specific STAT molecules by distinct cytokines represented a key aspect of our analysis. Thus, to assess age-dependent changes in IFN-γ, IL-12, or IL-4-specific signaling, we measured the activation of STAT1, STAT4, or STAT6, respectively. To assess IL-2- and IL-7-mediated cytokine signaling, STAT5 activation was measured (54). Furthermore, to address a potential role of JAK/STAT signaling in Th-2 biased infant immune responses, we compared the activation of JAK2 and STAT1 by IFN-γ to the activation of JAK3 and STAT6 in response to IL-4. The underlying immune mechanisms that contribute to the infant’s Th2 bias have only recently been discovered. Cytokines play a major role in deciding the fate of naïve CD4+ T cells toward the development of a specific Th cell lineage (16–21). The production of the Th1-defining cytokine IFN-γ by CD4+ T cells is critically dependent on IL-12 (22–24). However, not only do infant antigen-presenting cells produce less IL-12 than adults (12, 14, 25–29), naïve CD4+ T cells, that represent the majority of neonatal T cells, lack the expression of the IL-12Rβ2 (30–33). The preferential induction of IL-4, the prototype Th2 cytokine, versus IFN-γ in infant CD4+ T cells is further regulated by epigenetic modulations. INTRODUCTION Thus, the IL-12p35 gene is in a repressive chroma- tin state in infant dendritic cells (34, 35), and while the promoter of IFN-γ is hypermethylated in infant compared to adult CD4+ T cells (36, 37), gene loci associated with Th2 cytokines, such as IL-13, are hypomethylated (38).hf Citation: dela Peña-Ponce MG, Rodriguez- Nieves J, Bernhardt J, Tuck R, Choudhary N, Mengual M, Mollan KR, Hudgens MG, Peter- Wohl S and De Paris K (2017) Increasing JAK/STAT Signaling Function of Infant CD4+ T Cells during the First Year of Life. Front. Pediatr. 5:15. doi: 10.3389/fped.2017.00015 February 2017  |  Volume 5  |  Article 15 Frontiers in Pediatrics  |  www.frontiersin.org 1 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. Frontiers in Pediatrics  |  www.frontiersin.org Phosflow™ Analysisl Exclusion criteria included HIV-positive status of the mother, known in utero infections, treatment of the mother with immu- nosuppressive drugs, diagnosis of mother or child with immuno- suppressive disorder, life-threatening malformations of the infant or life expectancy <6 months. Infant blood samples were also excluded if the infant had a bleeding disorder or had a chronic infection. The Virology, Immunology, and Microbiology Core of the UNC Center for AIDS Research provided blood samples from healthy adults. Age, sex, and race of the adult donors were unknown. The study was approved by the UNC-CH Institutional Review Board, and informed parental consent was obtained. Institutional guidelines strictly adhere to the World Medical Association’s Declaration of Helsinki. Phosflow™ analysis was performed to measure the activation of JAK2 after IFN-γ stimulation or the activation of STAT proteins according to the manufacturer’s protocols (BD Biosciences) using Perm Buffer III (CD4+ T cells) or Perm Buffer IV (NK cells). Although most JAK and STAT molecules contain multiple tyrosine and/or serine phosphorylation sites, the analysis was limited to a single phosphorylation site. Briefly, 300–400 µL of whole blood was treated for 10 min with 10 µg/mL of human recombinant IFN-γ (Miltenyi Biotec) and samples were then stained with antibodies to total JAK2 or with antibodies to phosphorylated JAK2 (Tyr1008; clone D4A8, Cell Signaling Technology, Danvers, MA, USA). Phospho-JAK2 (pJAK2) was detected with anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor 647, Cell Signaling Technology). To detect the phospho- rylation of specific STAT proteins after stimulation with IFN-γ IL-4, IL-2, or IL-7, whole blood was incubated for 15 min with 10  µg/mL of recombinant IFN-γ, 0.1  µg/mL of recombinant IL-4 (R&D, Minneapolis, MN, USA) or 0.1  µg/mL human IL-2 or IL-7 (Miltenyi Biotec), respectively, and subsequently stained for phosphorylated STAT-1 (Tyr 701; clone 58D6, Cell Signaling Technology), STAT6 (Y641; clone pY641) or STAT5 (Y694; clone pY694, both BD Biosciences), respectively. Non- phosphorylated STAT1, STAT6, or STAT5 proteins were meas- ured in parallel. Unstimulated whole blood served as negative control. STAT4 phosphorylation (pSTAT4 Y693) was measured using the same protocol, except that PBMCs were stimulated for 45 min with IL-12 (75 ng/mL) in serum-free RPMI 1640 media. To distinguish CD4+ T cells with different antigen experience, blood samples were costained with CD3 (SP34-2)-PeCy7, CD4 (L200)-PerCPCy5.5, and CD27 (L128)-PE, and naïve, central memory, and effector/effector memory populations were defined as being CD27+, CD27low, or CD27−, respectively (57). Human Subjects j Study subjects were recruited from the University of North Carolina, Chapel Hill (UNC-CH) and the NC Children’s Hospital. CB samples from full-term infants were collected immediately after birth or obtained from the Carolinas Cord Blood Bank. Direct capillary or venous blood samples (0.5–3.0  mL) from infants age 2 weeks to 14 months were collected by nursing or phlebotomy personnel during routine physician visits. The infants consisted of similar numbers of males and females. The exact number of infants per age group is listed in Table 1; we had 70 CB samples, 35 samples from infants between the ages of 1 and 9 months and 64 samples from 1 year olds (10–14 months). The large number of infants (n = 169) was required because limited blood volumes (0.5–2.0 mL) from infants age 2 weeks to 1 year restricted the number of assays and parameters that could be analyzed. The infant participants were recruited from the North Carolina population that consists of about 46% Caucasians, 46% In addition to Th cell differentiation, cytokines regulate many other immune processes (17, 39–45). Cytokines encompass a large group of diverse soluble biological mediators that can act in an autocrine, paracrine, or endocrine fashion to exert their functions. Aberrations in cytokine signaling and/or gene defi- ciencies have been associated with serious clinical outcomes, such as severe immunodeficiency (46, 47), inflammatory immune disorders (48), and altered lymphopoiesis (49). The importance of cytokine signaling in host immunity is further underlined by the fact that several viruses have evolved to inhibit specific components of cytokine signaling pathways to limit the induction of antiviral immunity (50, 51). February 2017  |  Volume 5  |  Article 15 Frontiers in Pediatrics  |  www.frontiersin.org 2 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. Table 1 | Study population. Age No. of subjects by sex Male Female Unknown Total Birth 24 24 22a 70 1–3 months 3 7 0 10 4–6 months 6 11 2 19 7–9 months 2 4 0 6 10–14 months 39 24 1 64 Total 74 70 25 169 aCord blood samples obtained from the Carolinas Cord Blood Bank. CA, USA), IL-4Rα (hIL4R-M57), IL-2Rα (CD25-clone-4E3, Miltenyi Biotec, Auburn, CA, USA), or IL-2Rβ (CD122-clone- Mikβ). Cytokine receptor expression is reported as percentage of CD4+ T cell populations or quantitated on a per cell basis using a standard curve generated with Quantibrite PE beads (BD Biosciences). Phosflow™ Analysisl Cytokine concentrations and incubation times were determined in preliminary experiments (data not shown). Samples were acquired on a LSRII instrument analyzed with FlowJo Software. Cells positive for JAK2 or STAT phosphorylation are reported as percentage of CD4+ T cells after subtracting the values from the untreated samples. Antibodies were titrated prior to use and gating was based on FMO and isotype antibody controls. In addition, a subset of samples was analyzed on the Amnis® ImageStreamX instrument according to the manufacturer’s protocol. Sample Processing Cord blood from full-term infants was collected into CB col- lection bags containing CPD anticoagulant, whereas all other blood samples were collected into EDTA-containing blood tubes. Blood samples were processed within 24 h. Whole blood was used for Phosflow™ analysis of JAK2 and STAT proteins; for all other assays, peripheral blood mononuclear cells (PBMCs) were isolated by gradient density separation using Lymphocyte Separation Medium (MP Biomedicals, OH) as described (55, 56). Human Subjects The staining protocol was performed at 4°C. Samples were acquired on a LSRII instrument (BD Biosciences) by collecting a minimum of 300,000 events. The flow cytometry data were analyzed using FlowJo Software (Tree Star, Ashland, OR, USA), version 9.8. Antibodies were titrated prior to use and gating was based on Fluorescence Minus One (FMO) and isotype antibody controls. African-Americans, and 8% other groups, and within the ethnic categories, 10% represent Hispanics. CD4+ T Cell Enrichment CD4+ T cells were enriched by magnetic bead separation using the CD4+ T cell enrichment Kit from Stemcell Technologies (Vancouver, BC, Canada) according to the manufacturer’s instructions. The purity of CD4+ T cell populations was confirmed by flow cytometry and exceeded 96%. Cytokine Receptor Expression on Infant CD4+ T Cells Cytokine receptor expression was evaluated both by quantifying the number of receptors per CD4+ T cell and by determining the percentage of CD4+ T cells expressing each cytokine receptor. Two main comparisons were performed. First, we determined whether there was a significant change in cytokine receptor expression from birth to 1 year of age. Second, we tested whether the expression of cytokine receptors differed between 1-year-old infants and adults. These comparisons were conducted both for total CD4+ T cells and for CD4+ T cell subsets. The analysis of CD4+ T cells according to their phenotypic characterization as naïve [T(N); CD45RA+CCR7+], central memory [T(CM); CD45RA−CCR7+], or effector memory/effector [T(EM/Eff); CD45RA±CCR7−] CD4+ T cells was important because the majority of infant peripheral blood CD4+ T cells represent naïve CD4+ T cells and the relative frequencies of these CD4+ T cell population change with age and pathogen exposure. Thus, this analysis was necessary to accurately evaluate age-related differ- ences in infant CD4+ T cells from birth to 1 year, and equally critical for the comparison of infant and adult CD4+ T cell parameters.i Cord blood CD4+ T cells expressed significantly more IL-4Rα molecules per CD4+ T cell than adults (Table 2). Although there was an age-dependent decline in median IL-4Rα numbers/CD4+ T cell comparing birth to 1 year, these differences were not sta- tistically significant. The percentage of IL-4Rα+CD4+ T cells also did not vary substantially with age (Table 3). To initiate signaling upon IL-4 binding, the IL-4Rα chain dimerizes with the common γ-chain (γc); we did, however, not measure the common γ-chain, because previous studies have already shown that it is expressed at lower levels in infants (58).h The IFN-γ receptor consists of the IFN-γR1, promoting IFN- γ binding, and the IFN-γR2 that mediates signaling to the JAK kinases. The number of IFN-γR1 molecules per CD4+ T cell did not significantly differ between birth and 1 year (Table 2). CD4+ T cells in CB and in blood from 1-year-old infants expressed significantly higher numbers of IFN-γR1/CD4+ T cell than adults (Table 2; Figure 1A). In contrast, the percentage of CD4+ T cells expressing IFN-γR1 increased from birth to 1 year, and even at 1  year, the percentage of IFN-γR1+CD4+ T cells was significantly lower compared to adults (Table 3, Figure 1A). This difference was especially pronounced in the subset of effector memory/effector CD4+ T cells (Table 3; Figure 1B). RESULTS The phosphorylation of JAK3 in response to stimulation with IL-4 or IL-2 was measured by Western blot. CD4+ T cells were enriched as described above and either stimulated immediately with IL-4 or IL-2 or preactivated for 2  days prior to cytokine stimulation. For the latter, CD4+ T cells were stimulated at a concentration of 2–4 × 106 cells/mL with plate-bound anti-CD3 (10 µg/mL; clone SP34-2), precoated overnight in a 24-well plate, and cultured at 37°C and 5% CO2. Controls were set up in parallel with media only. After 48  h, to activate JAK3, CD4+ T cells were stimulated for 5 min with 0.1 µg/mL recombinant human IL-4 or IL-2 at 37°C in a water-bath followed by a 5-min centrifugation at 300 × g (total stimulation time: 10 min). Cells were lysed with 100 µL of Blue Loading Pack (Cell Signaling Technology) per 4 × 106 cells. Protein concentrations were determined using the Pierce 660 nm Assay (Thermo Scientific, Rockford, IL, USA). Samples were loaded onto 8% Novex® Tris-Glycine gels (Novex®, Life Technologies, NY, USA). The XCell SureLock™ Mini Cell was used for electrophoresis in Novex® Tris-Glycine SDS running buffer and for subsequent transfer using Novex® Tris-Glycine transfer buffer. Transfer PVDF membranes were obtained from Bio-Rad Laboratories (Hercules, CA, USA). All detection antibodies and reagents were obtained from Cell Signaling Technology unless noted otherwise. To determine JAK3 phosphorylation, the membranes were incubated with phospho-JAK3 antibody (Tyr980/981; clone D44E3; 1:1,000 dilution) in 1× TBST overnight, stripped using Restore Plus Western blot (Pierce/ Thermo Scientific, Rockville, IL, USA) and re-probed for non-phosphorylated JAK3 (clone D1H3; 1:1,000 dilution:) and beta (β)-Actin (clone 13E5; 1:50,000 dilution) as loading control. The membranes were then incubated with secondary anti-Rabbit IgG HRP-linked antibody at a dilution of 1:2,000 and developed using the Amersham™ ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Images were captured using the Bio- Rad ChemiDoc MP System and densitometric measurements were obtained using the Image Lab Software Version 5.2.1. Protein sizes were verified based on the NuPage HiMark Prestained Protein Standard (Life Technologies, Grand Island, NY, USA). Cytokine Receptor Expression on Infant CD4+ T Cells We did not observe significant changes in total expression levels of IFN-γR2 (Table 2), and although the frequencies of naïve and effector memory/effector IFN-γR2 CD4+ T cells seemed to decrease in the first year, the biological significance of the changes is ques- tionable given their very low frequencies (Table 3). Note that the relatively low frequencies of IFN-γR1 or IFN-γR2 CD4+ T cells in infants were not the result of poor staining, as IFN-γR- positive B cells could be easily enumerated in the same samples (Figure 1C). The analysis of the ratio of relative expression levels of IL-4Rα to IFN-γR1 or IL-4Rα to IFN-γR2 on infant CD4+ T Cytokine Receptor Expression y p p Peripheral blood mononuclear cells were stained with CD3 (SP34-2)-Pacific Blue, CD4 (L200)-PerCPCy5.5, CD8 (RPA-T8)- Alexa Fluor700, CD45RA (5H9)-FITC, and CCR7 (3D12)-PeCy7 to determine naïve [T(N): CD45RA+CCR7+], central memory [T(CM): CD45RA−CCR7+], and effector/effector memory cell [T(Eff/EM), CD45RA±CCR7−] T cell populations. All antibodies, unless noted otherwise, were purchased from BD Biosciences (San Jose, CA, USA). The expression of cytokine receptors on CD4+T cells was quantitated using PE-labeled antibodies: IFN-γR1 (CD119-GIR-208), IFN-γR2 (2HUB-159, Biolegend, San Diego, February 2017  |  Volume 5  |  Article 15 Frontiers in Pediatrics  |  www.frontiersin.org 3 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. r of mon Statistical Analysis Data were analyzed using GraphPad Prism Software (Version 6, 2014; La Jolla, CA, USA). Continuous measurements were compared between independent age groups using non- parametric Mann–Whitney tests. Statistical comparisons were only performed if the sample size was n ≥ 4 in both age groups. Associations between continuous variables was estimated using non-parametric Spearman rank correlation. To visualize the age-dependent changes in STAT activation in response to IL-4, IFN-γ, and IL-2, a LOESS trend curve was generated with a smooth parameter of 0.5. Only statistically significant p-values are shown in the figures. A 0.05 significance level was used with no adjustment for multiple hypothesis testing. February 2017  |  Volume 5  |  Article 15 Frontiers in Pediatrics  |  www.frontiersin.org 4 B 10 of cytokine receptors/CD4+ T cell (range) months 7–9 months 10–14 months Adult 71–429) 303 (271–451) 286 (201–662) 278 (179–368) 68–505) 314 (273–380) 283 (196–996) 281 (210–423) 30–350) 275 (250–459) 287 (196–331) 265 (172–391) 65–567) 516 (397–725) 333 (175–395) 304 (200–371) 85–1,119) n.d.** 834 (627–1,388) 653 (390–1,824) 85–1,638) n.d. 944 (668–2,756) 872 (430–3,293) 83–946) n.d. 776 (599–1,159) 718 (364–2,063) 08–817) n.d. 640 (594–909) 568 (392–1,792) 13–1,940) n.d. 630 (299–1,062) 635 (271–2,018) 210–3,842) n.d. 860 (413–1,367) 890 (332–2,213) 51–965) n.d. 451 (253–856) 507 (223–2,573) 48–1,547) n.d. 684 (355–3,409) 584 (289–1,873) 21–1,780) 1,446 (1,032–1,633) 1,121 (807–1,815) 501 (200–1,439) 78–2,821) 2,005 (1,782–2,155) 1,575 (1,019–2,497) 420 (238–1,170) 96–1,335) 1,266 (869–1,356) 981 (607–1,467) 452 (183–1,176) 13–1,661) 1,469 (947–1,699) 1,176 (911–2,119) 659 (220–1,827) 94–704) n.d. 289 (207–427) 266 (145–461) 77–3,302) n.d. 300 (201–540) 340 (171–671) 70–393) n.d. 272 (203–383) 225 (134–457) 94–519) n.d. 280 (212–347) 265 (130–457) 018) 213) 573) 873) 439) 170) 176) 827) 1) 1) 7) 7) Birth 0–14 cells expressing specific cytokine receptors (range) Bi 10– months 7–9 months 10–14 months Adult .2–0.4) 0.4 (0.3–0.6) 0.2 (0.1–4.0) 0.5 (0.1–1.2) .6–0.7) 0.7 (0.6–0.8) 0.4 (0.1–23.8) 0.9 (0.2–4.2) .1–0.3) 0.3 (0.2–0.5) 0.2 (0.1–4.8) 0.4 (0.1–1.7) .1–0.3) 0.6 (0.1–0.7) 0.2 (0.0–0.6) 0.2 (0.0–0.6) p .3–3.0) n.d.** 1.4 (0.5–3.3) 4.3 (1.3–10.0) .7–2.8) n.d. 1.5 (0.4–2.5) 0.8 (0.3–2.7) .1–2.9) n.d. 1.2 (0.4–2.3) 1.1 (0.3–5.6) .8–6.8) n.d. 6.3 (1.7–9.3) 12.6 (3.9–31.7) .3–0.7) n.d. 0.5 (0.2–0.8) 0.2 (0.1–1.9) .6–5.0) n.d. 0.4 (0.1–1.0) 0.3 (0.1–2.3) p .1–0.5) n.d. 0.3 (0.1–0.8) 0.1 (0.0–1.0) .1–0.5) n.d. 0.2 (0.0–0.7) 0.2 (0.0–4.2) p .1–14.0) 9.6 (8.6–11.7) 9.6 (7.9–13.8) 30.6 (16.2–41.4) p .4–16.4) 9.1 (9.0–10.6) 7.4 (2.9–14.7) 9.7 (4.6–23.1) .7–10.6) 6.4 (4.0–7.1) 8.1 (5.3–10.3) 36.8 (21.2–53.8) p 9.5–66.5) 42.2 (34.0–52.0) 44.2 (36.8–58.3) 47.4 (27.3–76.4) .0–0.3) n.d. cells mont Statistical Analysis 0.4 (0.1–2.7) 0.9 (0.1–4.3) .0–0.3) n.d. 0.2 (0.0–1.8) 0.2 (0.1–1.7) .0–0.2) n.d. 0.3 (0.1–2.2) 0.7 (0.1–4.4) .1–1.5) n.d. 1.9 (0.2–9.6) 1.8 (0.1–8.9) Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. A: A: B: C: D: gure 1 | Cytokine receptor expression in infants. (A) The changes in absolute numbers of IFN-γR1 on CD4+ T cells and the percentage of IFN-γ+ CD4+ ells with age. Each symbol represents an individual infant (open triangle); adults are shown by black cross symbols. The p values indicate statistically significant erences between 10–14 months old infants and adults. (B) The percentage of IFN-γ+ CD4+ T cells in naïve [T(N)], central memory [T(CM)], and effector memory/ ctor [T(EM/Eff)] CD4+ T cells of 10- to 14-month-old infants and adults. (C) A representative histogram of IFN-γR1-positive cells in an infant blood sample. Note t the majority of IFN-γR1-positive cells represent B cells. (D) The left graph shows the age-dependent increase in soluble CD25 (sCD25) plasma levels. The right ph illustrates the Spearman rank correlation between sCD25 plasma levels and IL-2Rα+CD4+ T cells using infants at birth and 1 year. Each symbol represents an vidual infant, with infants at different ages being characterized by specific symbols: cord blood (CB): open triangle, 1–3 months: yellow diamonds, 4–6 months: nge circles, 7–9 months: red hexagons, 10–14 months (1 year): blue triangles. Horizontal lines represent median values. The differences in plasma sCD25 levels ween CB and 1-year olds were determined by Mann–Whitney test. B: B: C: C: D: D: Figure 1 | Cytokine receptor expression in infants. (A) The changes in absolute numbers of IFN-γR1 on CD4+ T cells and the percentage of IFN-γ+ CD4+ T cells with age. Each symbol represents an individual infant (open triangle); adults are shown by black cross symbols. The p values indicate statistically significant differences between 10–14 months old infants and adults. (B) The percentage of IFN-γ+ CD4+ T cells in naïve [T(N)], central memory [T(CM)], and effector memory/ effector [T(EM/Eff)] CD4+ T cells of 10- to 14-month-old infants and adults. (C) A representative histogram of IFN-γR1-positive cells in an infant blood sample. Note that the majority of IFN-γR1-positive cells represent B cells. (D) The left graph shows the age-dependent increase in soluble CD25 (sCD25) plasma levels. The right graph illustrates the Spearman rank correlation between sCD25 plasma levels and IL-2Rα+CD4+ T cells using infants at birth and 1 year. Statistical Analysis Each symbol represents an individual infant, with infants at different ages being characterized by specific symbols: cord blood (CB): open triangle, 1–3 months: yellow diamonds, 4–6 months: orange circles, 7–9 months: red hexagons, 10–14 months (1 year): blue triangles. Horizontal lines represent median values. The differences in plasma sCD25 levels between CB and 1-year olds were determined by Mann–Whitney test. Figure 1 | Cytokine receptor expression in infants. (A) The changes in absolute numbers of IFN-γR1 on CD4+ T cells and Figure 1 | Cytokine receptor expression in infants. (A) The changes in absolute numbers of IFN-γR1 on CD4+ T cells and the percentage of IFN-γ+ CD4+ T cells with age. Each symbol represents an individual infant (open triangle); adults are shown by black cross symbols. The p values indicate statistically significant differences between 10–14 months old infants and adults. (B) The percentage of IFN-γ+ CD4+ T cells in naïve [T(N)], central memory [T(CM)], and effector memory/ effector [T(EM/Eff)] CD4+ T cells of 10- to 14-month-old infants and adults. (C) A representative histogram of IFN-γR1-positive cells in an infant blood sample. Note that the majority of IFN-γR1-positive cells represent B cells. (D) The left graph shows the age-dependent increase in soluble CD25 (sCD25) plasma levels. The right graph illustrates the Spearman rank correlation between sCD25 plasma levels and IL-2Rα+CD4+ T cells using infants at birth and 1 year. Each symbol represents an individual infant, with infants at different ages being characterized by specific symbols: cord blood (CB): open triangle, 1–3 months: yellow diamonds, 4–6 months: orange circles, 7–9 months: red hexagons, 10–14 months (1 year): blue triangles. Horizontal lines represent median values. The differences in plasma sCD25 levels between CB and 1-year olds were determined by Mann–Whitney test. February 2017  |  Volume 5  |  Article 15 Frontiers in Pediatrics  |  www.frontiersin.org 7 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. unstimulated CD4+ T cells in CB (19-fold; range: 4.9- to 25.4- fold), samples of 1 year olds (13-fold; range: 10.7- to 15.4-fold), and adults (2.5-fold; range: 2.2- to 11.6-fold).hfi cells did not reveal changes that would support a switch from a more Th2-biased response at birth versus a more balanced Th2/ Th1 response at 1 year. h p y An analysis of the IL-2R was included because T cell expan- sion is a critical step in T cell differentiation. Statistical Analysis The IL-2R consists of three distinct chains, the α, β, and γ-chain. The IL-2Rα chain (or CD25) is of special importance as it is a marker of regulatory T cells (Tregs), which occur in higher frequencies in infant compared to adult blood, but CD25 is also expressed on activated T cells that are more frequent in adult peripheral blood. Both, absolute numbers of IL-2Rα per CD4+ T cell and the percentage of IL-2Rα+CD4+ T cells were higher at 1 year of age than at birth (Tables 2 and 3; Figure 1D). At 1 year, the number of IL-2Rα molecules per CD4+ T cells exceeded that of adult CD4+ T cells (Table 2), whereas the percentage of IL-2Rα+CD4+ T cells was significantly lower compared to adults (Table  3). Whether or not these results reflected the dichotomy of the CD25 molecule as a marker of Treg or activated CD4+ T cells was not a focus of the study. However, soluble CD25 (sCD25) plasma levels were higher at 1 year com- pared to CB (Figure 1D), and there was a significant positive correlation between plasma sCD25 levels and the percentage of IL-2Rα+CD4+ T cells (Figure 1D). Although these results were indicative of increasing activated CD4+ T cells with age, higher sCD25 plasma levels (Figure 1D) and IL-2Rα+CD4+ T cells (Table 3) were already increased by 1–3 months and did not further increase from 3 to 14 months. The effective activation of pJAK3 by IL-4 in CB was confirmed by the fact that the downstream IL-4-specifc transcription factor STAT6 was activated in 64% (median value) of CB CD4+ T cells, although subject-to-subject variation at birth was high (range: 14–99%; Figure 2D). Despite the seemingly potent induction of the JAK/STAT signaling pathway by IL-4 in CB, median frequen- cies of pSTAT6+CD4+ T cells were lower compared to adults (93%; range: 60–98%). The activation of pSTAT6 significantly increased with age (Figure  2D), and by 4–6  months of age, pSTAT6 activation was comparable to 1-year-old infants and to adults (Figure 2D). The age-dependent increase in IL-4-induced pSTAT6 activation observed in the total CD4+ T cell populations appeared to be mainly reflective of changes in naive CD4+ T cells (Figure  2D). Although memory CD4+ T cells also responded with higher pSTAT6 activation by 4–6  months and at 1  year, pSTAT6 activation remained below [T(CM)] or within the lower range [T(EM/Eff)] of adult memory CD4+ T cells (Figure 2D). Age-Dependent Increase in IFN-γ-Induced JAK/STAT Signaling g g In contrast to JAK3 activation by IL-4, only a small fraction of CB CD4+ T cells induced JAK2 phosphorylation in response to IFN-γ stimulation (Figure 4A). The histograms in Figure 4B are representative for CB and adult CD4+ T cells and demonstrate that unstimulated CD4+ T cells in both age groups have similar low levels of pJAK2 and similar background level staining in FMO controls, but clearly differ in the levels of pJAK2 after IFN- γ stimulation. However, by 1 year of age, pJAK2 activation was even higher than in adult CD4+ T cells (Figure 4A). To more spe- cifically determine when infant CD4+ T cell acquired improved IFN-γ signaling capacity, we measured STAT1 phosphorylation in CD4+ T cells from birth to 1 year. As expected, the increase in pSTAT1 activation paralleled the observed increase in pJAK2+ CD4+ T cells (Figure  4C). In CB, only 1.3% (median; range: 0.0–10.7%) CD4+ T cells activated pSTAT1 upon IFN-γ stimula- tion (Figure 4D). Activation of pSTAT1 was already increased by 1–3  months of age (median: 26.1%) and by 4–6  months pSTAT1+CD4+ T cell frequencies were detected in about 40% CD4+ T cells (Figure 4D); representative histograms are shown in Figure S1 in Supplementary Material. Although there was a significant positive correlation between increasing infant age and higher frequencies of pSTAT1+CD4+ T cells, this relationship IL-4 Signaling Ability of Infant CD4+ T Cells As the Th2-bias of infant CD4+ T cells is well documented, we hypothesized that CB CD4+ T cells would activate the JAK/ STAT signaling pathway in response to IL-4 as effectively as adult CD4+ T cells. Upon binding of IL-4 to its receptor, JAK1 and JAK3 will be activated by engaging the IL-4Rα or the common γ chain, respectively. As the common γ chain is also part of the IL-2 receptor, we chose to measure JAK3 activation to maximize the number of parameters that could be analyzed considering the small sample volumes available. The limited blood volumes for 10- to 14-month-old infants also required that we pooled PBMCs from multiple infants (pool 1: n = 9; pool 2: n = 5) to enrich CD4+ T cells for Western blot analysis.h The protein levels of total JAK3 were comparable between CD4+ T cells isolated from CB, 1-year olds or adults (Figure 2A). Statistical Analysis However, as naïve cells represented the majority of infant CD4+ T cells, the reduced pSTAT6 activation by infant memory cells was masked in the total CD4+ T cell population. To test for the translocation of activated pSTAT6 to the nucleus, a critical event for downstream gene induction, we utilized Amnis® ImageStreamX analysis. CB and adult CD4+ T cells showed similar expression of unphosphorylated STAT6 (Figure 3). Upon IL-4-stimulation, activated pSTAT6 was clearly detectable in the nucleus of CB CD4+ T cells, but the fluorescence intensity was slightly stronger in CD4+ T cells of an 8-month-old infant and in adult CD4+ T cells (Figure 3). The expression levels of IL-2Rβ did not differ between infant CD4+ T cells at birth and or at 1 year, or between CD4+ T cells of 1-year-old infants and adults. We did not measure the expression of the common γ-chain (see above).hf The results highlight that naïve, central memory, and effector memory CD4+ T cells show age-dependent differences in cytokine receptor expression that are likely to influence the functional capacity of these CD4+ T cell subsets. Frontiers in Pediatrics  |  www.frontiersin.org Age-Dependent Increase in IFN-γ-Induced JAK/STAT Signaling With the caveat that exact frequencies of pJAK3+CD4+ T cells could not be quantified by Western blot, CD4+ T cells isolated from CB or 1-year-old infants were equally able to activate pJAK3 in response to IL-4 (Figures 2B,C). Thus, densitometry analysis showed no statistically significant differences in the increase of median pJAK3 densities in IL-4 stimulated compared to February 2017  |  Volume 5  |  Article 15 8 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. A D B C Figure 2 | JAK/STAT activation in infant CD4+ T cells in response to IL-4. (A) Representative Western blot gel for total JAK3 (upper band; empty white arrow) and the β-actin control (lower band, empty arrow head) using CD4+ T cell protein lysates from cord blood samples, 10- to 14-month-old infants and adults stimulated with IL-4 or IL-2 in comparison to media (M) only. (B) Representative Western blot showing phosphorylated JAK3 (pJAK; black arrow). (C) Western blot gels were analyzed by densitometry to determine relative changes in JAK3 activation in media or IL-4 stimulated CD4+ T cells. (D) The graphs show the frequencies of pSTAT6+ cells in total CD4+ T cells and in naïve [T(N)], central memory [T(CM)], and effector memory/effector [T(EM/Eff)] CD4+ T cell populations of at birth (n = 33), infants aged 4–6 (n = 6) and 10–14 (n = 11) months. The gray area in each graph represents the median frequencies (middle line) of pSTAT6+ CD4+ T cells in adults (n = 25), with the lower and upper line corresponding to the 25th and 75th percentile. Each symbol represents an individual subject, with the exception of (B): the samples for 10- to 14-month-old infants represent pools of nine or five subjects each. Statistical significant differences between two age groups were determined by non-parametric Mann–Whitney test. C A B D D D Figure 2 | JAK/STAT activation in infant CD4+ T cells in response to IL-4. (A) Representative Western blot gel for total JAK3 (upper band; empty white arrow) and the β-actin control (lower band, empty arrow head) using CD4+ T cell protein lysates from cord blood samples, 10- to 14-month-old infants and adults stimulated with IL-4 or IL-2 in comparison to media (M) only. (B) Representative Western blot showing phosphorylated JAK3 (pJAK; black arrow). (C) Western blot gels were analyzed by densitometry to determine relative changes in JAK3 activation in media or IL-4 stimulated CD4+ T cells. Age-Dependent Increase in IFN-γ-Induced JAK/STAT Signaling (D) The graphs show the frequencies of pSTAT6+ cells in total CD4+ T cells and in naïve [T(N)], central memory [T(CM)], and effector memory/effector [T(EM/Eff)] CD4+ T cell populations of at birth (n = 33), infants aged 4–6 (n = 6) and 10–14 (n = 11) months. The gray area in each graph represents the median frequencies (middle line) of pSTAT6+ CD4+ T cells in adults (n = 25), with the lower and upper line corresponding to the 25th and 75th percentile. Each symbol represents an individual subject, with the exception of (B): the samples for 10- to 14-month-old infants represent pools of nine or five subjects each. Statistical significant differences between two age groups were determined by non-parametric Mann–Whitney test. both IL-4 and IFN-γ-induced JAK/STAT signaling, we observed a more than 10-fold increase in the ratio of pSTAT1:pSTAT6 from 0.04 (median; range: 0.0–0.2) at birth to 0.5 (range: 0.4–0.9) at 1 year (Figure 5B). Whether or not these findings have direct implications for CD4+ Th cell differentiation, was outside the scope of the current study. To further probe CB CD4+ T cells for reduced signaling function in response to Th1-promoting cytokines, we tested whether STAT4 phosphorylation after in vitro IL-12 stimulation was also reduced. Indeed, only 0.42% (median value; range: 0.0–4.2%) CB CD4+ T cells responded with STAT4 phosphorylation (Figure 6A). Despite higher IL-12 signaling ability of CD4+ T cells in 1-year-old infants, median frequencies of pSTAT4+CD4+ T cells still accounted for <1% in 10- to 14-month-old infants (range: 0.1–1.7%) and remained significantly lower compared to pSTAT4+ frequencies in adult did not appear to be linear (Figure 4D). In fact, IFN-γ-induced pSTAT1 activation seemed to plateau at about 6 months of age (Figure 4D; Table S1 and Figure S2 in Supplementary Material). As was shown for total STAT6 expression, unphosphorylated STAT1 could be detected in both CB and adult CD4+ T cells (Figure  4E). However, consistent with the age-dependent increase in pSTAT1 activation, activated pSTAT1 was localized mainly in the cytoplasm in CB CD4+ T cells, whereas in samples from 6-month-old infants pSTAT1 was clearly detectable in the nucleus (Figure 4E). Compared to only a 1.4-fold increase in median frequencies of pSTAT6+CD4+ T cells from birth (63.8%) to 1 year (90.6%), median frequencies of pSTAT1+CD4+ T cells increased 30-fold in the first year (birth: 1.3%; 1 year: 38.0%) (Figure 5A). Frontiers in Pediatrics  |  www.frontiersin.org Age-Dependent Increase in IFN-γ-Induced JAK/STAT Signaling Furthermore, in samples for which sufficient blood volume was available to test February 2017  |  Volume 5  |  Article 15 Frontiers in Pediatrics  |  www.frontiersin.org 9 dela Peña-Ponce et al. Infant CD4+ T Cell Cytokine Signaling Figure 3 | Amnis®-ImageStreamX analysis of STAT6 activation. The images are representative examples of whole blood samples that were untreated (left panels) or stimulated with IL-4 for 15 min (right panels) and then stained and analyzed for total (unphosphorylated) STAT6 and activated pSTAT6. Each image row shows a single cell labeled with CD3 (blue), CD4 (pink), unphosphorylated STAT 6 (yellow), or phosphorylated STAT6 (pSTAT6; green). The first two rows represent examples of cord blood (CB) stained with STAT 6 or pSTAT6; followed by an 8-month old infant blood sample, and one adult donor blood. Channel 1 (“Ch1”) shows the brightfield image of the cell in relation to the 10-μm size marker. Figure 3 | Amnis®-ImageStreamX analysis of STAT6 activation. The images are representative examples of whole blood samples that were untreated (left panels) or stimulated with IL-4 for 15 min (right panels) and then stained and analyzed for total (unphosphorylated) STAT6 and activated pSTAT6. Each image row shows a single cell labeled with CD3 (blue), CD4 (pink), unphosphorylated STAT 6 (yellow), or phosphorylated STAT6 (pSTAT6; green). The first two rows represent examples of cord blood (CB) stained with STAT 6 or pSTAT6; followed by an 8-month old infant blood sample, and one adult donor blood. Channel 1 (“Ch1”) shows the brightfield image of the cell in relation to the 10-μm size marker. The activation of pJAK3 by IL-2 appeared to be comparable in CD4+ T cells from CB, blood of 1-year-old infants and adults (Figure 7A; see Figure 2A for controls). However, because we could not quantitate the exact number of pJAK3+CD4+ T cells by Western blot (Figure 7A), we measured the activation of the IL-2 specific transcription factor STAT5, downstream of JAK3. This analysis showed that frequencies of pSTAT5+CD4+ T cells were positively correlated (p  <  0.0001) with increasing age between birth and 1 year (Figure 7B). However, even at 1 year, median pSTAT5+CD4+ T cell frequencies (36%; range: 25–51%) remained significantly lower compared adults (median: 69%; range: 44–80%) (Figure 6B). CD4+ T cells (median: 3.3%; range: 0.2–18.2%) (Figure  6A). Consistent with this result, few CD4+ T cells with nuclear pSTAT4 localization could be detected in 1-year-old infants (Figure 6B). Age-Dependent Increase in IFN-γ-Induced JAK/STAT Signaling In addition, it appeared that the nuclear translocation might have been less efficient compared to nuclear localization of pSTAT6 or pSTAT1 in CD4+ T cells of 1-year-old infants stimulated with IL-4 or IFN-γ, respectively (Figure 6B). However, the tools for a more quantitative protein analysis of the Amnis®ImageStreamX data were not available. Considering that the IL-12Rβ2 chain is not expressed on naive CD4+ T cells (59, 60), we compared in pSTAT4+CD4+ T cell frequencies in memory CD4+ T cells of different age groups. Despite the low frequencies of CD4+ T cells that can be phenotypically characterized as T(CM) or T(EM/Eff) CD4+ T cells in CB, about 1 and 4%, respectively, could activate pSTAT4. By 1 year, the functional capacity of T(EM/Eff) CD4+ T cells to signal in response to IL12 was equivalent to adult T(EM/Eff) CD4+ T cells (Figures 6C,D), but the low frequencies of differ- entiated infant CD4+ T cells likely prevented the detection of this response within the total CD4+ T cell population (Figure 6A). Despite higher frequencies of memory CD4+ T cells in adults, IL-12Rβ2 expression is relatively low within the total CD4+ T cell population (Figure S3 in Supplementary Material). In contrast, NK cells (CD3−CD14−CD19−CD56+), even in infants, show more pronounced expression of IL-12Rβ2 (Figure S3 in Supplementary Material), with IL-12Rβ2 expression being highest on CD16+ NK cell subsets (Figure S3 in Supplementary Material). To determine whether this effect was specific for IL-2, we tested whether JAK/STAT signaling to the related common γ-chain cytokine IL-7, that also promotes proliferation, was impaired in infants as well. Compared to <1% median fre- quencies of pSTAT5+ CD4+ T cells activated by IL-2 in CB, IL-7 induced about 19% (range: 0.3–73.2%) pSTAT5+CD4+ T cells (Figure 7C). IL-7-induced frequencies of pSTAT5+CD4+ T cells increased to 72% (range: 62.1–87.3%) by 4–6 months and reached 90% (range: 45.2–95.5%) by 1 year, levels com- parable to those achieved in adults (median: 93.3%; range: 83.0–97.6%) (Figure 7C). The differential signaling capacity of infant CD4+ T cells to IL-2 or IL-7 was further supported by image analysis of blood samples from 6-month-old infants (Figure 7D). Although we could consistently detect pSTAT5 in the nucleus of samples stimulated with IL-7, activation and nuclear translocation of pSTAT5 in response to IL-2 was ­variable (Figure 7E).hf JAK/STAT Signaling by Infant CD4+ T Cells in Response to the γ Chain Cytokines IL-2 and IL-7 Median frequencies of adult (n = 12) pJAK2+CD4+ T cells with the 25th and 75th percentile are indicated by the gray shaded area. (B) Representative pJAK2 histograms from a CB sample and an adult blood donor. Note that baseline pJAK2 levels are similar in CB and adult blood, and in IFN-γ stimulated samples, the FMO controls also do not differ between CB and adult blood. In contrast, there is an induction of pJAK2 in adult, but notCB, CD4+ T cells after IFN-γ stimulation. (C) The association between pJAK2+ and STAT1+ CD4+ T cells in relation to age through 14 months. Note that the analysis only included infant samples that had sufficient volume to measure both parameters. (D) The associations between pSTAT1+CD4+ T cells and age through 14 months. Correlations were determined by Spearman rank test. (E) Representative examples of CB, 6-month-old infant and adult blood samples analyzed by Amnis®- ImageStreamX for the nuclear localization of STAT1 and pSTAT1 in unstimulated samples (left panels) or after IFN-γ stimulation (right panels). The legend is as described in Figure 3. Figure 4 | IFN-γ-induced JAK/STAT signaling. (A) The percentages of pJAK2+CD4+ T cells in cord blood (CB, n = 10), 4- to 6-month-old (n = 3), and 10- to 14-month-old (n = 10) infants. Median frequencies of adult (n = 12) pJAK2+CD4+ T cells with the 25th and 75th percentile are indicated by the gray shaded area. (B) Representative pJAK2 histograms from a CB sample and an adult blood donor. Note that baseline pJAK2 levels are similar in CB and adult blood, and in IFN-γ stimulated samples, the FMO controls also do not differ between CB and adult blood. In contrast, there is an induction of pJAK2 in adult, but notCB, CD4+ T cells after IFN-γ stimulation. (C) The association between pJAK2+ and STAT1+ CD4+ T cells in relation to age through 14 months. Note that the analysis only included infant samples that had sufficient volume to measure both parameters. (D) The associations between pSTAT1+CD4+ T cells and age through 14 months. Correlations were determined by Spearman rank test. (E) Representative examples of CB, 6-month-old infant and adult blood samples analyzed by Amnis®- ImageStreamX for the nuclear localization of STAT1 and pSTAT1 in unstimulated samples (left panels) or after IFN-γ stimulation (right panels). The legend is as described in Figure 3. JAK/STAT Signaling by Infant CD4+ T Cells in Response to the γ Chain Cytokines IL-2 and IL-7 The difference in IL-2 versus IL-7 signaling was mainly due to differential activation of naïve CD4+ T cells (Figures 8A,B). At 1  year, pSTAT5+ frequencies within the naïve CD4+ T cell population were still significantly lower (p < 0.0001) compared to adults after IL-2 stimulation, whereas IL-7-induced pSTAT5+ CD4+ T(N) cell frequencies were comparable between 1-year olds IL-2 is a key cytokine promoting the proliferation and expansion of CD4+ T cells. As pointed out above, IL-2 signals through the common γ chain of the IL-2R and activates the JAK3 kinase. February 2017  |  Volume 5  |  Article 15 Frontiers in Pediatrics  |  www.frontiersin.org 10 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. Figure 4 | IFN-γ-induced JAK/STAT signaling. (A) The percentages of pJAK2+CD4+ T cells in cord blood (CB, n = 10), 4- to 6-month-old (n = 3), and 10- to 14-month-old (n = 10) infants. Median frequencies of adult (n = 12) pJAK2+CD4+ T cells with the 25th and 75th percentile are indicated by the gray shaded area. (B) Representative pJAK2 histograms from a CB sample and an adult blood donor. Note that baseline pJAK2 levels are similar in CB and adult blood, and in IFN-γ stimulated samples, the FMO controls also do not differ between CB and adult blood. In contrast, there is an induction of pJAK2 in adult, but notCB, CD4+ T cells after IFN-γ stimulation. (C) The association between pJAK2+ and STAT1+ CD4+ T cells in relation to age through 14 months. Note that the analysis only included infant samples that had sufficient volume to measure both parameters. (D) The associations between pSTAT1+CD4+ T cells and age through 14 months. Correlations were determined by Spearman rank test. (E) Representative examples of CB, 6-month-old infant and adult blood samples analyzed by Amnis®- ImageStreamX for the nuclear localization of STAT1 and pSTAT1 in unstimulated samples (left panels) or after IFN-γ stimulation (right panels). The legend is as described in Figure 3. and adults (Figure 8) In T(CM) and T(EM/Eff) CD4+ T cells Consistent with the fact that CD4+ T cell differentiation into Figure 4 | IFN-γ-induced JAK/STAT signaling. (A) The percentages of pJAK2+CD4+ T cells in cord blood (CB, n = 10), 4- to 6-month-old (n = 3), and 10- to 14-month-old (n = 10) infants. JAK/STAT Signaling by Infant CD4+ T Cells in Response to the γ Chain Cytokines IL-2 and IL-7 Consistent with the fact that CD4+ T cell differentiation into memory T cells is associated with increased expression of CD25, the IL-2Rα, we noted an age-dependent positive correlation and adults (Figure 8). In T(CM) and T(EM/Eff) CD4+ T cells of 1-year-old infants, the activation of pSTAT5 remained below adult levels after both IL-2 and IL-7 stimulation (Figures 8A,B). February 2017  |  Volume 5  |  Article 15 Frontiers in Pediatrics  |  www.frontiersin.org 11 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. Figure 5 | Age-dependent changes in JAK/STAT signaling. (A) The relative increase (Δ) in median pSTAT6 or pSTAT1 activation in infant CD4+ T cells from birth to 1 year. (B) A positive correlation (Spearman rank test) between infant age and the pSTAT1:pSTAT6 ratio in infant blood samples that had sufficient blood volume to analyze both pSTAT1 and pSTAT6 activation in response to IFN-γ or IL-4 stimulation, respectively. Each symbol represents an individual infant; infants of different ages are represented by distinct color symbols as described in Figure 1. Figure 5 | Age-dependent changes in JAK/STAT signaling. (A) The relative increase (Δ) in median pSTAT6 or pSTAT1 activation in infant CD4+ T cells from birth to 1 year. (B) A positive correlation (Spearman rank test) between infant age and the pSTAT1:pSTAT6 ratio in infant blood samples that had sufficient blood volume to analyze both pSTAT1 and pSTAT6 activation in response to IFN-γ or IL-4 stimulation, respectively. Each symbol represents an individual infant; infants of different ages are represented by distinct color symbols as described in Figure 1. between IL-2-induced pSTAT5 activation and frequencies IL-2Rα+CD4+ T cells (Figure 8C). the relative activation of the JAK/STAT signaling pathway by different cytokines have to consider these factors. Assuming that adult CD4+ T cells are fully functional, the activation of specific STAT molecules in response to distinct cytokines could be considered as 100% response rate. Applying this assumption and by expressing the activation of STAT molecules in infant CD4+ T cells as a function of STAT activation by the same cytokines in adult CD4+ T cells, we can demonstrate cytokine- dependent differences in JAK/STAT signaling function in infant CD4+ T cells (see Figure S4 in Supplementary Material). JAK/STAT Signaling by Infant CD4+ T Cells in Response to the γ Chain Cytokines IL-2 and IL-7 Among the cytokines evaluated, CD4+ T cells at birth showed relatively strong activation of the JAK/STAT signaling in response to IL-4 and also to IL-7, whereas the induction of the JAK/STAT path- way in response to IL-2, IFN-γ and IL-12 was less developed. The obvious question arising is which factors govern these signaling differences. We plan to define such factors in future studies; their evaluation was outside the scope of the current study as the first step was to determine how CD4+ T cell JAK/STAT signaling function changes from birth to 1 year in response to distinct cytokines. Frontiers in Pediatrics  |  www.frontiersin.org DISCUSSION To develop novel prevention and intervention strategies aimed at reducing neonatal and infant morbidity and mortality, we first need to understand how the immune system develops in the healthy infant. Toward this goal, the current study defined the temporal development of the JAK/STAT signaling function in human infant CD4+ T cells from birth to 1 year of age in response to specific cytokines. We focused on cytokines important in Th cell differentiation, because increased susceptibility to infec- tious diseases in infants has been associated with Th2-biased responses. Our results suggest that the maturation of the JAK/STAT signaling function in infant CD4+ T cells might be regulated by common and cytokine-specific factors. Regardless of the cytokine tested, the activation of cytokine-specific STAT mol- ecules was significantly lower in CB CD4+ T cells compared to 1-year-old infants and to adults. During the first year of life, there was a statistically significant age-dependent increase in STAT activation, with the most pronounced changes occurring within the first 6 months (Table S1 and Figure S2 in Supplementary Material). The overall magnitude and the kinetics of changes in JAK/STAT signaling function, however, were distinct for each cytokine. In addition, the ability to activate the JAK/STAT pathway varied dependent on the differentiation status of CD4+ T cells.f y The differences in the age-dependent increase in STAT acti- vation by distinct cytokines can be partially explained by the preponderance of naïve versus more differentiated CD4+ T cells in infants compared to adults. This was most evident in IL-12- mediated JAK/STAT signaling. Thus, although median frequen- cies of IL-12-induced pSTAT4+ CD4+ T cells within the naïve CD4+ T cell population of CB or blood from 1-year-old infants were significantly lower than those in adult CD4+ T cells, median frequencies of pSTAT4+CD4+ T(EM/Eff) cells were within the range of those induced in adult CD4+ T cells by IL-12 (see Figure 6). However, as the majority of CD4+ T cells at 1 year still represent naïve CD4+ T cells, this response by memory CD4+ T As different cytokines exert distinct functions, the activation of the JAK/STAT signaling pathway will likely be regulated in a cytokine-dependent manner, too. Therefore, comparisons in February 2017  |  Volume 5  |  Article 15 12 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. Figure 6 | IL-12-induced pSTAT4 activation. DISCUSSION (A) The increase in pSTAT4 activation after IL-12 stimulation of CD4+ T cells at birth (n = 18) and 1 year (n = 17); adult values (n = 27) are indicated by the gray shaded area with median, 25th, and 75th percentile. (B) Representative composite images of CD4+ T cells from 6-month-old infants stimulated with media only (top row), or with (bottom row) IL-4, IFN-γ, or IL-12 and analyzed by Amnis®-ImageStreamX for the nuclear localization of their relevant transcription factor. Note the distinct nuclear localization of pSTAT6 and pSTAT1 that fills almost the whole nucleus in comparison to only partial pSTAT4 nuclear localization. (C) is analogous to (A), but shows the induction of pSTAT4 in T(N), T(CM), and T(EM/Eff) CD4+ T cells. The legend and the statistical comparisons are as described in Figures 1 and 2. (D) The relative frequencies of naïve [CD27+, T(N)], central memory [CD27low, T(CM)], and effector memory/effector [CD27−, T(EM/Eff)] CD4+ T cell populations of a representative adult and 1-year-old infant and the corresponding histograms of pSTAT4 activation after IL-12 stimulation (blue). pSTAT4 frequencies in unstimulated controls are shown by orange histograms. Figure 6 | IL-12-induced pSTAT4 activation. (A) The increase in pSTAT4 activation after IL-12 stimulation of CD4+ T cells at birth (n = 18) and 1 year (n = 17); adult values (n = 27) are indicated by the gray shaded area with median, 25th, and 75th percentile. (B) Representative composite images of CD4+ T cells from 6-month-old infants stimulated with media only (top row), or with (bottom row) IL-4, IFN-γ, or IL-12 and analyzed by Amnis®-ImageStreamX for the nuclear localization of their relevant transcription factor. Note the distinct nuclear localization of pSTAT6 and pSTAT1 that fills almost the whole nucleus in comparison to only partial pSTAT4 nuclear localization. (C) is analogous to (A), but shows the induction of pSTAT4 in T(N), T(CM), and T(EM/Eff) CD4+ T cells. The legend and the statistical comparisons are as described in Figures 1 and 2. (D) The relative frequencies of naïve [CD27+, T(N)], central memory [CD27low, T(CM)], and effector memory/effector [CD27−, T(EM/Eff)] CD4+ T cell populations of a representative adult and 1-year-old infant and the corresponding histograms of pSTAT4 activation after IL-12 stimulation (blue). pSTAT4 frequencies in unstimulated controls are shown by orange histograms. February 2017  |  Volume 5  |  Article 15 Frontiers in Pediatrics  |  www.frontiersin.org 13 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. DISCUSSION The response of CD4+ T cells to IL-2 was more variable with some cells showing pSTAT5 activation and nuclear localization (left images), whereas other cells remained unresponsive, indicated by lack of nuclear staining with pSTAT5 (right images). In contrast, the majority of CD4+ T cells showed pSTAT5 activation and nuclear translocation after IL-7 stimulation. of IL-2Rα+ and pSTAT5+ CD4+ T cell frequencies from birth to 1 year of age. cells was masked when only the total CD4+ T cell population was analyzed. These results emphasize the importance of comparing functional responses in relevant T cell subpopulations.f It should be emphasized though that the phenotypic charac- terization of CD4+ T cells as naïve or memory T cell was not suf- ficient to explain differences between CD4+ T cells in CB or blood samples collected from 1-year-old infants or adults. Again, this can be best illustrated when we express the activation of STAT molecules in naïve or memory infant CD4+ T cells as percent of adult CD4+ T cell responses in the same populations (Figure S5 in Supplementary Material). Thus, additional factors likely contrib- ute to age-dependent changes in JAK/STAT signaling function between infant and adult CD4+ T cells. Naïve and memory CD4+ T cells differ in the expression of specific cytokine receptor chains. Yet, the formation of the fully assembled cytokine receptor, which consists of multiple distinct chains, is required to induce the JAK/STAT signaling pathway. Thus, the differences in pSTAT4 activation between naïve and memory CD4+ T cells can be explained, at least partially, by the lack of the IL-12Rβ2 chain on naive CD4+ T cells (59, 60). Similarly, the differential activation of pSTAT5 by IL-2 and IL-7 was likely the result of distinct expression levels of the IL-2Rα and the IL-7Rα on naïve and memory CD4+ T cells. The IL-7Rα is highly expressed on recent thymic emigrants that are abundant in the naïve CD4+ T cell population in CB (61). In contrast, naïve CD4+ T cells lack the expression of the IL-2Rα chain and can therefore only form the intermediate affinity IL-2R consisting of the IL-2Rβ and the common γ-chain. As the number of activated and differentiated CD4+ T cells increases, the high affinity IL-2R complex (IL-2Rαβγ) can be formed and pSTAT5 activation will increase concurrently. DISCUSSION Figure 7 | Differential activation of pSTAT5 by IL-2 and IL-7. (A) Western blot analysis of pJAK3 activation as described in Figure 2C. (B) The age-dependent change in pSTAT5+ CD4+T cells from birth to 14 months of age with Spearman rank correlation values. Adult values are shown in comparison and were compared to 10- to 14-month-old infants by Mann–Whitney test. (C) Frequencies of pSTAT5+ CD4+T cells in response to in vitro IL-7 stimulation are shown for cord blood (n = 11), 4- to 6-month-old (n = 3), and 10- to 14-month-old (n = 7) infants. The gray area indicates median frequencies of adult pSTAT5+ CD4+T (n = 17) with borders representing the 25th and 75th percentiles. Each symbol represents an individual subject. Horizontal lines represent median values. Each age group is assigned a different symbol as described in Figure 1; Mann–Whitney test was used for between group comparisons. (D,E) Representative composite images of CD4+ T cells from 6-month-old infants stimulated with IL-7 (D) or IL-2 (E) and analyzed by Amnis®-ImageStreamX. The response of CD4+ T cells to IL-2 was more variable with some cells showing pSTAT5 activation and nuclear localization (left images), whereas other cells remained unresponsive, indicated by lack of nuclear staining with pSTAT5 (right images). In contrast, the majority of CD4+ T cells showed pSTAT5 activation and nuclear translocation after IL-7 stimulation. Figure 7 | Differential activation of pSTAT5 by IL-2 and IL-7. (A) Western blot analysis of pJAK3 activation as described in Figure 2C. (B) The age-dependent change in pSTAT5+ CD4+T cells from birth to 14 months of age with Spearman rank correlation values. Adult values are shown in comparison and were compared to 10- to 14-month-old infants by Mann–Whitney test. (C) Frequencies of pSTAT5+ CD4+T cells in response to in vitro IL-7 stimulation are shown for cord blood (n = 11), 4- to 6-month-old (n = 3), and 10- to 14-month-old (n = 7) infants. The gray area indicates median frequencies of adult pSTAT5+ CD4+T (n = 17) with borders representing the 25th and 75th percentiles. Each symbol represents an individual subject. Horizontal lines represent median values. Each age group is assigned a different symbol as described in Figure 1; Mann–Whitney test was used for between group comparisons. (D,E) Representative composite images of CD4+ T cells from 6-month-old infants stimulated with IL-7 (D) or IL-2 (E) and analyzed by Amnis®-ImageStreamX. Frontiers in Pediatrics  |  www.frontiersin.org DISCUSSION Indeed, we found a significant positive correlation between increasing frequencies While the current study examined JAK/STAT signaling in response to single cytokines only, in response to an in vivo stimu- lus, the cytokine milieu will be shaped by the interplay of multiple cytokines secreted by diverse cell populations. For example, IFN- γ, can promote the induction of the IL-12Rβ2 chain on naïve CD4+ T cells (59, 60). IL-2-induced pSTAT5 can transcriptionally regulate the expression of the IL-12Rβ and IL-4Rα (62, 63), and thereby could influence JAK/STAT signaling in response to IL-12 and IL-4. As our results show that IL-2-induced pSTAT5 activa- tion, even at 1 year of age, is significantly lower in infant compared February 2017  |  Volume 5  |  Article 15 Frontiers in Pediatrics  |  www.frontiersin.org 14 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. Figure 8 | CD4+ T cell differentiation in STAT5 activation. (A,B) The percentages of T(N), T(CM), and T(EM/Eff) CD4+ T cells that were activated pSTAT5 in response to IL-2 (A) or IL-7(B), respectively, in infant blood samples collected at birth and 1 year. Each symbol represents an individual infant; horizontal lines represent median values. The gray area indicates median frequencies of adult pSTAT5+ CD4+T cells (n = 17) with borders representing the 25th and 75th percentiles. (C) The correlation by Spearman rank test between pSTAT5+CD4+T cells and IL-2Rα+ CD4+ T cells in infant samples of different ages (colored symbols as explained in Figure 1). Note that we only included samples that were analyzed for both pSTAT5+ activation and IL-2Rα+ expression. Figure 8 | CD4+ T cell differentiation in STAT5 activation. (A,B) The percentages of T(N), T(CM), and T(EM/Eff) CD4+ T cells that were activated pSTAT5 in response to IL-2 (A) or IL-7(B), respectively, in infant blood samples collected at birth and 1 year. Each symbol represents an individual infant; horizontal lines represent median values. The gray area indicates median frequencies of adult pSTAT5+ CD4+T cells (n = 17) with borders representing the 25th and 75th percentiles. (C) The correlation by Spearman rank test between pSTAT5+CD4+T cells and IL-2Rα+ CD4+ T cells in infant samples of different ages (colored symbols as explained in Figure 1). Note that we only included samples that were analyzed for both pSTAT5+ activation and IL-2Rα+ expression. Figure 8 | CD4+ T cell differentiation in STAT5 activation. DISCUSSION (A,B) The percentages of T(N), T(CM), and T(EM/Eff) CD4+ T cells that were activated pSTAT5 in response to IL-2 (A) or IL-7(B), respectively, in infant blood samples collected at birth and 1 year. Each symbol represents an individual infant; horizontal lines represent median values. The gray area indicates median frequencies of adult pSTAT5+ CD4+T cells (n = 17) with borders representing the 25th and 75th percentiles. (C) The correlation by Spearman rank test between pSTAT5+CD4+T cells and IL-2Rα+ CD4+ T cells in infant samples of different ages (colored symbols as explained in Figure 1). Note that we only included samples that were analyzed for both pSTAT5+ activation and IL-2Rα+ expression. to adult CD4+ T cells, it is possible that reduced IL-2 signaling impedes the development of optimal Th1 responses in infants (Figure S4 in Supplementary Material). One could hypothesize that at birth IL-2-induced pSTAT5 would preferentially bind to the il4ra promoter because the ifnγ promoter of infant CD4+ T cells is hypermethylated (36, 37), but once the methylation status changes with age, pSTAT5 could also bind to the ifnγ and/or to the il12rβ2 promoters (Figure S4 in Supplementary Material). In fact, recent studies have provided evidence of age-dependent changes in epigenetic modifications (64, 65). statistically significant increase in the pSTAT1:pSTAT6 ratio from birth to 1 year in infant CD4+ T cells. However, to conclusively define the role of JAK/STAT signaling in Th cell differentiation, future studies should examine signaling events downstream of STAT activation, such as the interaction of specific STAT molecules with promoter regions of genes important in Th1 cell differentia- tion (e.g., tbx, il12rβ) and/or changes in epigenetic modification of these genes (Figure S4 in Supplementary Material). Furthermore, cytokine signals only represent one of the signals driving Th cell differentiation. Pathogen type, costimulatory signals by antigen- presenting cells, antigen concentration, and signals through the T cell receptor all influence CD4+ T cell differentiation [e.g., Ref. (19, 20, 66–78)].h i Some of our results suggest that the JAK/STAT signaling of infant CD4+ T cells might contribute to the Th2-biased response of infants. Frontiers in Pediatrics  |  www.frontiersin.org ETHICS STATEMENT Study subjects were recruited from the University of North Carolina, Chapel Hill (UNC-CH) and the NC Children’s Hospital. Cord blood samples from full-term infants were collected imme- diately after birth or obtained from the Carolinas Cord Blood Bank. Direct capillary or venous blood samples (0.5–3.0 mL) from infants aged 2 weeks to 14 months were collected by nursing or phlebotomy personnel during routine physician visits. The infant participants were recruited from the North Carolina population that consists of about 46% Caucasians, 46% African-Americans, and 8% other groups, and within the ethnic categories, 10% rep- resent Hispanics. Exclusion criteria included HIV-positive status of the mother, known in utero infections, treatment of the mother with immunosuppressive drugs, diagnosis of mother or child with immunosuppressive disorder, life-threatening malformations of the infant or life expectancy <6 months. Infant blood samples were also excluded if the infant had a bleeding disorder or had a chronic infection. The Virology, Immunology, and Microbiology Core of the UNC Center for AIDS Research provided blood samples from healthy adults. Age, sex, and race of the adult donors were unknown. The study was approved by the UNC-CH Institutional Review Board and informed parental consent was obtained. Institutional guidelines strictly adhere to the World Medical Association’s Declaration of Helsinki. t g p The above conclusion that major changes in JAK/STAT signal- ing function occurred within the first 6 months does not rule out, however, that changes occurred very early, maybe even within the first month of life. Although we were able to obtain a relatively large number (n = 169) of healthy infant blood samples, with about equal numbers of CB samples (n = 70) and blood from 1-year-old infants (n  =  64), blood samples from infants age 0.5 month to 1 year amounted to only about half this number (n = 35). Furthermore, <5% of all infant samples represented longitudinal samples. The sample size in our study, however, is comparable to other studies of immune ontogeny. For example, in three different studies examining the ontogeny of Toll-like receptor-mediated cytokine responses in human infants was defined using longitudinal samples from 35 (87), 30 (88), or 28 infants (89). Only two of these three studies included samples within the first year of life, with one of them sampling at 3-month intervals (88), and the other also being able to obtain samples at 2 and 6 weeks of age (89). ETHICS STATEMENT The difficulties in obtaining samples from healthy infants and blood volume restrictions that limit the analy- sis scope of such samples constitute a main and common problem for investigators and have severely hampered the progress in our understanding of immune maturation. Despite our limitations in sample size, and by applying conservative non-parametric statistical analysis tools, we were able to, nonetheless, demon- strate broad stages of the temporal development of JAK/STAT signaling function in infant CD4+ T cells using. Furthermore, trends detected in the analysis of cross-sectional samples were consistent with age-dependent changes observed in longitudinal sample pairs (Figure S6 in Supplementary Material).if DISCUSSION We observed relatively strong JAK/STAT signaling responses to the Th2 cytokine IL-4 and poorer responses to the Th1-driving cytokines IFN-γ and IL-12 in CB, and there was a The fact that the most pronounced changes for all cytokines examined occurred within the first 6 months hint at a role of February 2017  |  Volume 5  |  Article 15 15 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. common developmental factors in the regulation and maturation of the JAK/STAT signaling function in infant CD4+ T cells. A key factor might be the establishment of the infant’s microbiome. The importance of the maternal and infant microbiome on infant health has become the subject of intense investigation in recent years [review examples; Ref. (79–83)]. The infant requires a qualitatively distinct response from adults to allow the transition from the relatively tolerogenic milieu in the uterus to exposure to multiple environmental and pathogenic stimuli after birth. Part of this early development is the establishment of the infant’s microbiome that may come at the cost of a more suppressive or Th2-prone response. In fact, a recent study demonstrated that arginase 2-producing CD71 cells suppressed immune activa- tion in response to host flora in neonates to allow colonization with normal flora (84). Similarly, the “layered immune system hypothesis” suggests that Tregs, induced to tolerate maternal or other antigens transmitted to the fetus in utero, may persist long after birth and mediate tolerogenic responses (85, 86).h study. The data highlight how little we still understand about the multiple factors that initiate, drive, and regulate CD4+ T cell responses in the early postnatal period and the complex interac- tions between these factors. A deeper insight into postnatal devel- opment of immune cells and their functions will be instrumental for the design of novel therapeutic interventions, will inform the timely implementation of novel pediatric vaccines, and thus aid in more effectively combating childhood diseases. AUTHOR CONTRIBUTIONS KDP, SP-W, and MP-P designed the study. JB and SP-W oversaw all clinical aspects of the study (e.g., IRB approval, consenting, sample collection). MP-P, J-R-N, RT, NC, and MM performed the experiments. MP-P, J-R-N, KM, MH, and KDP analyzed the data. MP-P, SP-W, KM, and KDP wrote the manuscript. ACKNOWLEDGMENTS We would like to thank our nurses Kristin Allyne and Cynthia Clark. Our special gratitude goes to the parents and guardians of the infants who participated in this study. We would also like to thank Kaylan Rao, Maggie Conner, and Reyad Queijan for technical assistance. In summary, the current study identified age-related differ- ences in cytokine signaling and established the time frame during which the JAK/STAT signaling function develops in infant CD4+ T cells in response to specific cytokines. These data provide the foundation for future studies identifying the molecular mecha- nisms that regulate JAK/STAT signaling in infant CD4+ T cells, including, but not limited to, epigenetic regulation (90), the role of miRNAs (91, 92), and the regulation of JAK kinases and STAT proteins by suppressors of cytokine signaling molecules (93, 94). These mechanistic studies were outside the scope of the current Frontiers in Pediatrics  |  www.frontiersin.org SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fped.2017.00015/ full#supplementary-material. Figure S1 | Age-dependent increase in pSTAT1 activation. Relative frequencies of naïve [CD27+, T(N)], central memory [CD27low, T(CM)], and effector memory/effector [CD27−, T(EM/Eff)] CD4+ T cell populations of a representative cord blood sample (top row), 1-year-old infant (middle row), and an adult (bottom row) and the corresponding histograms of pSTAT1 activation after IFN-γ stimulation (blue) are shown. pSTAT1 frequencies in unstimulated controls are shown by orange histograms. Note that y-axis scales differ between the age groups. Figure S2 | Correlation between infant age and STAT activation. From left to right, the correlations between infant age (x-axis) and STAT phosphorylation in response to IL-4 (pSTAT6), IFN-γ (pSTAT1), and IL-2 (pSTAT5) are graphed, with each symbol representing an individual infant and green lines representing LOESS curves with a 0.5 smooth factor. These graphs are meant to complement Table S1 in Supplementary Material and demonstrate that pSTAT6 and pSTAT1 activation plateau by about 6 months, whereas there is still a further, although slight, increase in pSTAT5 activation from 6 months to 1 year. Figure S5 | Changes in STAT activation in infant CD4+ T cell subpopulation from birth to 1 year. Analogous to Figure S4 in Supplementary Material, median frequencies of pSTAT positive (colored circles) and negative (white) naïve [T(N)], central memory [T(CM)], and effector memory/ effector [T(EM/ Eff)] CD4+ T cells are expressed at fraction of median frequencies of adult CD4+ T cell subpopulations at birth (CB, cord blood) and 1 year of age (10–14 months). Each square consists of 10 × 10 circles, with each circle presenting 1%. The CD4+ T cell subpopulations are listed on the column top and the cytokine with its relevant transcription factor are listed on the left. The color coding is as described in Figure S4 in Supplementary Material. Figure S3 | Expression of IL-12Rb2 on CD4+ T cells. (A) CD3+CD4+ T cells of a representative adult donor were stained for CD3+CD4+ T cells expression immediately after cell isolation (grey histogram) and after incubation for 48 h with a cytokine cocktail consisting of IL-2, IL-12, and IL-15. Note the upregulation of the IL-12Rb2 upon cytokine stimulation (blue histogram) compared to CD3+CD4+ T cells cultured in media only (orange histogram). (B) To validate the staining, CD3-negative cells of the same donor were analyzed for IL-12Rβ2 expression on NK cells, defined as CD3-CD14−CD20−CD56+. Figure S4 | Summary of age-related differences in STAT signaling and conceptual interpretation. Median frequencies of pSTAT positive Figure S4 | Summary of age-related differences in STAT signaling and conceptual interpretation. Median frequencies of pSTAT positive FUNDING The complex interplay between these factors and other immune cells was outside the scope of the current study. (colored circles) and negative (white) total CD4+ T cells expressed at fraction of median frequencies of adult CD4+ T cells, as measured at birth (CB, cord blood), 4–6 and 10–14 months. Each square consists of 10 × 10 circles, with each circle presenting 1%. The age is listed on the left, the cytokine and the cytokine-specific transcription factor are listed on the top of each column. IL-4-induced pSTAT6 frequencies are shown in red. The strong Th2 bias at birth is represented with the red bar/arrow on the left that is balanced with increasing age by an increasing Th1 response (green arrow on right). The most pronounced changes in STAT activation occurred in the time from birth to 6 months; in the subsequent time period from 6 months to 1 year only minor changes were noted. This is indicated by the dashed line. We propose that the establishment of the microbiome was a key factor driving these changes. In addition, other developmentally regulated factors, such as decreasing methylation levels on the ifnγ promoter region, likely contributed to an increase in STAT signaling. Increasing exposure to danger signals (pathogens, environmental stimuli, pediatric vaccines, etc.) drive the activation of CD4+ T cells and results in a changing ratio of naïve and memory CD4+ T cells. Activated and more differentiated CD4+ T cells express higher levels of certain cytokine receptors (e.g., IL-2Rα, IL-12Rβ) allowing for more effective pSTAT5 activation and signaling. We propose that at birth pSTAT5 may predominantly activate the il4 and il4ra genes, while with increasing age pSTAT5 will be able to activate to also activate ifnγ and il12rβ genes and thereby, promote Th1 responses. This figure is meant to summarize our data and present some factors that may have contributed to the observed age-related changes in JAK/ STAT signaling function and present a potential role of IL-2 in driving Th1 development in infants. Other factors are likely important in postnatal development of JAK/STAT signaling; this figure is by no means exhaustive. The complex interplay between these factors and other immune cells was outside the scope of the current study. Immunology, the UNC Chapel Hill Lineberger Cancer Center (NCI Center Core Support Grant P30 CA06086), and the Office of the Director, National Institutes of Health, award number 1S10 OD017984-01A1. SUPPLEMENTARY MATERIAL Note that IL-12Rβ2 expression, in contrast to the low expression on CD3+CD4+ T cells, was detectable on the majority of NK cells, even immediately after cell isolation. (C) The expression of the IL-12Rβ2 on CD3+CD4+ T cells (left histogram) and NK cells (right histogram) of a representative cord blood sample. (D) IL-12Rβ2 expression differs among NK cell subsets (adult donor example). Figure S6 | Age-dependent changes in STAT activation in longitudinal infant blood samples. (A) The frequencies of pSTAT6, pSTAT1, and pSTAT5+ CD4+ T cells after stimulation of longitudinal samples from the same infant with IL-4, IFN-γ, or IL-2, respectively. Samples from the same infant are represented by the same symbol and longitudinal data points are connected by a black line. (B) Representative histograms of samples shown in panel (A) are depicted. FUNDING Funding was provided by the NIH/NIAID 1R01 AI100067 to KDP. Studies were supported by the Center for AIDS Research (NIH grant 2 P30 AI050410) and by the UNC Flow Cytometry Core that is supported by the Department of Microbiology and February 2017  |  Volume 5  |  Article 15 16 Infant CD4+ T Cell Cytokine Signaling dela Peña-Ponce et al. (colored circles) and negative (white) total CD4+ T cells expressed at fraction of median frequencies of adult CD4+ T cells, as measured at birth (CB, cord blood), 4–6 and 10–14 months. Each square consists of 10 × 10 circles, with each circle presenting 1%. The age is listed on the left, the cytokine and the cytokine-specific transcription factor are listed on the top of each column. IL-4-induced pSTAT6 frequencies are shown in red. The strong Th2 bias at birth is represented with the red bar/arrow on the left that is balanced with increasing age by an increasing Th1 response (green arrow on right). The most pronounced changes in STAT activation occurred in the time from birth to 6 months; in the subsequent time period from 6 months to 1 year only minor changes were noted. This is indicated by the dashed line. We propose that the establishment of the microbiome was a key factor driving these changes. In addition, other developmentally regulated factors, such as decreasing methylation levels on the ifnγ promoter region, likely contributed to an increase in STAT signaling. Increasing exposure to danger signals (pathogens, environmental stimuli, pediatric vaccines, etc.) drive the activation of CD4+ T cells and results in a changing ratio of naïve and memory CD4+ T cells. Activated and more differentiated CD4+ T cells express higher levels of certain cytokine receptors (e.g., IL-2Rα, IL-12Rβ) allowing for more effective pSTAT5 activation and signaling. We propose that at birth pSTAT5 may predominantly activate the il4 and il4ra genes, while with increasing age pSTAT5 will be able to activate to also activate ifnγ and il12rβ genes and thereby, promote Th1 responses. This figure is meant to summarize our data and present some factors that may have contributed to the observed age-related changes in JAK/ STAT signaling function and present a potential role of IL-2 in driving Th1 development in infants. Other factors are likely important in postnatal development of JAK/STAT signaling; this figure is by no means exhaustive. REFERENCES 6. Le J, Prensky W, Yip YK, Chang Z, Hoffman T, Stevenson HC, et al. Activation of human monocyte cytotoxicity by natural and recombinant immune inter- feron. J Immunol (1983) 131:2821–6. 6. 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Very rare, exclusive, hadronic decays in QCD factorization
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© 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/). ⋆e-mail: m.koenig@uni-mainz.de Talk based on work in collaboration with Stefan Alte, Yuval Grossman and Matthias Neubert [1–4]. d on work in collaboration with Stefan Alte, Yuval Grossman and Matthias Neubert [1–4]. d on work in collaboration with Stefan Alte, Yuval Grossman and Matthias Neubert [1–4]. , 129 EPJ Web of Conferences QCD@Work 2016 00014 (2016) , 129 EPJ Web of Conferences QCD@Work 2016 00014 (2016) DOI: 10.1051/ 129 epjconf/2016 00014 Very rare, exclusive, hadronic decays in QCD factorization Matthias König1,⋆ 1Mainz Institute for Theoretical Physics, JGU Mainz Abstract. We study exclusive hadronic decays of the electroweak bosons Z, W and h in the framework of QCD factorization. We show that the theory uncertainties in these channels are remarkably small compared to past applications of the QCD factorization framework. While the branching ratios are small, many of the modes are accessible at future colliders. The Higgs decays exhibit interesting dependences on the couplings due to the interferences of different diagram topologies, making the h →Vγ decays possible probes of the quark Yukawa couplings and the h →VZ decays probes of the coupling between the Higgs boson, a photon and a Z-boson. 2 QCD factorization In the framework of QCD factorization, the amplitude for a hard exclusive process can be written as a convolution of a hard scattering function with a hadronic function, where the result will formally be an expansion of the scale separation parameter λ ∼ΛQCD/μhard. While the original derivation of the factorization formula dates back to the early 1980s, it can be rephrased in the language of Soft- Collinear Effective Theory (SCET) [14–17]. The factorization formula for the decays of h/Z →MV the final state meson M and the gauge boson V can be written as: iA =  q  dx T q H(x, μ) φq M(x, μ) + O(λ) , (1) (1) where T q H(x, μ) is the hard-scattering kernel, φq M(x, μ) is the light-cone distribution amplitude (LCDA) and the sum runs over the active quark flavors. Power corrections of higher orders in λ are tiny in our case due to the high scale μhard. The LCDAs are expanded in the set of Gegenbauer polynomials The LCDAs are expanded in the set of Gegenbauer polynomials φq M(x, μ) = 6x¯x ⎡⎢⎢⎢⎢⎢⎢⎣1 +  n=2,4,... aM,q n (μ)C(3/2) n (2x −1) ⎤⎥⎥⎥⎥⎥⎥⎦, (2) (2) where aM,q n (μ) are the scale-dependent Gegenbauer moments [9, 13, 18–21]. By evolving these pa- rameters from the hadronic scale to the high scale using the renormalization group, large logarithms of the form αs log λ are resummed to all orders. It is noteworthy that the evolution to an arbitrarily high scale lets all moments aM,q n vanish, decreasing the sensitivity of our prediction to these poorly-known hadronic parameters. 1 Introduction The Standard Model (SM) of particle physics is in remarkable agreement with observation and the discovery of the Higgs boson in 2012 [5, 6] is arguably the biggest triumph of particle physics in recent times. Still many questions remain unanswered and thus the SM has to be extended. When doing so, the Higgs sector is usually modified, making it a crucial experimental task to measure the parameters in this sector as accurately as possible. The exclusive hadronic decays of the form h →Mγ, h →MZ and h →MW can be used to probe several different couplings of the Higgs boson to the SM fermions and gauge bosons [2, 7, 8]. Even within the SM, particle physics still faces the challenge of obtaining rigorous control over non-perturbative effects in QCD at low energy scales. The framework of QCD factorization is a well- established approach to hard exclusive processes with indiviual final state hadrons [9–13]. Within this framework, amplitudes are given as convolutions of hard-scattering functions with non-local hadronic matrix elements, which encode the non-perturbative physics at the low scale. The amplitudes will formally be given as expansions in the ratio of the two scales. Past applications struggled with the fact that this factorization scale was not high enough for power corrections to be neglected and that it was difficult to disentangle these uncertainties from those in the poorly-known hadronic input parameters. In our case, the scale is set by the decaying heavy boson and power corrections are well under control. The decays Z →Mγ and W →Mγ can be used to test that factorization approach in a theoretically clean environment [1, 3]. DOI: 10.1051/ 129 epjconf/2016 00014 , 129 EPJ Web of Conferences QCD@Work 2016 00014 (2016) , 129 EPJ Web of Conferences 00014 (2016) QCD@Work 2016 Z γ M Z γ M Z γ M Z γ M Z γ M Figure 1. A subset of the diagrams contributing to Z →Mγ at O(αs). Additional mirrored diagrams exist. Figure 1. A subset of the diagrams contributing to Z →Mγ at O(αs). Additional mirrored diagrams exist. 3 Radiative hadronic decays of Z-bosons Decay mode Branching ratio Z0 →π0γ (9.80 + 0.09 −0.14 μ ± 0.03 f ± 1.02φ) · 10−12 Z0 →ηγ (2.36 + 0.02 −0.04 μ ± 1.19 f ± 0.04φ) · 10−10 Z0 →η′γ (6.68 + 0.08 −0.11 μ ± 0.49f ± 0.12φ) · 10−9 Z0 →ρ0γ (4.19 + 0.04 −0.06 μ ± 0.16f ± 0.44φ) · 10−9 Z0 →ωγ (2.89 + 0.03 −0.05 μ ± 0.15f ± 0.38φ) · 10−8 Z0 →φγ (8.63 + 0.08 −0.13 μ ± 0.41f ± 0.92φ) · 10−9 Z0 →J/ψ γ (8.02 + 0.14 −0.15 μ ± 0.20f + 0.39 −0.36 φ) · 10−8 Z0 →Υ(1S ) γ (5.39 + 0.10 −0.10 μ ± 0.08f + 0.11 −0.08 φ) · 10−8 Z0 →Υ(4S ) γ (1.22 + 0.02 −0.02 μ ± 0.13f + 0.02 −0.02 φ) · 10−8 Z0 →Υ(nS ) γ (9.96 + 0.18 −0.19 μ ± 0.09f + 0.20 −0.15 φ) · 10−8 where Q(′) are combinations of the meson decay constants and the quark couplings to the Z-boson and the photon. Evaluating diagrams like the ones shown in figure 1, we find the hard scattering coefficients: where Q(′) are combinations of the meson decay constants and the quark couplings to the Z-boson and the photon. Evaluating diagrams like the ones shown in figure 1, we find the hard scattering coefficients: C(±) n (mV, μ) = 1 + CFαs(μ) 4π c(±) n mV μ + O(α2 s) , with c(±) n mV μ = 2 (n + 1)(n + 2) −4Hn+1 + 3 ⎛⎜⎜⎜⎜⎝log m2 V μ2 −iπ ⎞⎟⎟⎟⎟⎠ + 4H2 n+1 −4(Hn+1 −1) ± 1 (n + 1)(n + 2) + 2 (n + 1)2(n + 2)2 −9 . (5) (5) This is the leading result in our expansion in the scale separation parameter λ, with subleading cor- rections starting at O(m2 V/m2 Z), that we can safely neglect. We present our phenomenological results in table 1. Similar results are obtained for the W →Mγ decays. We omit a discussion of these for briefness and refer the reader to ref. [1] for details. 3 Radiative hadronic decays of Z-bosons We can now write down the amplitude for the decays Z →Mγ. We will neglect the modes Z →η(′)γ here since the flavor-singlet components lead to non-trivial matching conditions that are too involved to explain here [3]. The amplitude of a Z-boson decaying into a meson M with momentum k and a photon with momentum q can be written as: iA = ± eg 2cW ⎡⎢⎢⎢⎢⎢⎣iϵμναβ kμqνεα Zε∗β γ k · q FM 1 −(ϵ⊥ Z · ϵ⊥∗ γ )FM 2 ⎤⎥⎥⎥⎥⎥⎦. (3) (3) We can express the form factors as sums over the Gegenbauer moments and the hard scattering coefficients: We can express the form factors as sums over the Gegenbauer moments and the hard scattering coefficients: FM 1 = QM ∞  n=0 C(+) 2n (mZ, μ)aM 2n(μ) , FM 2 = −Q′ M ∞  n=0 C(−) 2n+1(mZ, μ)aM 2n+1(μ) , (4) (4) 2 , 129 EPJ Web of Conferences QCD@Work 2016 00014 (2016) DOI: 10.1051/ 129 epjconf/2016 00014 Decay mode Branching ratio Z0 →π0γ (9.80 + 0.09 −0.14 μ ± 0.03 f ± 1.02φ) · 10−12 Z0 →ηγ (2.36 + 0.02 −0.04 μ ± 1.19 f ± 0.04φ) · 10−10 Z0 →η′γ (6.68 + 0.08 −0.11 μ ± 0.49f ± 0.12φ) · 10−9 Z0 →ρ0γ (4.19 + 0.04 −0.06 μ ± 0.16f ± 0.44φ) · 10−9 Z0 →ωγ (2.89 + 0.03 −0.05 μ ± 0.15f ± 0.38φ) · 10−8 Z0 →φγ (8.63 + 0.08 −0.13 μ ± 0.41f ± 0.92φ) · 10−9 Z0 →J/ψ γ (8.02 + 0.14 −0.15 μ ± 0.20f + 0.39 −0.36 φ) · 10−8 Z0 →Υ(1S ) γ (5.39 + 0.10 −0.10 μ ± 0.08f + 0.11 −0.08 φ) · 10−8 Z0 →Υ(4S ) γ (1.22 + 0.02 −0.02 μ ± 0.13f + 0.02 −0.02 φ) · 10−8 Z0 →Υ(nS ) γ (9.96 + 0.18 −0.19 μ ± 0.09f + 0.20 −0.15 φ) · 10−8 Table 1. Predicted branching fractions for various Z →Mγ decays, including error estimates due to scale dependence (subscript “μ”) and the uncertainties in the meson decay constants (“ f”), and the shape parameters of the LCDA (“φ”). Table 1. Predicted branching fractions for various Z →Mγ decays, including error estimates due to scale dependence (subscript “μ”) and the uncertainties in the meson decay constants (“ f”), and the shape parameters of the LCDA (“φ”). 4 Exclusive hadronic decays of the Higgs boson In order to investigate the hadronic decays of the Higgs boson and understand to what extend new physics can manifest itself in them, we work in an effective Lagrangian framework, where we allow a rescaling of the relevant parameters in the SM as well as including higher-dimensional operators: LHiggs eff = κW 2m2 W v h W+ μ W−μ + κZ m2 Z v h ZμZμ −  f mf v h ¯f  κf + i˜κ f γ5  f + α 4πv κγγ h FμνFμν −˜κγγ h Fμν ˜Fμν + 2κγZ sWcW h FμνZμν −2˜κγZ sWcW h Fμν ˜Zμν . (6) (6) 3 DOI: 10.1051/ 129 epjconf/2016 00014 , 129 EPJ Web of Conferences QCD@Work 2016 00014 (2016) Figure 2. Left diagram: The dependence of Br(h →Υ(1S )γ) on the rescaling of the b-quark Yukawa coupling under the assumption that all other parameters take their SM values. Right diagram: An upper bound on Br(h → Υ(1S )γ) constrains the b-quark Yukawa to the orange region in the κb–˜κb plane, whereas the measurement of Br(h →¯bb) constrains only the absolute value κ2 b + ˜κ2 b, giving the blue band. The overlap region yields the allowed range for the b-quark Yukawa coupling in the CP-plane. Figure 2. Left diagram: The dependence of Br(h →Υ(1S )γ) on the rescaling of the b-quark Yukawa coupling under the assumption that all other parameters take their SM values. Right diagram: An upper bound on Br(h → Υ(1S )γ) constrains the b-quark Yukawa to the orange region in the κb–˜κb plane, whereas the measurement of Br(h →¯bb) constrains only the absolute value κ2 b + ˜κ2 b, giving the blue band. The overlap region yields the allowed range for the b-quark Yukawa coupling in the CP-plane. In the SM, the parameters κW, κZ and κf are equal to 1 whereas all other κ’s vanish. In the SM, the parameters κW, κZ and κf are equal to 1 whereas all other κ’s vanish. In the following, we only briefly state the main results of our analysis and direct the interested f In the following, we only briefly state the main results of our analysis and direct the interested reader towards refs. [2, 4]. 4.1 Radiative hadronic decays of the Higgs The case of h →Vγ is similar to the Z-boson decays, with the difference of an important additional contribution, where the Higgs decays into a pair of photons or into a photon and a Z-boson. Here, either the off-shell photon or Z-boson converts to the final state meson. The hγγ- and hγZ-couplings are generated at 1-loop in the SM or can occur at tree-level in our effective Lagrangian [22]. Generally, these “indirect” contributions are dominant over the ones that directly involve the Yukawa couplings of the valence quarks of the vector meson V. When one wants to use these decays to probe said Yukawa couplings, it is useful to normalize the branching ratios to the branching ratio of h →γγ, since all NP effects in the indirect contribution will affect h →γγ in the same way (up to small corrections due to the off-shellness of the photon and the Z-boson diagram). Expanding in small parameters, we find: Br(h →Vγ) Br(h →γγ) = Γ(h →Vγ) Γ(h →γγ) = 8π α2 EM(mV) αEM(0) Q2 V f 2 V m2 V ⎛⎜⎜⎜⎜⎝1 −m2 V m2 h ⎞⎟⎟⎟⎟⎠ 2 |1 −ΔV|2 + rCP −˜ΔV 2 1 + |rCP|2 . (7) (7) The parameters ΔV and ˜ΔV contain the direct contributions and the corrections to the indirect contri- butions due to the off-shellness of the photon and the additional diagram with the Z-boson: (∼) ΔV = (∼)κ VFV direct + indirect corrections . (∼) ΔV = (∼)κ VFV direct + indirect corrections . (8) (8) The direct form factors FV direct can written in a similar form to the ones in eq. (4). The parameter rCP vanishes in the SM and contains the various CP-odd couplings to the indirect amplitude and the h →γγ rate. The detailed expressions for both FV and rCP can be found in ref. [2]. Table 2 quotes our SM branching ratios. Note that in the case of h →Υ(1S ) γ the direct and indirect contributions cancel almost exactly, suppressing this decay by three orders of magnitude. This cancellation leads to a spectacular sensitivity of this branching ratio on κb and ˜κb, as demonstrated in 4 DOI: 10.1051/ 129 epjconf/2016 00014 , 129 EPJ Web of Conferences QCD@Work 2016 00014 (2016) QCD@Work 2016 Table 2. 4.1 Radiative hadronic decays of the Higgs Predicted branching fractions for various h →Vγ decays, including error estimates due to the uncertainties in the meson decay constants (“ f”), the direct contribution (“direct”) and the branching ratio Br(h →γγ). Decay mode Branching ratio h →ρ0γ (1.68 ± 0.02 f ± 0.08h→γγ) · 10−5 h →ωγ (1.48 ± 0.03 f ± 0.07h→γγ) · 10−6 h →φγ (2.31 ± 0.03 f ± 0.11h→γγ) · 10−6 h →J/ψ γ (2.95 ± 0.07f ± 0.06direct ± 0.14h→γγ) · 10−6 h →Υ(1S ) γ (4.61 ± 0.06 f + 1.75 −1.21 direct ± 0.22h→γγ) · 10−9 h →Υ(2S ) γ (2.34 ± 0.04 f + 0.75 −0.99 direct ± 0.11h→γγ) · 10−9 h →Υ(3S ) γ (2.13 ± 0.04 f + 0.75 −1.21 direct ± 0.10h→γγ) · 10−9 h Z Z h Z γ h Z Figure 3. Diagrams contributing to the h →ZM decays. The suppression from the loops in the second diagram is lifted by the near on-shell photon. uncertainties in the meson decay constants ( f ), the direct contribution ( direct ) and the branching ratio Br(h →γγ). Decay mode Branching ratio h →ρ0γ (1.68 ± 0.02 f ± 0.08h→γγ) · 10−5 h →ωγ (1.48 ± 0.03 f ± 0.07h→γγ) · 10−6 h →φγ (2.31 ± 0.03 f ± 0.11h→γγ) · 10−6 h →J/ψ γ (2.95 ± 0.07f ± 0.06direct ± 0.14h→γγ) · 10−6 h →Υ(1S ) γ (4.61 ± 0.06 f + 1.75 −1.21 direct ± 0.22h→γγ) · 10−9 h →Υ(2S ) γ (2.34 ± 0.04 f + 0.75 −0.99 direct ± 0.11h→γγ) · 10−9 h →Υ(3S ) γ (2.13 ± 0.04 f + 0.75 −1.21 direct ± 0.10h→γγ) · 10−9 h Z Z h Z γ h Z Figure 3. Diagrams contributing to the h →ZM decays. The suppression from the loops in the second diagram is lifted by the near on-shell photon. figure 3 for the cases of ˜κb vanishing (left diagram) and not vanishing (right diagram). When compared to the branching ratio of h →¯bb, even information on the CP-phase of the Yukawa coupling of the b-quark can be obtained. figure 3 for the cases of ˜κb vanishing (left diagram) and not vanishing (right diagram). When compared to the branching ratio of h →¯bb, even information on the CP-phase of the Yukawa coupling of the b-quark can be obtained. 5 Conclusions Exclusive hadronic decays of heavy electroweak bosons are a theoretically clean playground for the framework of QCD factorization. The hard scale μhard is set by the decaying bosons and thus very high, rendering the impact of power corrections of O(ΛQCD/μhard) tiny. Additionally, the hadronic uncertainties are comparably small thanks to the renormalization group evolution of the LCDA. The price to pay is the smallness of the branching ratios. For the Z-decays, the most promising experi- mental environment would be a future lepton collider running at √s = m2 Z, whereas the Higgs decays are an interesting physics target for the high-luminosity run of the LHC or a future 100 TeV proton collider. This report skips over a large amount of technical and phenomenological details, like a detailed derivation of the factorization theorem, the renormalization group evolution of the hadronic param- eters and the special case of flavor-singlet mesons as well as the W± →M±γ and h →M±W∓ decays [1–3]. We nevertheless hope to convince the reader that the physics opportunities in a dedi- cated program for the decays considered here are compelling, both within and beyond the standard model. Acknowledgements M.K is supported by the DFG Graduate School Symmetry Breaking in Fundamental Interactions (GRK 1581) and wants to thank the organizers of the workshop for the hospitality and a very interest- ing conference. 4.2 Weak radiative Higgs decays Weak Higgs decays of the form h →VZ can be used to probe the coupling of the Higgs to a photon and a Z-boson, both loop-induced and from the couplings κγZ and ˜κγZ. There are three important contributions, shown in figure 3. While naive dimensional analysis suggests that the tree-level diagram involving the hZZ gauge coupling is the dominant amplitude, the diagram with the loop-induced hγZ vertex is equally important. This is because the loop-suppression is overcome by the photon propagator being almost on its pole. For pseudoscalar mesons, the photon diagram does not exist and thus the decays h →PZ can only serve as a SM reference values. The current limits on the parameters κγZ and ˜κγZ from CMS [23] and ATLAS [24] imply upper bounds on the decay rates of 9 and 11 times the SM value, respectively, both at 95% confidence level. The stronger bound from CMS implies the constraint (under the assumption that all other parameters are at their SM value)  |κγZ −2.395|2 + |˜κγZ|2 < 7.2 . (9) (9) Table 3 quotes our branching ratios both for the SM along with the maximal deviation when the bounds (9) are saturated. Of course it should be noted, that NP effects in κγZ and ˜κγZ will also show in the direct h →γZ measurement. 5 5 DOI: 10.1051/ 129 epjconf/2016 00014 , 129 EPJ Web of Conferences QCD@Work 2016 00014 (2016) Table 3. Predicted branching fractions for various h →Vγ decays, including error estimates due to the uncertainties in the meson decay constants (“ f”), the direct contribution (“direct”) and the branching ratio Br(h →γγ). 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